Abstract

   We investigated the functions of microRNAs in the therapeutic effects
   of noni (Morinda citrifolia L.) fruit juice on mouse models of acute
   gouty arthritis induced with monosodium urate (MSU). Compared with the
   model group (treated with MSU), mice in both the positive control group
   (treated with both MSU and colchicine) and noni fruit juice group
   (treated with MSU and noni fruit juice) showed a significantly
   decreased degree of paw swelling in 5 days, as well as the contents of
   two types of proinflammatory cytokines (i.e., NALP3 and TNF-α). Based
   on the next-generation sequencing technology, a total of 3896 microRNAs
   (234 known and 3662 novel) were identified in mice treated with noni
   fruit juice. A large amount of differentially expressed miRNAs were
   identified in the noni fruit juice group, suggesting the significant
   effects of noni fruit juice on the mice with acute gouty arthritis,
   while the different patterns of change in the numbers of both
   upregulated and downregulated miRNAs in both noni fruit juice and
   positive control groups indicated that the mice of acute gouty
   arthritis may be regulated by differential mechanisms between the
   treatments of noni fruit juice and colchicine. The target genes of
   microRNAs involved in the pathogenesis and pathology of acute gouty
   arthritis in mice were identified and further annotated by both Gene
   Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)
   enrichment analyses. Our results revealed the therapeutic effects of
   noni fruit juice on acute gouty arthritis in mice with a group of
   microRNAs involved in the pharmacological mechanisms of noni fruit
   juice, providing scientific evidence to support both the agricultural
   cultivation and pharmacological significance of noni plants.

   Keywords: acute gouty arthritis, mice, monosodium urate, Morinda
   citrifolia, microRNA, noni fruit juice

1. Introduction

   Gout is a chronic disease in animals caused by the deposition of
   monosodium urate (MSU) crystal with typical symptoms of acute,
   self-limiting inflammatory arthritis affecting the joints in lower
   limbs [[48]1], showing abnormally high levels of serum uric acids due
   to the disorders of purine metabolism. Gouty arthritis is one of the
   most common rheumatic diseases with rising incidence worldwide probably
   due to the increased incidence of comorbidities and use of causative
   medications as well as the change of lifestyle, i.e., diet and living
   habits [[49]2,[50]3]. For example, as recognized as the most common
   form of inflammatory arthritis, gouty arthritis shows a prevalence of
   0.9–2.5% in Europe, 3.9% in the USA, over 6% in some Oceanic–Pacific
   ethnic groups [[51]4], and 4.5–6.8% in Australia [[52]5], and is
   predicted with an increasing mortality rate [[53]6], affecting ~3.9% of
   people worldwide and causing significant clinical burden globally
   [[54]7,[55]8].

   Acute arthritis is recognized as the most common initial symptom of
   primary gout with typical symptoms including redness, swelling, and
   severe pain in the affected joints and surrounding tissues [[56]9]. In
   the occurrence of acute gouty arthritis, the MSU crystals are formed
   due to the supersaturation of uric acid in the affected joints to
   interact with local microenvironment, triggering the innate immune
   response to induce the inflammation cascades and ultimately causing
   severe inflammation in the joints. In this process, a large number of
   inflammatory factors and cellular contents are released as key factors
   involved in the pathogenesis of acute gouty arthritis [[57]10,[58]11].
   As a member of the family of the nucleotide-binding oligomerization
   domain-like receptors (NLRs), cytokine NALP3, also known as nod-like
   receptor protein 3 (NLRP3), is encoded by gene NLRP3, playing an
   important role in the MSU-mediated gout inflammation and immune
   regulation [[59]12]. As one of the proinflammatory factors, tumor
   necrosis factor-α (TNF-α) also plays important roles in the
   pathogenesis of osteoarticular inflammation, while a large amount of
   TNF-α is recovered in patients of gout. Molecular studies have shown
   that the polymorphism in the promoter region of TNF-α gene promotes the
   development of gouty arthritis [[60]13]. Furthermore, the TNF-α is
   involved in the expression of pro-IL-1β mRNA and IL-1β protein in gout,
   triggering the MSU-induced inflammation in mice [[61]14]. Moreover,
   studies have shown that TNF-α induces the proliferation of synovial
   fibroblast-like cells and increases the RNA expression in synovial
   cells, ultimately leading to inflammation [[62]15].

   The pathogenetic mechanisms of acute gouty arthritis have recently been
   widely explored at the molecular, genetic, and genomic levels. The
   progress in these areas has significantly advanced the identification
   of potential therapeutic targets in the treatment of acute gouty
   arthritis with many inflammatory mediators identified as being
   regulated by microRNAs (miRNAs), which could be potentially used as
   biomarkers for the diagnosis and prognosis of acute gouty arthritis
   [[63]16,[64]17]. Furthermore, the promising possibility has been raised
   to apply genetic testing in personalized treatment of patients with
   gout [[65]18]. For example, as one of the important proinflammatory
   cytokines strongly associated with the inflammation of gout, the IL-1β
   has been identified as the target of several miRNAs
   [[66]19,[67]20,[68]21,[69]22,[70]23,[71]24], though the regulatory
   roles of these miRNAs still need to be experimentally verified in vivo
   [[72]25]. As another proinflammatory cytokine, the TNF-α has been
   regulated by various types of miRNAs involved in several signaling
   pathways
   [[73]19,[74]22,[75]23,[76]24,[77]26,[78]27,[79]28,[80]29,[81]30,[82]31,
   [83]32,[84]33,[85]34,[86]35,[87]36].

   Acute gouty arthritis is generally treated with one or two of the
   nonsteroidal anti-inflammatory drugs (NSAIDs), colchicine, or
   corticosteroids, while the xanthine oxidase inhibitor therapy is used
   to prevent the recurrent gout [[88]1,[89]2,[90]3]. Although these drugs
   have shown curative effects in a short term, their effectiveness still
   needs improvement. Furthermore, these common medicines used for a long
   time in treatment of gout should be taken with precaution due to their
   severe side effects including gastrointestinal and renal toxicities
   [[91]37,[92]38,[93]39,[94]40]. Therefore, it is clinically important to
   develop and identify alternative medicines to treat acute gouty
   arthritis without toxic and side effects [[95]2]. Notably, traditional
   Chinese medicines (e.g., extracted agents from some herbal plants) have
   achieved satisfactory effects in treating gout with low toxicity and
   reduced side effects [[96]41,[97]42]. Similar effects have been
   realized by other natural products including curcumin, polydatin, and
   pterostilbene in treating gout [[98]43,[99]44,[100]45,[101]46]. Several
   species of plants have been identified as having the anti-gout
   potential due to their high inhibitory effects on xanthine oxidase
   [[102]47]. In particular, the effects of fruit or fruit juice intake on
   treating gout have been complex and controversial [[103]48].

   As a member of Rubiaceae and a native tropical shrub in Asia and
   Polynesia, noni (Morinda citrifolia L.) is also known as Indian
   mulberry in India, mengkudu in Malaysia, painkiller bush in the
   Caribbean, cheese fruit in Australia, and ba ji tian in China
   [[104]49]. In the early 1990s, due to its wide applications in health
   and food industries, the culture of noni plants showed significant
   contributions in agriculture, industry, and pharmacology, globally
   spreading from Polynesian and Hawaiian cultures [[105]50,[106]51].
   Recently, the complete chloroplast genome of noni was sequenced,
   providing the foundation for further molecular studies of this
   economically important plant [[107]52]. Noni fruits have been
   historically recognized as a traditional medicinal herb for over 2000
   years due to their promising medicinal properties [[108]53]. For
   example, nearly all parts of noni plants (i.e., roots, stem, bark,
   fruits, and leaves) have been used as curative or preventive treatments
   of various acute and chronic diseases [[109]54,[110]55], probably
   functioning by stimulating the immune system [[111]56]. In 2003,
   Tahitian noni juice was approved as a novel type of food by the Health
   and Consumer Protection Directorate General of the European Commission
   [[112]55]. Bioassays in vitro have shown that the molecular mechanisms
   of the therapeutic effects of the Tahitian noni juice on gout are
   attributed to its inhibitory effect on the xanthine oxidase [[113]57].
   Furthermore, studies have shown that noni fruits contain polyphenolic
   compounds (e.g., quercetin, rutin, scopoletin, and kaempferol) with
   potent antioxidant and neuroprotective effects in animals [[114]58].
   Noni has also been demonstrated as a novel natural product with
   prophylactic and therapeutic effects on many human diseases [[115]59]
   and used as therapeutic agent in antipsychotic, anxiolytic, sedative,
   and hypnotic treatments [[116]55,[117]60]. Due to the wide spectrum of
   claimed medical and therapeutic utilities, it is urgent to obtain
   scientific evidence to verify the pharmacological effectiveness of noni
   fruit juice. Therefore, it is important to investigate and confirm the
   therapeutic effects of noni fruit juice and further establish its wide
   medical applications.

   In this study, we investigated the potential therapeutic effects of
   noni fruit juice on acute gouty arthritis in vivo. We established the
   mouse model of acute gouty arthritis with the treatment of MSU. Our
   goals were to evaluate the therapeutic effects of noni fruit juice on
   acute gouty arthritis in mice by assessing the phenotypic
   characteristics (i.e., the paw swelling degree in mice), the contents
   of two types of proinflammatory cytokines (i.e., NALP3 and TNF-α), and
   the expression patterns of miRNAs, to ultimately illustrate the
   potential pharmacological mechanisms of noni fruit juice on acute gouty
   arthritis in mice. We further investigated the potential regulatory
   roles of miRNAs in the biological processes of the treatment of noni
   fruit juice on acute gouty arthritis in mice based on both Gene
   Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)
   enrichment analyses of the target genes of the differentially expressed
   miRNAs in mice treated with noni fruit juice.

2. Materials and Methods

2.1. Experimental Animals

   A total of 40 male Kunming mice (~5 weeks old with an average body
   weight of 25 ± 3 g) were purchased from the Liaoning Changsheng
   Biotechnology Co., Ltd. (Shenyang, China) with an animal certificate
   number of SCXK (Liao) 2015-0001. The selection of this type of mice was
   based on the previous studies on the acute gouty arthritis in mice
   [[118]61]. These mice were randomly and evenly divided into four
   groups, i.e., one normal control group and three experimental groups,
   including the model group (treated with MSU), the positive control
   group (treated with both MSU and colchicine), and the noni fruit juice
   group (treated with both MSU and noni fruit juice). The establishment
   of the positive control group was based on the results of previous
   studies showing that the application of colchicine was effective on the
   treatment of acute gouty arthritis [[119]2,[120]21]. After one week of
   adaptive feeding, the right hind feet of the 30 mice in the three
   experimental groups were first disinfected with medical ethanol (75%)
   and then injected subcutaneously with 0.05 mL of MSU (Sigma, St. Louis,
   MO, USA) crystal suspension (2.5%) with sterile syringes on both the
   ventral and dorsal sides of the foot, respectively. In one hour,
   compared with the 10 untreated mice in the normal control group, the
   mice treated with MSU showed evident swelling in their right hind feet
   with no evident liquid overflow, indicating the successful construction
   of the mouse model of acute gouty arthritis ([121]Figure 1). For the
   next five days, the mice in the noni fruit juice group and the positive
   control group were fed with noni fruit juice (7.8 mL/kg d^−1) and
   colchicine solution (Sigma, St. Louis, MO, USA) (0.975 mg/kg d^−1),
   respectively, while the mice in the model and the normal control groups
   were fed with saline water (7.8 mL/kg d^−1). All mice were fed with
   oral gavage needles. For the entire experiment, the mice were kept in
   cages with natural sunlight, constant temperature and humidity, and
   which were regularly cleaned and disinfected. After the experiments,
   the mice were euthanized with inhalation of carbon dioxide gas.

Figure 1.

   [122]Figure 1
   [123]Open in a new tab

   The ventral and dorsal views of the same mouse showing the right hind
   paw before (A,B) and 1 h after (C,D) the application of monosodium
   urate (MSU). The mice treated with MSU showed evident swelling in their
   right hind feet, indicating the successful construction of the mouse
   model of acute gouty arthritis. Bar = 4.15, 5.36, 4.85, and 5.48 mm in
   (A–D), respectively.

2.2. Production of the Noni Fruit Juice

   Fresh and mature noni fruits were obtained from the Fiji Pacific Noni
   Biotechnology Co., Ltd. (Hainan, China). These fruits were locally
   picked and kept frozen at −20 °C (Nadi, Fiji). Fruits thawed at room
   temperature were washed with sterile water, air-dried, cut into blocks
   each of ~5 mm thickness, and crushed by a fruit crusher. The fruit pulp
   was heated to ~25 °C, mixed with pectinase (0.225 g/L) while stirring,
   then inoculated with Lactobacillus plantarum (1%) to incubate for 4 h
   in an incubator (Shanghai Yiheng Scientific Instrument Co., Ltd.,
   Shanghai, China) at 40 °C. Then, the fruit pulp was filtered with the
   20-mesh filter cloth and fermented for 20–30 days at 38 °C. The
   supernatant was collected with a siphon, filtered, sterilized for 20
   min at 80–82 °C, and aseptically collected as the fermented noni fruit
   juice.

2.3. Measurement of the Paw Swelling in Mice

   To evaluate the effectiveness of noni fruit juice on treating acute
   gouty arthritis in mice, the phenotypic characteristics were observed
   by measuring the paw thickness (i.e., the distance between the ventral
   and dorsal sides of the paw including the foot pad) of mice with a
   digital vernier caliper before and after (1 and 5 days) the treatments.
   The paw swelling degree of mice was calculated as the difference
   between the thickness of the right hind paw before and after (1 and 5
   days) the treatments in three experimental and the control groups of
   mice. Data were presented as mean ± standard deviation and were
   statistically analyzed using SPSS 19.0. with the significant difference
   of ANOVA set at p-value ≤ 0.05.

2.4. Measurement of the Contents of NALP3 and TNF-α

   Due to their direct involvement in the pathogenesis of acute gouty
   arthritis, the proinflammatory cytokines NALP3 and TNF-α were chosen to
   assess the effectiveness of noni fruit juice on the treatment of acute
   gouty arthritis in mice. In 5 days, the blood sample (~1.0 mL) was
   collected from the eyeballs of all mice and incubated in an
   electrothermal incubator (Shanghai Yiheng Scientific Instrument Co.,
   Ltd., Shanghai, China) for 1 h at 37 °C, kept for 3 h at 4 °C, and
   centrifuged for 10 min at 3000 rpm to separate the serum (~0.5 mL) from
   the red blood cells. The supernatant was collected for the
   enzyme-linked immunosorbent assay (ELISA) analysis to detect the
   contents of NALP3 and TNF-α using the NALP3 and TNF-α ELISA kits
   (Shanghai Le Pure Biological Technology Co., Ltd., Shanghai, China),
   respectively, in accordance with the manufacturer’s instructions with
   the concentration of the cytokines measured at 450 nm by the microplate
   enzyme reader (Tecan Austria Gmbh, Salzburg, Austria). Data were
   presented as mean ± standard deviation and were statistically analyzed
   using SPSS 19.0. with the significant difference of ANOVA set at
   p-value ≤ 0.05.

2.5. High-Throughput Sequencing and Analysis of microRNAs

   In order to explore the functions of small RNAs in the pathogenesis and
   treatment of acute gouty arthritis mediated by MSU and noni fruit
   juice, respectively, we randomly selected a representative mouse serum
   sample from each of the four groups of mice to identify the miRNAs
   using the next-generation sequencing platform BGISEQ-500 (BGI,
   Shenzhen, China) [[124]62,[125]63]. To extract the total RNA, the
   QIAzol Lysis Reagent (1000 μL) was added to the serum sample (200 μL),
   vortexed evenly, and kept for 5 min at room temperature. Then, a total
   of 200 μL chloroform/isoamyl alcohol (24:1) was added to the reaction,
   vortexed or shaken vigorously for 15 s, and incubated for 2–3 min at
   room temperature. The reaction was centrifuged for 8 min at 12,000× g
   and 4 °C (5427R, Eppendorf Co., Ltd., Beijing, China). The supernatant
   was collected and added with absolute ethanol of twice the volume of
   the supernatant and mixed well. The mixed solution was filtered through
   the column for purification, and washed once with buffer RWT (700 μL)
   and twice with buffer RPE (500 μL). The solution was centrifuged for 2
   min at 12,000× g and room temperature, and the purification column was
   moved to a new collection tube with 20 μL RNA-free water, incubated for
   1 min at room temperature, and finally centrifuged for 2 min at 12,000×
   g and room temperature to elute RNA. The concentration of the eluted
   RNA was measured by the Agilent 2100 Bioanalyzer (Agilent Technologies
   Co., Ltd., Beijing, China). To construct the libraries for
   high-throughput sequencing of microRNAs, a total of 200–1000 ng of RNA
   sample was used to filter the small RNAs and to separate RNA segments
   of different sizes by PAGE gel and the stripes of 18–30 nt (14–30 ssRNA
   Ladder Marker, TAKARA) were recycled. The connection 3′adaptor system
   was prepared to complete the adaptor ligation with the reaction
   condition of 70 °C for 2 min and 25 °C for 2 h; the RT-Primer was added
   with the reaction conditions of 65 °C for 15 min and ramp to 4 °C at a
   rate of 0.3 °C/s; the 5′adaptor mix system was added with the reaction
   condition of 70 °C for 2 min and 25 °C for 1 h. The RT-PCR reaction was
   conducted as follows: The First Strand Master Mix and Super Script II
   (Invitrogen) reverse transcription were prepared with the reaction
   condition of 42 °C for 1 h and 70 °C for 15 min; and several rounds of
   PCR amplification with PCR Primer Cocktail and PCR Master Mix were
   performed to enrich the cDNA fragments with the following reaction
   conditions: 95 °C for 3 min, 15–18 cycles of “98 °C for 20 s, 56 °C for
   15 s, and 72 °C for 15 s”, and extension at 72 °C for 10 min; 4 °C
   hold. The PCR products were purified with PAGE gel and the recycled
   products were dissolved in EB solution. The double-stranded PCR
   products were heat denatured and circularized by the splint oligo
   sequence. The single strand circle DNA (ssCir DNA) was formatted as the
   final library, which was validated on the Agilent 2100 Bioanalyzer. The
   library was amplified with phi29 to make DNA nanoball (DNB) containing
   more than 300 copies of one molecule. The DNBs were loaded into the
   patterned nanoarray and single-end 50 base reads were generated in the
   way of combinatorial probe-anchor synthesis (cPAS).

   The clean sequencing tags were annotated using Bowtie2
   ([126]http://www.bowtie-bio.sourceforge.net/bowtie2/ (accessed on 21
   April 2019)) based on various small RNA databases including miRbase
   ([127]http://www.mirbase.org/ (accessed on 21 April 2019)), Rfam
   ([128]https://rfam.xfam.org/ (accessed on 21 April 2019)), siRNA
   ([129]http://web.mit.edu/sirna/ (accessed on 21 April 2019)), piRNA
   ([130]https://www.pirnadb.org/ (accessed on 21 April 2019)), and snoRNA
   ([131]http://snoopy.med.miyazaki-u.ac.jp (accessed on 21 April 2019)).
   Based on the results of these annotations, the unknown tags were
   further annotated to predict novel miRNAs using miRDeep and miRA
   [[132]64,[133]65,[134]66].

2.6. Identification and Analysis of Differentially Expressed Genes of
microRNAs

   A stringent algorithm was developed to identify the differentially
   expressed genes (DEGs) of miRNAs in mice according to Audic and
   Claverie [[135]67]. By using the false discovery rate (FDR) method, the
   FDR ≤ 0.001 and the absolute value of Log2Ratio(Fold Change) ≤ 1 were
   set as the default thresholds to evaluate the significant difference in
   gene expression. The hierarchical clustering of differentially
   expressed miRNAs was constructed using the function “pheatmap” of R
   package ([136]http://www.r-project.org (accessed on 25 April 2019)).
   The miRNAs were further processed to predict their target genes jointly
   by both miRanda [[137]68] and TargetScan [[138]69]. The functional
   enrichment analyses of the target genes of differentially expressed
   miRNAs were further conducted based on Gene Ontology (GO;
   [139]http://www.geneontology.org/ (accessed on 25 April 2019)) database
   using WEGO software [[140]70] and Kyoto Encyclopedia of Genes and
   Genomes (KEGG; [141]http://www.genome.jp/kegg/ (accessed on 25 April
   2019)) database [[142]71], respectively.

3. Results and Discussion

3.1. Paw Swelling Degree in Mice

   The phenotypic characteristics of the mice treated with either
   colchicine or noni fruit juice showed significant changes in comparison
   to those of the model group. The results of the paw swelling degree in
   the mice of these three experimental groups in 1 and 5 days after the
   treatments were shown in [143]Table 1. In one day, the paw swelling
   degree in mice of the colchicine group was significantly decreased in
   comparison to that of the model group, while the significantly
   decreased paw swelling degree was observed in 5 days in the noni fruit
   juice group. Overall, the noni fruit juice showed less effectiveness
   than that of the colchicine in the treatment of swollen paws in mice.
   These results suggested that the therapeutic effects of the noni fruit
   juice on acute gouty arthritis started slower with probably a long-term
   effect in comparison to that of the colchicine [[144]72].

Table 1.

   The paw swelling (mm) in mice of the three experimental and the control
   groups before and after (1 and 5 days) treatments. Data are presented
   as mean ± standard deviation with the paw swelling degree (i.e., the
   difference of the paw thickness before and after the treatment) given
   in parentheses (n = 10).
   Group of Mice Day 0 Day 1 Day 5
   Normal control group 1.69 ± 0.08 1.72 ± 0.12 (0.12 ± 0.08) ** 1.71 ±
   0.08 (0.04 ± 0.02) **
   Model group 1.70 ± 0.10 3.70 ± 0.25 (2.01 ± 0.22) 2.89 ± 0.27 (1.19 ±
   0.24)
   Colchicine group 1.70 ± 0.11 3.50 ± 0.21 (1.80 ± 0.16) * 2.31 ± 0.25
   (0.61 ± 0.27) **
   Noni fruit juice group 1.74 ± 0.07 3.65 ± 0.16 (1.91 ± 0.16) 2.68 ±
   0.20 (0.94 ± 0.21) *
   [145]Open in a new tab

   Symbols “*” and “**” indicate the significant difference at p < 0.05
   and p < 0.01, respectively, in comparison to the model group.

   The phenotypic observations of the reduced paw swelling degree in mice
   are generally used to evaluate the therapeutic effects of medicines on
   acute gouty arthritis. For example, the ethanol extracts from one of
   the traditional Chinese medicinal herbal plants (Gnaphalium
   pensylvanicum Willd.) have been shown to reduce the paw swelling in
   mice likely by decreasing the serum uric acid and inhibiting the
   xanthine oxidase [[146]73]. Similarly, the combination of ethanol
   extracts from a few traditional Chinese medicinal herbal plants showed
   therapeutic effects on acute gouty arthritis by attenuating the degree
   of ankle swelling and inflammation in mice [[147]9]. These results are
   consistent with our findings showing the therapeutic effects of noni
   fruit juice on acute gouty arthritis in mice. The molecular mechanisms
   of the therapeutic effects of noni fruit juice on acute gouty arthritis
   in mice were further explored by evaluating the change of the two main
   proinflammatory cytokines, i.e., NALP3 and TNF-α (below).

3.2. Contents of NALP3 and TNF-α

   Due to their direct involvement in the pathogenesis of acute gouty
   arthritis, the levels of both proinflammatory cytokines of NALP3 and
   TNF-α were detected to evaluate the effectiveness of noni fruit juice
   on the treatment of acute gouty arthritis in mice. In 5 days after the
   treatments, the contents of NALPs and TNF-α were presented in
   [148]Table 2.

Table 2.

   Contents of NALP3 and TNF-α detected in the serum of three experimental
   and the control groups of mice 5 days after treatments. Data are
   presented as mean ± standard deviation (n = 10). Symbols “*” and “**”
   indicate the significant difference at p < 0.05 and p < 0. 01,
   respectively, in comparison to the model group.
   Group of Mice          NALP3 (pg/mL)    TNF-α (pg/mL)
   Normal control group   86.09 ± 6.92 **  37.07 ± 4.49 **
   Model group            128.90 ± 9.31    71.94 ± 9.76
   Colchicine group       102.73 ± 7.25 ** 43.09 ± 3.76 **
   Noni fruit juice group 110.19 ± 8.25 *  62.06 ± 7.29 *
   [149]Open in a new tab

   In comparison to the model group, the contents of both NALP3 and TNF-α
   in mice of the normal control, the colchicine, and the noni fruit juice
   groups were significantly decreased. These findings were in accordance
   with the molecular pathways of the generation of acute gouty arthritis,
   showing that upon the recognition of MSU, the molecular structure of
   NALP3 was altered to activate the NALP3 inflammasome, ultimately
   generating a large amount of TNF-α [[150]12]. Similar to the results of
   the treatment of noni fruit juice on the paw swelling degree in mice,
   these results of the change of both NALP3 and TNF-α in mice
   demonstrated again the therapeutic effects of noni fruit juice on
   treating acute gouty arthritis in mice. Overall, the noni fruit juice
   was less effective than colchicine in reducing the levels of NALPs and
   TNF-α in mice with acute gouty arthritis. Furthermore, our results were
   consistent with those previously reported in the treatment of acute
   gouty arthritis. For example, the expression of both NALP3 and TNF-α,
   which were involved in the NLRP3/apoptosis-associated speck-like
   protein/caspase-1 pathways, was significantly decreased in mice of
   acute gouty arthritis treated with the alcohol extract from Polygonum
   cuspidatum [[151]74]. Similarly, a recent study demonstrated that the
   chemical extracts from the traditional Chinese medicinal herbal plant
   (i.e., Dendrobium loddigesii Rolfe) showed anti-gout activities in rat
   model of acute gouty arthritis [[152]75]. Specifically, the chemical
   extracts inhibited the acute gouty arthritis in rats and showed
   anti-inflammatory effects as demonstrated by reduced levels of
   proinflammatory cytokines including IL-1β and TNF-α. Furthermore,
   studies have shown that the combined ethanol extracts from a few
   traditional Chinese herbal medicinal plants decreased the levels of
   TNF-α but not of IL-1β in the rat model of acute gouty arthritis
   [[153]9]. These results suggested that the noni fruit juice showed
   therapeutic effects on acute gouty arthritis and that the chemical
   extracts of different herbal plants may be involved in their
   therapeutic effects on acute gouty arthritis with different molecular
   mechanisms. As one of the proinflammatory mediators involved in the
   pathogenesis of acute gouty arthritis, TNF-α has been constantly shown
   with increased expression in the subjects of acute gouty arthritis
   [[154]22]. Notably, the polysaccharides extracted from noni fruits have
   been shown to decrease the levels of TNF-α in mice treated with acetic
   acid [[155]49]. These results suggested that the noni fruit juice
   extracted in our study may contain the same or similar chemical
   contents as the polysaccharides as reported previously
   [[156]49,[157]76]. Future studies are necessary to further identify and
   characterize the functional substances in noni fruit juice [[158]77].

   Studies have shown that the proinflammatory cytokines (e.g., IL-1β and
   TNF-α) are regulated by various types of miRNAs
   [[159]19,[160]22,[161]37,[162]78,[163]79,[164]80]. Similarly, several
   miRNAs have been identified as potential markers associated with NALP3
   in the development of acute gouty arthritis [[165]17,[166]80]. These
   results strongly indicated that the noni fruit juice showed its
   therapeutic effects on acute gouty arthritis by inhibiting the
   TLR/MyD88/NF-kB pathways (i.e., reducing the content of TNF-α). Further
   studies are necessary to investigate the mechanism of noni fruit juice
   in regulating the expression of these cytokines in the occurrence of
   acute gouty arthritis. We further explored the potential regulatory
   functions of miRNAs in the treatment of noni fruit juice on mice of
   acute gouty arthritis (below).

3.3. Small RNA Sequencing

3.3.1. Characteristics of Small RNAs

   To investigate the therapeutic effects of noni fruit juice on acute
   gouty arthritis in mice, miRNAs were sequenced using the
   next-generation sequencing platform BGISEQ-500 technology. The
   low-quality tags were removed prior to further data analyses
   ([167]Table 3). The results of the length distribution analysis showed
   that the composition of the small RNAs generally ranged from 18 to 30
   bases in length with the miRNAs of 21–22 bases.

Table 3.

   Statistics of small RNA sequencing in three experimental and the
   control groups of mice based on BGISEQ-500 technology. The percentage
   of clean tag is calculated as (clean tag counts/raw tag counts) × 100%.
   The percentage of mapped tag is calculated as (mapped tag counts/clean
   tag counts) × 100%.
   Sample Raw Tag Clean Tag (%) Q20 of Clean Tag (%) Mapped Tag (%)
   Normal control group 29,268,292 23,957,783 (81.86) 99.20 21,749,074
   (90.78)
   Model group 26,562,125 18,683,543 (70.34) 99.30 15,904,741 (85.13)
   Colchicine group 27,814,896 25,478,925 (91.60) 99.20 24,180,100 (94.90)
   Noni fruit juice group 28,571,428 24,142,747 (84.50) 99.30 22,911,510
   (94.90)
   [168]Open in a new tab

   There was a total of 92,262,998 (~82.22%) valid reads (out of
   112,216,741 raw tags) obtained in four groups of mice with 84,745,425
   clean tags (~91.85%) identified as various types of RNAs ([169]Figure
   S1; Table S1). The raw sequencing data were deposited in the Sequence
   Read Archive (SRA) at the National Center for Biotechnology Information
   (NCBI) ([170]http://www.ncbi.nlm.nih.gov/sra (accessed on 7 April
   2021)) under the BioProject accession number of PRJNA719968. In both
   the noni fruit juice and normal control groups, most small RNAs with
   higher proportions ranged from 10 to 33 bases with the most abundant
   small RNAs of 20 and 31 bases. In the model group, the length
   distributions of most small RNAs ranged from 18 to 22 bases with the
   most abundant small RNAs of 20 bases, while the length distributions of
   most small RNAs ranged from 29 to 33 bases with the most abundant small
   RNAs of 31 bases in the colchicine group. A total of 271, 350, 331, and
   234 known miRNAs and a total of 2146, 1396, 581, and 3662 miRNAs were
   identified as novel in the normal control, the model, the colchicine
   control, and the noni fruit juice groups, respectively ([171]Table 3
   and [172]Table S2).

3.3.2. Differentially Expressed microRNAs

   Differentially expressed miRNAs ([173]Table S3) were identified based
   on the values of FDR ≤ 0.001 and |Log2Ratio(Fold Change)| ≤ 1 in the
   six pairwise comparisons among the three experimental and the control
   groups of mice in order to evaluate the variation in miRNA gene
   expression profiles in response to MSU (the model group), colchicine
   (the positive control group), and noni fruit juice in mice ([174]Figure
   2). The heatmaps based on intersection and union of miRNAs in four
   groups of mice ([175]Figure S2) and differentially expressed miRNAs in
   six pairwise comparisons of four groups of mice ([176]Figure S3) were
   generated to show the hierarchical clustering of these miRNAs.

Figure 2.

   [177]Figure 2
   [178]Open in a new tab

   Differentially expressed microRNAs in the six pairwise comparisons of
   the three experimental and the normal control groups of mice.

   The results showed that the highest numbers of differentially expressed
   miRNAs were revealed in the noni fruit juice group ([179]Figure 2). Two
   of the six pairwise comparisons of experimental and control groups,
   i.e., the normal control group vs. noni fruit juice group and the model
   group vs. noni fruit juice group showed two of the highest number of
   upregulated miRNAs of 2624 and 2963, respectively. The highest number
   of downregulated miRNAs (3460) were revealed in the comparison between
   the noni fruit juice group vs. colchicine group, while the lowest
   numbers of upregulated miRNAs of 130, 275, and 191 were revealed in the
   pairwise groups of the normal control group vs. colchicine group, the
   model group vs. colchicine group, and the noni fruit juice group vs.
   colchicine group, respectively. The other three pairwise comparisons
   (i.e., the normal control group vs. model group, the normal control
   group vs. colchicine group, and the model group vs. colchicine group)
   showed relatively smaller numbers of differentially expressed miRNAs of
   2670, 2205, and 1635, respectively. Evidently, the large amount of
   differentially expressed miRNAs were identified in the noni fruit juice
   groups, indicating the significant effects of noni fruit juice on the
   mice with acute gouty arthritis. The large number of down-regulated
   miRNAs suggested the additive effects of both noni fruit juice and
   colchicine on the mice with acute gouty arthritis, while the different
   patterns of change in the numbers of both upregulated and downregulated
   miRNAs in both noni fruit juice and colchicine groups suggested that
   the mice of acute gouty arthritis may be regulated by differential
   mechanisms between the treatments of noni fruit juice and colchicine.
   Further studies are needed to identify the explicit functions of these
   miRNAs in the development and therapeutic treatment of acute gouty
   arthritis in mice.

3.3.3. Target Genes of microRNAs in Mice

   A total of 13,958 target genes of the miRNAs in the experimental and
   control groups of mice were predicted based on both miRanda and
   TargetScan with a total of 13,552 target genes identified in all four
   groups of mice ([180]Figure 3; [181]Table S4). Results showed that in
   the pairwise comparisons, a total of 133 and 14 target genes were
   uniquely identified in the noni fruit juice and the model groups,
   respectively, while a total of 38 and 252 target genes were exclusively
   revealed in the colchicine and the model groups, respectively.
   Furthermore, when the four groups of mice were compared, a total of 10,
   43, 3, and 8 target genes annotated by a total of 10 novel and 1 known,
   60 novel, 3 known, and 9 novel and 1 known miRNAs were exclusively
   identified in the normal control, the noni fruit juice, the colchicine,
   and the model groups of mice, respectively. These results suggested
   that the therapeutic effects of noni fruit juice on acute gouty
   arthritis in mice were probably regulated by different molecular
   mechanisms from those of colchicine. Future studies are necessary to
   investigate the explicit functions of these miRNAs and their target
   genes in the pathology and pathogenesis of acute gouty arthritis and
   the pharmacological mechanisms of noni fruit juice in treating acute
   gouty arthritis in mice.

Figure 3.

   [182]Figure 3
   [183]Open in a new tab

   Target genes of microRNAs predicted by both TargetScan and miRanda in
   four groups of mice.

3.3.4. Enrichment Analyses of Target Genes of differentially Expressed
microRNAs

   Gene Ontology (GO) enrichment analysis was performed to annotate the
   functions of target genes of the differentially expressed miRNAs
   identified in the six pairwise comparisons among the three experimental
   and the control groups of mice. Results showed that a total of ~78,000
   (ranging from 78,445 to 78,864), ~56,000 (ranging from 56,319 to
   56,744), and ~16,000 (ranging from 16,841 to 16,953) target genes were
   annotated in three categories of GO database, i.e., cellular component,
   molecular function, and biological process, respectively ([184]Figure
   4; [185]Table S4). Similar patterns of gene annotation were observed in
   the six pairwise comparisons of the experimental and control groups in
   mice, with the greatest number of genes mapped to the category of
   biological process of GO terms, while the category of molecular
   function contained the least number of genes annotated. In the category
   of biological process of GO database, five GO terms showed the highest
   numbers of functional genes annotated (~6500–9100), including cellular
   process, single-organism process, metabolic process, biological
   regulation, and regulation of biological process, while the other 21
   terms each contained less than 5200 genes annotated ranging from 16 to
   5016. In the category of cellular component in GO database, three GO
   terms (i.e., cell, cell part, and organelle) showed the highest numbers
   of functional genes annotated (~8200–9800), while the other 16 terms
   each contained less than 5700 genes annotated ranging from 11 to 5693.
   In the category of molecular function, two GO terms (i.e., binding and
   catalytic activity) were revealed to contain the largest numbers of
   annotated genes (~3800–8100), while the other 18 terms each contained
   less than 900 genes annotated ranging from 3 to 890.

Figure 4.

   [186]Figure 4
   [187]Open in a new tab

   Functional annotations based on the Gene Ontology (GO) database of the
   differentially expressed microRNAs identified in the six pairwise
   comparisons of the three experimental and the control groups in mice,
   including normal control group vs. model group (A), normal control
   group vs. colchicine group (B), normal control group vs. noni fruit
   juice group (C), model group vs. noni fruit group (D), model group vs.
   colchicine group (E), and noni fruit juice group vs. colchicine group
   (F). Comparable results were revealed among the six pairwise
   comparisons of the experimental and control groups of mice, showing the
   largest and the lowest numbers of genes annotated in the categories of
   cellular component and biological process of the GO database,
   respectively.

   As one of the major pathways involved in the pathology of acute gouty
   arthritis, the formation of NLRP3 (NALP3) inflammasome complex and the
   regulations of NLRP3 inflammasome complex assembly were further
   investigated based on the GO annotation of the target genes of
   differentially expressed miRNAs identified in the six pairwise
   comparisons of the experimental and control groups of mice ([188]Table
   4; [189]Table S5). In the categories of both cellular component and
   biological process in GO database, many up- and downregulated novel
   miRNAs were involved in the formation of NLRP3 inflammasome complex and
   the positive and negative regulations of NLRP3 inflammasome complex
   assembly. These results indicated that the metabolic pathways of NLRP3
   inflammasome complex was affected in mice treated with noni fruit
   juice, suggesting that these differentially expressed miRNAs of these
   GO terms may be related to the metabolic pathways of the NLRP3
   inflammasome complex. These results were consistent with those based on
   the pathogenetic investigations in our study and with those reported
   previously [[190]17]. Further studies are necessary to investigate the
   detailed regulatory functions of these novel miRNAs in the occurrence
   of acute gouty arthritis and the pharmacological mechanisms of noni
   fruit juice in treating acute gouty arthritis in mice.

Table 4.

   Expression patterns of microRNAs targeting the genes involved in the
   formation of NLRP3 (NALP3) inflammasome complex and the positive and
   negative regulations of NLRP3 inflammasome complex assembly based on
   the Gene Ontology (GO) annotation in the six pairwise comparisons among
   the experimental and control groups of mice. Data are presented as
   number of (known)/(novel) miRNAs. Symbols “↑” and “↓” indicate up- and
   down-regulation, respectively.
   GO Term and Gene Control vs. Model Groups Control vs. Noni Fruit Juice
   Groups Control vs. Colchicine Groups Model vs. Noni Fruit Juice Groups
   Noni Fruit Juice vs. Colchicine Groups Model vs. Colchicine Groups
   Formation of NLRP3 inflammasome complex (GO: 0072559)
   [191]NM_026960.4 (2↑)/(9↑, 13↓) (3↓)/(21↑, 8↓) (3↓)/(2↑, 17↓)
   (3↓)/(26↑, 8↓) (1↑)/(28↓) (3↓)/(2↑, 11↓)
   [192]NM_009807.2 (0) /(1↑, 1↓) (0)/(5↑) (0)/(1↓) (0)/(6↑, 1↓) (0)/(6↓)
   (0)/(1↓)
   [193]XM_006532857.1 (2↑, 1↓)/(36↑, 95↓) (2↓)/(117↑, 53↓) (5↓)/(10↑,
   115↓) (1↑, 3↓)/(147↑, 27↓) (4↓)/(13↑, 174↓) (4↓)/(17↑, 63↓)
   Positive regulation of NLRP3 inflammasome complex assembly (GO:
   1900226)
   [194]XM_006503857.2 (0)/(5↑, 3↓) (0)/(9↑, 2↓) (0)/(1↑, 8↓) (0)/(10↑,
   4↓) (0)/(14↓) (0)/(7↓)
   [195]XM_006535624.3 (1↑, 1↓)/(16↑, 21↓) (1↑, 1↓)/(41↑, 12↓) (1↓)/(27↓)
   (0)/(44↑, 12↓) (1↓)/(1↑, 57↓) (1↓)/(4↑, 20↓)
   [196]NM_153564.2 (0)/(3↑, 13↓) (1↑)/(21↑, 6↓) (0)/(15↓) (1↑, 1↓)/(28↑,
   3↓) (1↓)/(1↑, 27↓) (1↓)/(2↑, 5↓)
   [197]NM_021297.3 (5↑)/(72↑, 125↓) (3↑, 3↓)/(193↑, 71↓) (5↓)/(8↑, 139↓)
   (1↑, 4↓)/(226↑, 57↓) (1↑, 4↓)/(14↑, 253↓) (6↓)/(23↑, 86↓)
   Negative regulation of NLRP3 inflammasome complex assembly (GO:
   1900227)
   [198]NM_022432.4 (3↑, 2↓)/(54↑, 99↓) (1↓)/(143↑, 59↓) (3↓)/(9↑, 132↓)
   (1↑, 3↓)/(170↑, 39↓) (1↓)/(11↑, 200↓) (3↓)/(14↑, 78↓)
   [199]NM_019453.2 (5↑, 1↓)/(45↑, 73↓) (2↑)/(115↑, 46↓) (5↓)/(10↑, 88↓)
   (1↑, 4↓)/(124↑, 35↓) (4↓)/(11↑, 148↓) (6↓)/(15↑, 59↓)
   [200]Open in a new tab

   The metabolic pathway enrichment analysis of the target genes of
   differentially expressed miRNAs was further performed based on the KEGG
   database to reveal their participation in the molecular mechanisms of
   the therapeutic effects of noni fruit juice on acute gouty arthritis in
   mice. The results showed the 44 different KEGG pathways in the six
   pairwise comparisons of the experimental and control groups in mice
   were involved in seven categories of KEGG terms, including cellular
   processes, environmental information processing, genetic information
   processing, human diseases, metabolism, and organismal systems
   ([201]Figure 5). The highest numbers of differentially expressed miRNAs
   (ranging from 5649 to 5696) were enriched in the category of human
   diseases, followed by the categories of organismal systems (ranging
   from 4658 to 4709) and metabolism (ranging from 3148 to 3178). The
   lowest numbers of differentially expressed miRNAs (ranging from 1428 to
   1444) were mapped in the category of genetic information processing,
   while the categories of cellular processes and environmental
   information processing contained ~2400 (ranging from 2426 to 2441) and
   ~2800 (ranging from 2823 to 2835) differentially expressed miRNAs,
   respectively. These results of the KEGG enrichment analysis based on
   the target genes of the differentially expressed miRNAs indicated that
   many metabolic pathways were affected in mice treated with noni fruit
   juice, suggesting that these differentially expressed miRNAs may be
   involved in the metabolic pathways related to acute gouty arthritis in
   mice. These results were consistent with the results based on the
   pathogenetic investigations in our study and with those reported
   previously [[202]17]. Further studies are required to reveal the
   explicit functions of these novel miRNAs in the occurrence of acute
   gouty arthritis and the pharmacological mechanisms of noni fruit juice
   in the treatment of acute gouty arthritis in mice.

Figure 5.

   [203]Figure 5
   [204]Open in a new tab

   Metabolic pathway enrichment analysis based on the Kyoto Encyclopedia
   of Genes and Genomes (KEGG) database of the differentially expressed
   microRNAs identified in the six pairwise comparisons of experimental
   and control groups of mice, including normal control group vs. model
   group (A), normal control group vs. colchicine group (B), normal
   control group vs. noni fruit juice group (C), model group vs. noni
   fruit group (D), model group vs. colchicine group (E), and noni fruit
   juice group vs. colchicine group (F). Comparable results were revealed
   among the six pairwise comparisons of the experimental and control
   groups of mice, showing the highest and the lowest numbers of
   differentially expressed miRNAs enriched in the categories of human
   diseases and genetic information processing of the KEGG database,
   respectively. The six color blocks (i.e., red, blue, green, purple,
   orange, and yellow) represent six categories of metabolic pathways,
   i.e., cellular processes, environmental information processing, genetic
   information processing, human diseases, metabolism, and organismal
   systems, annotated by the KEGG database, respectively. Number of genes
   represents the target genes of the differentially expressed miRNAs.

   In the top 20 enriched pathway terms mapped in the KEGG database, over
   900 functional genes were annotated in the metabolic pathways among the
   six pairwise comparisons of the experimental and control groups of
   mice, while less than 300 genes were mapped on other 19 pathway terms
   ([205]Figure 6). Future studies are needed to explicitly investigate
   the functions of these genes, in particular the genes annotated in the
   metabolic pathways, in the occurrence of acute gouty arthritis and the
   treatment of noni fruit juice on acute gouty arthritis in mice.

Figure 6.

   [206]Figure 6
   [207]Open in a new tab

   Scatter plots of the top 20 enriched pathway terms based on the Kyoto
   Encyclopedia of Genes and Genomes (KEGG) database of the differentially
   expressed microRNAs identified in the six pairwise comparisons of the
   three experimental and the control groups of mice, including normal
   control group vs. model group (A), normal control group vs. colchicine
   group (B), normal control group vs. noni fruit juice group (C), model
   group vs. noni fruit group (D), model group vs. colchicine group (E),
   and noni fruit juice group vs. colchicine group (F). A total of over
   900 functional genes were annotated in the metabolic pathways among the
   six pairwise comparisons of the experimental and control groups of
   mice. The rich factor is the ratio of target gene numbers of the
   differentially expressed miRNAs annotated in each pathway term to all
   gene numbers annotated in this pathway term. The greater value of Rich
   Factor indicates the greater degree of enrichment. The Q-values are
   corrected p-values ranging from 0~1 with the lower Q-value representing
   the greater level of enrichment.

   Studies have shown that two miRNAs (i.e., mir-30a-5p and miR-15b-5p)
   are involved in osteoarthritis by combining with two long noncoding
   RNAs (i.e., LINC00461 and LINC00662) to increase the inflammation and
   three types of cytokines (i.e., IL-6, IL-8, and TNF-α), respectively
   [[208]25,[209]29,[210]81]. These results were consistent with the
   findings revealed in our study based on the KEGG enrichment analysis,
   showing that these two miRNAs were responsive to the treatments of noni
   fruit juice and colchicine with up- and downregulated expressions in
   model mice and mice treated with both noni fruit juice and colchicine,
   respectively ([211]Table 5). These results demonstrated the therapeutic
   effects of noni fruit juice on acute gouty arthritis in mice.
   Furthermore, studies have shown that other four miRNAs (i.e.,
   miR-122-5p, miR-146a-5p, miR-17-5p, and miR181c-5p) are involved in
   uric acid nephropathy, acute pneumonia, LPS-induced inflammatory
   injury, and poststroke inflammation, by combining with long noncoding
   RNAs of ANRIL, SNHG16, NEAT1, and Malat1, respectively
   [[212]26,[213]28,[214]33,[215]82]. Similarly, our results showed that
   these four miRNAs responded to the treatments of noni fruit juice and
   colchicine, showing up- and downregulated expressions in model mice and
   mice treated with both noni fruit juice and colchicine, respectively,
   indicating the therapeutic effects of noni fruit juice on acute gouty
   arthritis in mice. Further studies are necessary to verify the detailed
   functions of these miRNAs in the development and treatment of acute
   gouty arthritis in mice.

Table 5.

   Expression patterns of known microRNAs involved in various types of
   inflammation based on the Kyoto Encyclopedia of Genes and Genomes
   (KEGG) enrichment of the six pairwise comparisons of the three
   experimental and the control groups of mice. Symbols “↑” and “↓”
   indicate up- and downregulation, respectively.
   Medical Condition Control vs. Model Groups Control vs. Noni Fruit Juice
   Groups Control vs. Colchicine Groups Model vs. Noni Fruit Juice Groups
   Model vs. Colchicine Groups Noni Fruit Juice vs. Colchicine Groups
   Osteoarthritis miR-30a-5p ↑ miR-30a-5p ↓ miR-30a-5p ↓ miR-30a-5p ↓
   miR-30a-5p ↓
   Osteoarthritis miR-15b-5p ↑ miR-15b-5p ↑ miR-15b-5p ↓ miR-15b-5p ↓
   miR-15b-5p ↓
   Uric acid nephropathy miR-122-5p ↑ miR-122-5p ↑ miR-122-5p ↑ miR-122-5p
   ↓
   Acute pneumonia miR-146a-5p ↑ miR-146a-5p ↓
   LPS-induced inflammatory injury miR-17-5p ↓ miR-17-5p ↓ miR-17-5p ↓
   miR-17-5p ↓ miR-17-5p ↓
   Poststroke inflammation miR-181c-5p ↓ miR-181c-5p ↓ miR-181c-5p ↓
   miR-181c-5p ↓
   [216]Open in a new tab

   Based on the KEGG enrichment analysis, we examined the expression
   patterns of both known and novel miRNAs identified in our study
   targeting five types of cytokines, including the two types of cytokines
   (i.e., the TNF-α and NALP3) examined in our pathogenetic investigations
   of acute gouty arthritis of this study, involved in the major metabolic
   pathways related to both acute gouty arthritis, i.e., toll-like
   receptor 4 (TLR4), NF-kB, and NLRP3, and gouty arthritis, i.e., p53 and
   forkhead-box class O (FoxO) ([217]Table 6; [218]Table S6). Our results
   showed that a large number of novel miRNAs were involved in these
   pathways related to both acute gouty arthritis and gouty arthritis,
   showing altered levels of accumulation of these cytokines. These
   results were consistent with those based on the pathogenetic
   investigations in our study and those reported previously [[219]17]. To
   date, the understanding of the molecular mechanisms of miRNAs
   regulating the development of acute gouty arthritis remain unclear
   [[220]83]. Further studies are necessary to verify the detailed
   regulatory functions of these novel miRNAs in the occurrence of acute
   gouty arthritis in mice and to further explore the pharmacological
   mechanisms of noni fruit juice in the treatment of acute gouty
   arthritis.

Table 6.

   Expression patterns of microRNAs in the pairwise comparisons among the
   three experimental and the control groups of mice targeting the five
   main proinflammatory cytokines involved in the major metabolic pathways
   related to acute gouty arthritis based on the Kyoto Encyclopedia of
   Genes and Genomes (KEGG) enrichment. Data are presented as number of
   (known)/(novel) miRNAs. Symbols “↑” and “↓” indicate up- and
   downregulation, respectively.
   Cytokine Control vs. Model Groups Control vs. Noni Fruit Juice Groups
   Control vs. Colchicine Groups Model vs. Noni Fruit Juice Groups Noni
   Fruit Juice vs. Colchicine Groups Model vs. Colchicine Groups
   Interleukin-6 (IL-6) (2↑)/(4↑, 2↓) (2↓)/(9↑, 1↓) (2↓)/(1↑, 4↓)
   (3↓)/(9↑, 4↓) (1↑)/(11↓) (3↓)/(4↓)
   TNF-α (4↑)/(52↑, 89↓) (3↓)/(98↑, 354↓) (4↓)/(3↑, 119↓) (5↓)/(116↑, 43↓)
   (2↓)/(5↑, 150↓) (5↓)/(12↑, 81↓)
   NALP3 (2↑, 1↓)/(37↑, 99↓) (2↓)/(118↑, 57↓) (5↓)/(10↑, 120↓) (1↑,
   3↓)/(149↑, 27↓) (4↓)/(13↑, 177↓) (4↓)/(17↑, 64↓)
   Interleukin-1β (IL-1β) (3↑, 1↓)/(18↑, 39↓) (1↑)/(49↑, 21↓) (2↓)/(2↑,
   41↓) (1↑, 3↓)/(57↑, 11↓) (2↓)/(3↑, 65↓) (3↓)/(8↑, 21↓)
   Monocyte chemoattractant protein-1 (MCP-1) (1↑)/(11↑, 18↓) (1↓)/(27↑,
   10↓) (1↓)/(1↑, 20↓) (2↓)/(31↑, 8↓) (0)/(34↓) (2↓)/(11↓)
   [221]Open in a new tab

4. Conclusions

   In this study, we investigated the therapeutic effects of noni fruit
   juice on treating the acute gouty arthritis in mice induced with MSU.
   Results showed that in comparison to the model group, the degree of paw
   swelling in mice was significantly decreased in both the positive
   control (treated with colchicine) and the noni fruit juice groups,
   while the contents of two proinflammatory cytokines NALP3 and TNF-α in
   serum of mice in both the positive control and the noni fruit juice
   groups were significantly decreased. These results demonstrated the
   therapeutic effects of noni fruit juice on the treatment of acute gouty
   arthritis in mice. Furthermore, based on the high-throughput sequencing
   technology, a group of differentially expressed miRNAs were identified
   involved in the pathogenetic mechanisms of acute gouty arthritis in
   mice. Results of the functional enrichment analyses of the target genes
   of differentially expressed miRNAs based on both GO and KEGG databases
   revealed the regulatory effects of these miRNAs on the treatment of
   acute gouty arthritis by noni fruit juice, indicating the potential
   therapeutic and pharmacological significance of noni fruit juice on the
   treatment of acute gouty arthritis in mice. These results demonstrate
   that these miRNAs are involved in the regulation of pathogenetic
   mechanisms of acute gouty arthritis, providing evidence to support the
   pharmacological effects of noni fruit juice on the treatment of acute
   gouty arthritis in mice. It is noted that future studies are necessary
   to investigate the explicit functions of these miRNAs in the regulation
   of the pathogenetic mechanisms of and the pharmacological effects of
   noni fruit juice on the treatment of acute gouty arthritis in mice. Due
   to their promising therapeutic effects in treatment of acute gouty
   arthritis, we foresee a prospective future of the agricultural
   cultivation and nutritional development of noni plants.

Acknowledgments