Abstract Purpose This study was the first to screen the active compounds of Jian Aikang Concentrated Pill (JAKCP) with network pharmacology, predict its potential targets, screen the signaling pathways, and combine with cellular experimental validation to explore the potential mechanism of JAKCP for the treatment of acquired immunodeficiency syndrome (AIDS). Methods The main compounds and targets of Chinese herbs in JAKCP were identified by TCMSP; the targets of AIDS were collected from Genecards, Online Mendelian Inheritance in Man (OMIM), Disgenet, Therapeutic Target Database (TTD) and Drugbank; the network of “Chinese herbs-active compounds-targets” for JAKCP was constructed by Cytoscape, and protein–protein interaction (PPI) network was constructed using STRING to generate the intersection targets, Metascape was conducted to analyze the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), and the network of “main active compounds-core targets-pathways” was constructed by Cytoscape. Finally, the effect of JAKCP on the survival rate of HIV pseudovirus-infected MT-4 cells was investigated by CCK-8 assay, and the predicted targets were verified by ELISA, qPCR and Western blot. Results A total of 147 active compounds of JAKCP were screened covering 351 targets and 416 AIDS disease targets were obtained, besides 140 intersection targets and 321 KEGG pathways were collected. Ultimately, quercetin, kaempferol, stigmasterol, beta-sitosterol, epigallocatechin gallate were identified as the important compounds, the core targets are HSP90AA1, IL-10, IL-6, TNF, IL-1β, TP53, and IL-1ɑ, and the biological pathways and processes mainly include T cell activation, regulation of DNA-binding transcription factor activity and apoptotic signaling pathway. Experiments on the targets of “T cell activation” demonstrated that JAKCP promotes the survival of HIV pseudovirus-infected MT-4 cells. Also, JAKCP down-regulated mRNA and protein levels of IL-1ɑ, IL-1β, and IL-6 while up-regulated mRNA and protein levels of IL-2, IL-6ST, and IL-10 in vitro. Conclusion JAKCP exerted regulatory immune functions through multi-component, multi-target and multi-pathway, thereby providing novel ideas and clues for the treatment of AIDS. Keywords: Jian Aikang Concentrated Pill, network pharmacology, HIV/AIDS, multi-pathway, mechanism Introduction Acquired immunodeficiency syndrome (AIDS) is an acquired immunodeficiency syndrome caused by human immunodeficiency virus (HIV) infection that severely damages the human immune system. It is usually divided into two types, HIV-1 and HIV-2, based on geographical distribution differences. Among them, HIV-1 is the main pathological type worldwide, and HIV-2 is mainly distributed in West African populations.[32]1 In recent years, AIDS is in a low epidemic trend with decreasing morbidity and mortality, but the population base still continues to grow,[33]2,[34]3 life expectancy always lags behind the general population,[35]4 AIDS still threatens human health. AIDS is a malignant infectious disease that is transmitted mainly through blood, sexual, mother-to-child and intravenous drug use. Infected infants, intravenous drug users, homosexuals, and patients with hemophilia are at high risk for the disease.[36]5 With the continuous progress of the disease, AIDS is mostly co-infected with opportunistic infections and malignancies, which seriously threaten human life. There is no curable drug or preventable vaccine for AIDS. Combination of antiretroviral therapy (cART) is currently the most effective regimen for the treatment of AIDS. Clinical combination drugs generally use two nucleoside or nucleotide reverse transcriptase inhibitors (NRTIs) plus protease inhibitors. Abacavir and tenofovir are potent drugs with anti-HIV activity while with significant nephrogenic and osteo-toxic characteristics; Ritonavir has the highest genetic activity among the enhanced protease inhibitors with the highest genetic resistance barrier.[37]6 Clinically, for patients diagnosed with HIV, cART should be started as soon as possible from the same day to 14 days after diagnosis,[38]7 and patients with advanced HIV are also recommended to start cART quickly to reduce mortality.[39]8,[40]9 Results of several studies have demonstrated significant viral load suppression 10 or 12 months after early rapid initiation of cART,[41]10–12 with a viral suppression rate of 85% at 1 year.[42]13 With the introduction of cART and prophylactic therapy for opportunistic infections, despite the clinical benefits for HIV-infected patients, resulting in significant improvements in life expectancy and quality of life,[43]14,[44]15 the lifelong persistence, toxicity,[45]16 long-term adverse effects,[46]17 drug resistance and high compliance[47]18 of their medications make clinical treatment more difficult and prognostic. Different from the single target of Western medicine, Traditional Chinese medicine (TCM) often shows different curative effects on various immune diseases by means of “multi-component”, “multi-target” and “multi-pathway”. So we try TCM, which is a complementary and alternative approach in the treatment of AIDS. JAKCP consists of five herbs, namely Renshen (RS), Lingzhi (LZ), Aiye (AY), Hongjingtian (HJT) and Gouqizi (GQZ), TCM contains a variety of compounds with medicinal value, pharmacological studies have shown that ginseng, wolfberry can modulate inflammation and immune responses, and are involved in multiple pathways in infectious and immune diseases.[48]19,[49]20 According to TCM theory, JAKCP is closely related to the spleen and kidney function. Clinically, TCM is effective in the treatment of AIDS,[50]21 with an 8-year survival rate of 87%.[51]22 JAKCP has been used to treat thousands of HIV/AIDS patients in “The Project of Treatment of HIV/AIDS Patients with Traditional Chinese Medicine” in Henan Province, China.[52]23 Our research group has conducted many studies on JAKCP and confirmed that JAKCP has a positive effect on AIDS immune function; however, JAKCP as a traditional Chinese medicine preparation covers compounds with multiple targets, and how these compounds synergistically exert their therapeutic effects are still unclear and in-depth research is required. The holistic view of TCM coincides with network pharmacology, which is characterized by networks and systems, and TCM network pharmacology is a promising methodology, with current research trends centering on the combination of network pharmacology prediction, experimental validation and clinical efficacy.[53]24 For the first time, we used an integrated strategy combining network pharmacology and experimental validation to explore the therapeutic potential and mechanism of JAKCP, with the aim of providing a reference for subsequent pharmacological studies and clinical treatment of AIDS. Materials and Methods Screening of JAKCP Candidate Active Compounds, Targets Prediction and Network Construction Through the TCM Systematic Pharmacology Platform (Traditional Chinese Medicine Systems, TCMSP, [54]http://tcmspw.com/tcmsp.php)[55]25 and relevant literature reports, the chemical composition of the herbs in the JAKCP formulae was collected. TCMSP database is conducted to predict the compounds, set Oral Bioavailability (OB) ≥ 30% and Drug-Likeness (DL)≥ 0.18 to determine the drug-forming properties of the compounds,[56]26,[57]27 and preliminary screening of active ingredients was performed to obtain active compounds and their protein targets of action. The published literature reports combined with PubChem database,[58]28 SwissTargetPrediction database were also used to supplement the known targets of the active compounds which were not predicted.[59]29 After the screening, the protein target names were standardized on the UniProt protein database ([60]https://www.uniprot.org), restricting humans as species. The “Chinese herbs-active compounds-targets” network was constructed and analyzed mainly by the software Cytoscape 3.6.0,[61]30 where the node indicates the component or target, the edge indicates the relationship between them, and the “degree” value of a node demonstrates the number of connections to that node in the network, and the higher the degree value, the more likely the target is to be the key targets for the compound.[62]31 The network analyzer built into Cytoscape 3.6.0 software was performed to analyze network characteristic parameters, including degree, betweenness and closeness, to investigate the more important components and targets in JAKCP and the relationships between them. Prediction of AIDS-Related Targets The prediction of targets of AIDS is available in 5 below disease gene database: the human gene annotation database (GeneCards, [63]https://www. genecards.org),[64]32 the Online Mendelian Inheritance in Man (OMIM, [65]https://omim.org/),[66]33 Disgenet (v7.0),[67]34 Therapeutic Target Database (TTD, [68]http://db.idrblab.net/ttd/)[69]35 and Drugbank database ([70]https://www.drugbank.ca).[71]36 Set “Acquired Immunodeficiency Syndrome” as a keyword, the targets were limited to “Homo sapiens”, and the search and screening were performed to summarize the obtained disease therapeutic targets and remove duplicate values. The potential target genes in HIV are the intersection of the above 5 databases. STRING-Based Protein–Protein Interaction (PPI) Network Construction and Intersection Target Screening The Venny online tool ([72]http://bioinformatics.psb.ugent.be/webtools/Venn/) was selected to calculate the intersection between JAKCP and AIDS targets to further elucidate the potential mechanism of JAKCP on AIDS. PPI networks were constructed by STRING (version 10.5, [73]https://string-db.org/), and the intersection targets of JAKCP and AIDS were input into STRING to generate PPI networks. Then, Cytoscape 3.6.0 was conducted to visualize the network of these targets.[74]37 BisoGenet-Based PPI Network Construction and Key Targets Screening PPI networks were constructed by the built-in plugin BisoGenet in Cytoscape,[75]38 and the predicted targets of JAKCP and AIDS were imported into BisoGenet separately to generate PPI networks for each. The intersection networks of drug PPI network and disease PPI networks were extracted by the Merge function in Cytoscape 3.6.0 and the attribute values of each node in the intersection network were analyzed using CytoNCA. The median of connectivity M1 is calculated and all nodes with connectivity greater than 3 times M1 are selected as “Hit hubs”. The attributes of each node in the Hit hubs network are calculated to archive the 5 median M2, C2, B2, L2 of degree centrality (DC), closeness centrality (CC), betweenness centrality (BC) and local average connectivity (LAC). All nodes whose node attributes satisfy >M2, C2, B2, L2 at the same time are selected, and the final target obtained by screening 2–3 times is used as the key targets. Gene Ontology (GO) and Pathway Enrichment Analysis The intersection targets of JAKCP and AIDS were recorded into Metascape ([76]http://metascape.org/gp/index.html), set P < 0.01,[77]39 performed GO analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) of their major biological processes and metabolic pathways and enriched them with the online drawing tool Omicshare ([78]https://www.omicshare.com/) to visualize the data. Preparation of JAKCP-Containing Serum JAKCP consisted of 3g rhizomes of Panax Ginseng C. A. Mey, 15g dry fruiting body of Ganoderma, 3g foliage of Folium Artemisiae Argyi, 5g root of Rhodiolae Crenulatae Radix et Rhizoma and 5g fruit of Lycii Fructus, all raw drugs and drug processing were provided by Henan Provincial Institute of Traditional Chinese Medicine. Forty healthy SPF-grade male SD rats were randomly divided into the JACKP-containing serum group and the blank serum control group, with 20 rats in each group. JACKP was administered by gavage to rats at a dose of 6.3g/Kg/d according to the equivalent dose conversion factor for 70 kg adults and rats,[79]40 and an equal volume of saline was given by gavage to the blank serum control group; the drug was administered continuously for 7d, divided into two daily doses in the morning and evening with an interval of 12h. After 2h of the last dose,[80]41 10% chloral hydrate was injected for anesthesia, and the blood was taken under aseptic conditions via the abdominal aorta. Blood was left at room temperature for 2h, centrifuged at 2500rpm for 30min, the supernatant was taken and mixed with the same group; inactivated at 56°C and 30 min in a water bath, filtered and de-bacterized by 0.22um microporous filter, and stored in a −80°C refrigerator for backup. Cell Culture MT-4 cells and 293T cells were selected for the following experiments, and all cells were derived from our laboratory—“Key Laboratory of Viral Diseases Prevention and Treatment with TCM of Henan Province”, and were authenticated by STR profile and kept in our laboratory at 37°C in a 5% CO2 incubator. MT-4 cells were grown in RPMI1640 medium containing 10% FBS and passaged by half change; 293T cells were cultured in DMEM medium supplemented with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin, and passaged once every 1–2 d. Preparation and Identification of HIV Pseudovirus and Establishment of AIDS Cell Model The dual plasmid system-PNL4-3-Luc-R-E-plasmid (containing HIV backbone protein gene) and JRFL plasmid (containing HIV envelope protein gene) were used to prepare HIV pseudoviruses. Lip2000 was used for cell co-transfection, and both plasmids were transfected into 293T cells, and supernatants were collected after 48h of culture to obtain transfected virus. After MT-4 cells was infected with this virus, the HIV-specific protein P24 antigen was determined by chemiluminescence, and HIVAg/Ab > 1 indicated that the virus had HIV activity and could identify the virulence of HIV pseudovirus.[81]42 Finally, the identified identified pseudovirus stock solution and 1640 complete medium containing MT-4 cells were divided into different groups according to the fold dilution method. The expression of fluorescent firefly luciferase gene and the effect on cell viability were measured to determine the optimal modeling concentration for HIV pseudovirus infection of MT-4 cells.[82]43 Cell Viability Assay CCK-8 assay was performed to evaluate the effect of JACKP-containing serum on the viability of HIV pseudovirus-infected MT-4 cells. MT-4 cells were inoculated into 96-well plates at a density of 3 × 10^4 cells/100 ul/well, incubated for 24 h with HIV pseudovirus 50 ul/well, and incubated successfully for 48 h. Cells were treated with different concentrations of JACKP-containing serum at 5%, 10%, 15% and 20% for 24 h, 48 h and 72 h. Then incubated with 10 µL of CCK-8 solution (5 mL; Solarbio, China) for 120 min. Absorbance was measured at 450 nm using a SpectraMax^® i3 multifunctional enzyme marker (Molecular Devices, America). Cell viability was calculated using SPSS 23.0 software. ELISA Quantitative Analysis MT-4 cells were inoculated in 6-well plates at a density of 1 × 10^6 cells/well, incubated at 37°C with 5% CO[2] for 24 h. The supernatant was discarded and 1 mL/well of titrated HIV-1 virus supernatant was added and incubated for 48 h. High, medium and low concentrations of serum containing JACKP and positive control drug lentinan (800ug/mL) 2mL/well were added to each well, then incubated for 24 h. The cells and supernatant of each well were collected, washed with PBS solution and stored at −80°C for analysis. ELISA assay was performed according to the reagent manufacturer’s instructions to determine the level of inflammatory cytokine IL-1ɑ (E-EL-H0088c, Elabscience, China), IL-1β (EK101BHS-96, MULTI SCIENCES, China), IL-2 (EK102HS-96, MULTI SCIENCES, China), IL-6 (EK106HS-96, MULTI SCIENCES, China), IL-6ST (E-EL-H6015, Elabscience, China) and IL-10 (EK110HS-96, MULTI SCIENCES, China) in the cells. qPCR Assay The supernatant was poured out and washed with PBS solution after collecting each group of cells. First, total RNA was extracted using FastPure^® Cell/Tissue Total (Nanjing, China) according to the manufacturer’s instructions, and then a nucleic acid protein assay was conducted to assess RNA purity and concentration. Finally, cDNA strands were prepared from total RNA (1 μg) using HiScript^® III RT SuperMix for qPCR (+gDNA wiper) (Nanjing, China). The cDNA was then amplified with specific primers in ChamQ Universal SYBR qPCR Master Mix reagent (Nanjing, China). PCR reaction system consisted of 10 µL 2×ChamQ Universal SYBR qPCR Master Mix, 1 µL each of upstream and downstream primer sequences, 2 µL template cDNA and 6µLRNase-free ddH2O. GAPDH was set as an internal reference and the relative expression of each target was calculated using the 2-ΔΔCt method in ABI Prism SDS 2.0.3. Western Blot Assay The cells were lysed with RIPA lysis solution for 30 min and then transferred to centrifuge tubes. 12,000 rpm/min centrifugation was performed for 10 min and the supernatant was extracted. Quantitative protein concentration was determined by BCA protein assay kit. Protein electrophoresis buffer was added to the SDS-PAGE gel in 20 µg spots, and after electrophoresis, the proteins on the SDS-PAGE gel were transferred onto PVDF membranes, sealed with 5% skim milk powder for 1 h at room temperature, and then washed three times with PBST solution. Rabbit anti-IL-1ɑ, IL-1β, IL-2, IL-6, IL-6ST, IL-10 and β-Tubulin antibodies were added respectively, incubated overnight at 4°C, then the membrane was washed again, the corresponding secondary antibodies were added. ECL reagents were used for luminescence development. The protein bands were imaged by chemiluminescence imaging system, and the grayscale values of protein bands were analyzed by Image-Pro Plus 6.0 software, and the relative expression of each group of proteins was compared using β-Tubulin as an internal reference. Statistical Analysis All data were expressed as mean ± SD, and the results were analyzed using GraphPad Prism 8 and SPSS 23.0 software. The paired-samples t-test was performed to compare quantitative data between groups, and P < 0.05 was considered a statistically significant difference between groups. Results Network of Chinese Herbs, Compounds and Targets in JAKCP According to TCMSP and literature reports, the compounds in each Chinese medicine were initially extracted: 190 species in Renshen, 242 species in Lingzhi, 135 species in Aiye, 188 species in Gouqizi and 35 species of Hongjingtian; After screening by conditions (OB≧30%, DL≧0.18) in ADME, A total of 147 active pharmaceutical components were achieved: 22 species of Renshen, 61 species of Lingzhi, 9 species of Aiye, 45 species of Gouqizi and 10 species of Hongjingtian, Quercetin, Kaempferol, Stigmasterol, Beta-sitosterol and Mandenol were the active compounds crossed by more than two herbal medicines. The 147 active compounds are obtained and listed respectively ([83]Supplementary Material 1), some of them are shown in [84]Table 1, and the TCMSP crossover active compounds are reflected and shown in [85]Table 2, [86]Figure 1 and [87]Supplementary Material 2. The action targets of 5 kinds of Chinese herbs are predicted by the method of TCMSP target prediction model combined with literature search: 118 for Renshen, 40 for Lingzhi, 187 for Aiye, 208 for Gouqizi and 254 for Hongjingtian. Finally, information of targets of the 5 Chinese herbs were combined and the duplicate values were removed to archive a total of 351 targets. Table 1. Information on Some of the 147 Compounds Contained in 5 Chinese Herbs in JAKCP Herbs Molecule ID Molecule Name OB (%) DL CAS Number RS MOL005314 Celabenzine 101.8 0.49 53938-08-2 MOL005308 Aposiopolamine 66.65 0.22 N/A MOL005401 Ginsenoside Rg5_qt 39.56 0.79 186763-78-0 MOL000422 Kaempferol 41.88 0.24 520-18-3 MOL004492 Chrysanthemaxanthin 38.72 0.58 26989-20-8 LZ MOL011221 Ganoderic acid V 30.19 0.8 86377-50-6 MOL000359 Beta-sitosterol 36.91 0.75 5779-62-4 MOL000279 Cerevisterol 37.96 0.77 516-37-0 MOL011258 Ganosporelactone B 31.21 0.33 138008-05-6 MOL011287 Lucidone A 37.22 0.64 97653-92-4 AY MOL000098 Quercetin 46.43 0.28 73123-10-1 MOL000449 Stigmasterol 43.83 0.76 83-48-7 MOL002883 Ethyl oleate (NF) 32.4 0.19 1191-41-9 MOL001040 (2R)-5,7-dihydroxy-2-(4-hydroxyphenyl) chroman-4-one 42.36 0.21 N/A MOL005720 24-methylenecyloartanone 41.11 0.79 N/A HJT MOL000422 Kaempferol 41.88 0.24 520-18-3 MOL001002 Ellagic acid 43.06 0.43 476-66-4 MOL013083 Skimmin 38.35 0.32 93-39-0 MOL000073 Epicatechin 48.96 0.24 35323-91-2 GQZ MOL000449 Stigmasterol 43.83 0.76 83-48-7 MOL008400 Glycitein 50.48 0.24 40957-83-3 MOL003578 Cycloartenol 38.69 0.78 469-38-5 MOL000098 Quercetin 46.43 0.28 73123-10-1 MOL008400 Glycitein 50.48 0.24 40957-83-3 [88]Open in a new tab Note: N/A represents there was no number of CAS. Abbreviations: RS, Renshen (Panax Ginseng C. A. Mey); LZ, Lingzhi (Ganoderma); AY, Aiye (Folium Artemisiae Argyi); HJT, Hongjingtian (Rhodiolae Crenulatae Radix et Rhizoma); GQZ, Gouqizi (Lycii Fructus). Table 2. Crossover Active Compounds of RS, LZ, AY, HJT and GQZ Herbs in JAKCP No. Molecule ID Cross Compounds Herbs A1 MOL000449 Stigmasterol RS、AY、GQZ A2 MOL000358 Beta-sitosterol RS、LZ、AY、GQZ B1 MOL000422 Kaempferol RS、HJT B2 MOL001494 Mandenol AY、GQZ C1 MOL000098 Quercetin AY、GQZ [89]Open in a new tab Note: A1, A2, B1, B2, C1 are the crossover active compounds of 5 kinds of Chinese herbs in JAKCP. Abbreviations: RS, Renshen (Panax Ginseng C. A. Mey); LZ, Lingzhi (Ganoderma); AY, Aiye (Folium Artemisiae Argyi); HJT, Hongjingtian (Rhodiolae Crenulatae Radix et Rhizoma); GQZ, Gouqizi (Lycii Fructus). Figure 1. [90]Figure 1 [91]Open in a new tab Crossover active compounds of RS (Renshen), LZ (Lingzhi), AY (Aiye), HJT (Hongjingtian) and GQZ (Gouqizi) herbs in JAKCP. In this figure, the blue area represent candidate compounds of RS, the red area represent candidate compounds of LZ, the green area represent candidate compounds of AY, the yellow area represent candidate compounds of HJT, the Orange area represent candidate compounds of GQZ. Finally, Quercetin, Kaempferol, Stigmasterol, Beta-sitosterol and Mandenol were the active compounds crossed by more than two herbal medicines. The relationship network of active compounds of the 5 Chinese herbs and their action targets was drawn and analyzed using Cytoscape 3.6.0 ([92]Figure 2 and [93]Supplementary Material 3), and a total of 432 nodes (containing 285 targets and 147 active compounds) with 1312 edge relationships were generated. The size of the nodes in the figure represents the corresponding degree values, and the larger area of the nodes represents the larger degree values, indicating the more biological functions involved and their higher biological importance. The top 5 compounds in this network in terms of degree value were Quercetin, Kaempferol, Stigmasterol, Beta-sitosterol and Epigallocatechin gallate, they were identified as important active compounds. In addition, different herbal medicines containing the same active ingredients exhibit more interactions and a considerable number of targets are modulated by multiple compounds. For example, Quercetin, a common active compound of Aiye and Gouqizi, could modulate multiple targets (eg, CASP9, IL10, IL6, PPARG). It is hypothesized that the active compounds of JAKCP may affect multiple targets to effectively treat AIDS. In this network, the relationship between 5 Chinese herbs, active compounds and targets and the potential pharmacological effects of JAKCP are visualized. Figure 2. [94]Figure 2 [95]Open in a new tab Network of the relationship between five kinds of Chinese herbs, active compounds and targets in JAKCP. All nodes were visualized in degree value, the larger the node, the darker the color and the higher the degree value. The five kinds of Chinese herbs are RS (Renshen), LZ (Lingzhi), AY (Aiye), HJT (Hongjingtian) and GQZ (Gouqizi), among them, the hexagons in the bright yellow area marked with RS represent main compounds contained in RS; the hexagons in the green area marked with LZ represent main compounds contained in LZ; the hexagons in the purple area marked with AY represent main compounds contained in AY; the hexagons in the blue area marked with HJT represent main compounds contained in HJT; the hexagons in the dark yellow area marked with GQZ represent main compounds contained in GQZ; The red diamonds in the middle area represent the targets that all compounds intersect. A1, A2, B1, B2, C1 are the crossover active compounds of five kinds of Chinese herbs in JAKCP. Prediction of AIDS-Related Targets The number of targets of AIDS retrieved from the 5 disease databases of Genecards, OMIM, Disgenet, TTD and DrugBank were 343, 21, 29, 0 and 80, respectively. The obtained targets were summarized and duplicate targets were removed and 416 targets were obtained finally. Acquisition of AIDS and JAKCP Intersection Targets The information of targets of the active compounds in 5 Chinese herbs in JAKCP and the targets of AIDS were genetically mapped through the online Venny 2.1.0 platform to get drug-disease intersection targets, indicating the relationship between the 5 Chinese herbs of JAKCP and AIDS, and identifying 140 target genes which affected by AIDS and regulated by RS, AY, LZ, GQZ and HJT ([96]Table 3, [97]Figure 3 and [98]Supplementary Material 4). Table 3. Information on the 140 Intersection Targets of AIDS and JAKCP No. Target UniProt ID No. Target UniProt ID No. Target UniProt ID No. Target UniProt ID 1 VIM [99]P08670 36 OPRM1 [100]P35372 71 AKT1 [101]P31749 106 CYP1B1 [102]Q16678 2 VEGFA [103]P15692 37 NR3C1 [104]P04150 72 IKBKB [105]O14920 107 CYP1A2 [106]P05177 3 VCAM1 [107]P19320 38 NR1I2 [108]O75469 73 IFNG [109]P01579 108 CXCL8 [110]P10145 4 TP53 [111]P04637 39 NOS3 [112]P29474 74 ICAM1 [113]P05362 109 CXCL10 [114]P02778 5 TNF [115]P01375 40 NOS2 [116]P35228 75 HSPB1 [117]P04792 110 CTSD [118]P07339 6 TLR4 [119]O00206 41 NFKBIA [120]P25963 76 HSPA5 [121]P11021 111 CRP [122]P02741 7 TGFB1 [123]P01137 42 MYC [124]P01106 77 HSP90AA1 [125]P07900 112 COMT [126]P21964 8 STAT3 [127]P40763 43 MPO [128]P05164 78 SERPINE1 [129]P05121 113 CDKN2A [130]P42771 9 STAT1 [131]P42224 44 MMP9 [132]P14780 79 HCK [133]P08631 114 CDKN1A [134]P38936 10 SP1 [135]P08047 45 MMP2 [136]P08253 80 GSTP1 [137]P09211 115 CDK4 [138]P11802 11 SOD1 [139]P00441 46 MDM2 [140]Q00987 81 FYN [141]P06241 116 CD40LG [142]P29965 12 SLPI [143]P03973 47 MAPK8 [144]P45983 82 FOS [145]P01100 117 CD4 [146]P01730 13 HIF1A [147]Q16665 48 MAPK3 [148]P27361 83 FGF2 [149]P09038 118 CCND1 [150]P24385 14 SELE [151]P16581 49 MAPK14 [152]Q16539 84 FGF1 [153]P05230 119 CCL2 [154]P13500 15 RELA [155]Q04206 50 MAPK1 [156]Q16539 85 FASLG [157]P48023 120 CAT [158]P04040 16 RAF1 [159]P04049 51 MAP2K1 [160]Q02750 86 FAS [161]P25445 121 CASP9 [162]P55211 17 PTGS2 [163]P35354 52 MAP2 [164]P11137 87 ESR1 [165]P03372 122 CASP8 [166]Q14790 18 PTGS1 [167]P23219 53 KDR [168]P35968 88 ERBB2 [169]P04626 123 CASP3 [170]P42574 19 PRKACA [171]P17612 54 JUN [172]P05412 89 EGFR [173]P00533 124 CASP1 [174]P29466 20 PPARG [175]P37231 55 IRF3 [176]Q14653 90 EGF [177]P01133 125 BCL2L1 [178]Q07817 21 PIK3CG [179]P48736 56 IL6 [180]P05231 91 DPP4 [181]P27487 126 BCL2 [182]P10415 22 PIK3CA [183]P42336 57 IL2 [184]P60568 92 DHFR [185]P00374 127 BAX [186]Q07812 23 PGR [187]P06401 58 IL1B [188]P01584 93 CYP3A7 [189]P24462 128 BAK1 [190]Q16611 24 PDGFB [191]P01127 59 IL1A [192]P01583 94 CYP3A4 [193]P08684 129 APP [194]P05067 25 ALOX5 [195]P09917 60 IL10 [196]P22301 95 ACHE [197]P22303 130 TOP2A [198]P11388 26 EDNRB [199]P24530 61 IL6ST [200]P40189 96 PLAU [201]P00749 131 TIMP1 [202]P01033 27 EDN1 [203]P05305 62 HTR2A [204]P40189 97 PLAT [205]P00750 132 SPP1 [206]P10451 28 DRD2 [207]P14416 63 HRAS [208]P01112 98 PDGFRB [209]P09619 133 SLC6A4 [210]P31645 29 CXCL11 [211]O14625 64 GAA [212]P10253 99 PCNA [213]P12004 134 SLC6A3 [214]Q01959 30 CDK1 [215]P06493 65 FLT1 [216]P17948 100 NR3C2 [217]P08235 135 RXRG [218]P48443 31 CDH1 [219]P12830 66 FGFR1 [220]P11362 101 MMP7 [221]P09237 136 RB1 [222]P06400 32 CCNA2 [223]P20248 67 FASN [224]P49327 102 MMP1 [225]P03956 137 PTEN [226]P60484 33 BRAF [227]P15056 68 F3 [228]P13726 103 LDLR [229]P01130 138 PRKDC [230]P78527 34 BIRC5 [231]O15392 69 ELANE [232]P08246 104 INSR [233]P06213 139 PRKCA [234]P17252 35 BAD [235]Q92934 70 AHSA1 [236]O95433 105 AR [237]P10275 140 AHR [238]P35869 [239]Open in a new tab Figure 3. [240]Figure 3 [241]Open in a new tab The venn diagram of 140 intersection targets of AIDS (Acquired immunodeficiency syndrome) and JAKCP (Jian Aikang Concentrated Pill). Construction of PPI Network of Intersection Targets for JAKCP and AIDS by STRING The 140 intersecting targets of JAKCP and AIDS are demonstrated by STRING ([242]Figure 4 and [243]Supplementary Material 5), which covers 134 nodes and 823 edges, indicating that JAKCP exerts therapeutic effects on AIDS through multiple protein targets, and the first 6 degrees of targets STAT3, PIK3CA, TP53, MAPK3, MAPK1 and AKT1 are the main targets among the intersecting targets. Figure 4. [244]Figure 4 [245]Open in a new tab PPI network diagram of intersection targets for JAKCP and AIDS. Edges represent protein–protein interactions, edge thickness indicates the strength of data support, the larger the node, the higher the degree value. The circles with different colors in the figure represent the target names with different degree values. Construction of PPI Network of the Key Targets for JAKCP and AIDS by BisoGenet PPI networks of JAKCP and AIDS were constructed by running the BisoGenet function in Cytoscape 3.6.0, and it was found that the potential targets of JAKCP could interact with 9082 targets directly or indirectly, and there were 197,204 interactions between these targets. At the same time, the PPI network of AIDS-related targets was drawn, showing that as many as 9091 targets were directly or indirectly related to them, and as many as 195,305 kinds of interrelationships between these targets, and the intersection network of the two is shown in [246]Supplementary Material 6. The values of network topological characteristics of the above PPI intersection network were calculated, and a total of 122 key targets were obtained through three screening, and the screening strategy is shown in [247]Figure 5 and [248]Supplementary Material 7. Some regions with high density in the PPI complex network are called community or module. Modules are considered to be biologically significant sets, and the interaction relationships are analyzed by molecular complex detection algorithms to obtain modules ([249]Figure 6 and [250]Supplementary Material 8) after obtaining the core PPI network. According to the P value, the biological processes with three best scores were retained in PPI network and Module respectively and their functions were described, shown in [251]Table 4. Figure 5. [252]Figure 5 [253]Open in a new tab The strategy of key targets in the treatment of AIDS by JAKCP. DC is Degree Centrality, BC is Betweenness Centrality, CC is Closeness Centrality and LAC is Local Average Connectivity-based method, they are four important indicators used in the strategy of screening effective compounds, 122 key targets were finally obtained after three screenings of the above indicators. Figure 6. [254]Figure 6 [255]Open in a new tab Potential Module network within the core targets-PPI network. Different colored circles represent different target categories. The size of the circular shape represents the magnitude of the degree value, and the lines between them represent the tightness of the connection between different targets. Table 4. Potential Module Function Description of PPI Network of AIDS and JAKCP (Top 3) Entry Functional Description lgp hsa05167 Kaposi sarcoma-associated herpesvirus infection −55.2 hsa04060 Cytokine-cytokine receptor interaction −23.10 hsa04215 Apoptosis −13.30 [256]Open in a new tab GO and KEGG Pathway Enrichment Analysis Gene enrichment analysis was performed using Metascape for targets associated with JAKCP and AIDS, including BP (biological process), CC (cellular component), MF (molecular function) and KEGG pathways, results were saved and bubble charted was plotted using Omicshare online platform, more detailed pathway analysis data are provided in [257]Figure 7 and [258]Supplementary Material 9. The top 20 significantly enriched GO terms are shown in [259]Figure 7A, where biological processes closely related to AIDS pathogenesis include apoptotic signaling pathway, T cell activation and positive regulation of cytokine production. These processes directly serve as links to CD4+ T cell differentiation, proliferation and apoptosis. It demonstrates that JAKCP has a direct regulatory role in the production and apoptosis of AIDS T lymphocytes. The top 20 significantly KEGG pathways enrichment are shown in [260]Figure 7D, where signaling pathway closely related to AIDS pathogenesis include cytokine–cytokine receptor interaction, JAK-STAT signaling pathway and Apoptosis. Figure 7. [261]Figure 7 [262]Open in a new tab GO and KEGG analysis for the major targets of JAKCP, (A) lists the top 20 significantly enriched biological processes in the GO analysis, namely GO-BP; (B) lists the top 20 significantly enriched cellular component in the GO analysis, namely GO-CC; (C) lists the top 20 significantly enriched molecular function in the GO analysis, namely GO-MF; (D) shows the top 20 signaling pathways in the KEGG pathway enrichment analysis. The Y-axis in the figure represents the top 20 biological functions, such as BP, CC, MF and KEGG, the X-axis represents the ratio of the number of target pathway genes to the total number of genes, the size of the bubble area represents the number of enriched genes, and the color of the bubble Represents the significance of enrichment, that is, the magnitude of the q-value value. Construction of “Main Active Compounds-Core Targets-Pathways” Network by Cytoscape The “main active compounds-core targets-pathways” network diagram is shown in [263]Figure 8 and [264]Supplementary Material 10. The important pathways are “T cell activation, Regulation of DNA-binding transcription factor activity and Apoptotic signaling pathway”, according to the degree ranking. The core targets are “HSP90AA1, IL-10, IL-6, TNF, IL-1B, TP53 and IL-1ɑ”. Therefore, we selected a certain pathway and target from the “main active compounds-core targets-pathways” system for experimental validation. After screening, the main active compounds in the JAKCP complex were “Quercetin, Ginsenoside rh2 and Epigallocatechin gallate”, which could act on “IL-1ɑ, IL-1β, IL-2, IL-6, IL-6ST and IL-10” and regulate the “T-cell activity” pathway. Based on this, we hypothesized that the mechanism of JAKCP for AIDS treatment is through the cytokines IL-1ɑ, IL-1β, IL-2, IL-6, IL-6ST and IL-10 of “T-cell activity” pathway. Consequently, vitro experiments were performed to verify this hypothesis. Figure 8. [265]Figure 8 [266]Open in a new tab The network of “main active compounds-core targets-pathways”. All nodes were visualized in degree value, the larger the node, the darker the color and the higher the degree value. A2, B1, C1 are the crossover active compounds of five kinds of Chinese herbs in JAKCP, A2 is Beta-sitosterol, B1 is Kaempferol, C1 is Quercetin, the other red circles represent the important effective compounds finally screened out, the blue diamonds in the middle represent the corresponding targets, and the green arrows around represent the corresponding pathways. Construction of AIDS Cell Model HIV pseudovirus infected MT-4 cells 48h after P24 antigen was measured by chemiluminescence method, and HIV Ag:1157.31 > 1 was determined, then the virus has the ability to infect MT-4 cells. The optimal HIV pseudovirus titer was determined by the multiplicative dilution method to be 1:4. Thus, an available cell model was constructed at this virus titer. Effect of JAKCP on the Survival Rate of HIV Pseudovirus Infected MT-4 Cells in vitro In order to verify the efficacy of JAKCP on AIDS, survival rate of HIV-pseudovirus-infected MT-4 cells treated with different concentrations of JACKP-containing serum was first determined. It demonstrated that JAKCP had a promotional effect on the survival of HIV pseudovirus-infected MT-4 cells in a concentration dependent manner. Cell survival curves are shown in [267]Figure 9 and [268]Supplementary Material 11. Follow-up experiments were performed using High (15%), Medium (10%) and Low (5%) doses of JACKP-containing serum. Figure 9. [269]Figure 9 [270]Open in a new tab Cell viability effects of JAKCP treatment on MT-4 cells with HIV pseudovirus, Drug concentration-cell viability curves were generated based on the cell viability assay. (A) The OD value of JAKCP treatment on MT-4 cells with HIV pseudovirus. (B) The cell survival rate of JAKCP treatment on MT-4 cells with HIV pseudovirus. All data was presented as mean ± standard deviation, ^###:p<0.001, compared with control group; **:p < 0.01, compared with model group. Expression of IL-1ɑ, IL-1β, IL-2, IL-6, IL-6ST and IL-10 by ELISA It was reported that numerous inflammatory cytokines have been reported to be associated with the pathogenesis of AIDS.[271]44,[272]45 Our ELISA results showed that the expression levels of IL-1ɑ, IL-1β and IL-6 in the JAKCP-containing serum group were significantly lower than those in the model group, while the expression levels of IL-2, IL-6ST and IL-10 were significantly higher than those in the model group ([273]Figure 10 and [274]Supplementary Material 12). These results indicated that JAKCP decreased the expression levels of pro-inflammatory cytokines and increased the expression levels of anti-inflammatory cytokines. JAKCP may play a role in regulation of inflammatory factors. Figure 10. [275]Figure 10 [276]Open in a new tab Expression levels of IL-1ɑ, IL-1β, IL-2, IL-6, IL-6ST and IL-10 in serum from JAKCP among groups Con (Control), Mod (Model), P (Positive), H (High), M (Medium) and L (Low). Each histogram is marked with its corresponding target name, which is IL-1ɑ, IL-1β, IL-2, IL-6, IL-6ST and IL-10. All data was presented as mean ± standard deviation, ^#:p < 0.05,^###:p<0.001, compared with control group;*:p < 0.05,**:p < 0.01, ***:p<0.001, compared with model group. mRNA Expression of IL-1ɑ, IL-1β, IL-2, IL-6, IL-6ST and IL-10 by qPCR The mRNA expression levels of several core targets on the T-cell activity pathway, including IL-1ɑ, IL-1β, IL-2, IL-6, IL-6ST and IL-10, were verified by qPCR. It was found that JAKCP-containing serum decreased the levels of IL-1ɑ, IL-1β and IL-6 and increased the levels of IL-2, IL-6ST and IL-10 ([277]Figure 11 and [278]Supplementary Material 13). Figure 11. [279]Figure 11 [280]Open in a new tab The expression of IL-1ɑ, IL-1β, IL-2, IL-6, IL-6ST and IL-10 mRNA levels were examined by qPCR treated with JAKCP among groups Con (Control), Mod (Model), P (Positive), H (High), M (Medium) and L (Low). Each histogram is marked with its corresponding target name, which is IL-1ɑ, IL-1β, IL-2, IL-6, IL-6ST and IL-10. All data was presented as mean ± standard deviation,^#:p < 0.05, ^##:p<0.01, ^###:p<0.001, compared with control group; *:p < 0.05,**:p < 0.01, compared with model group. Protein Expression of IL-1ɑ, IL-1β, IL-2, IL-6, IL-6ST and IL-10 by Western Blot Targets including IL-1ɑ, IL-1β, IL-2, IL-6, IL-6ST and IL-10 were measured by protein blotting analysis. JAKCP-containing serum decreased the protein expression of IL-1ɑ, IL-1β and IL-6 and increased the protein expression of IL-2, IL-6ST and IL-10 ([281]Figure 12 and [282]Supplementary Material 14). Figure 12. [283]Figure 12 [284]Open in a new tab The expression of IL-1ɑ, IL-1β, IL-2, IL-6, IL-6ST and IL-10 protein levels were examined by Western blot among groups Con (Control), Mod (Model), P (Positive), H (High), M (Medium) and L (Low).(A) Western blot analysis showed the optical density ratio of IL-1ɑ/Tubulin, IL-1β/Tubulin, IL-2/Tubulin, IL-6/Tubulin, IL-6ST/Tubulin and IL-10/Tubulin on MT-4 cells with HIV pseudovirus among different groups.(B) The expression of IL-1ɑ, IL-1β, IL-2, IL-6, IL-6ST and IL-10 protein levels was analyzed by images of the Western-blot results in different groups. All data was presented as mean ± standard deviation, ^#:p < 0.05, ^##:p<0.01, ^###:p<0.001, compared with control group;*:p < 0.05,**:p < 0.01,***:p<0.001,****:p<0.0001, compared with model group. Discussion Although cART can reduce AIDS-related morbidity and mortality and has the dual advantages of immunology and virology, cART has also shown some clinical negative effects, which greatly reduce the quality of life of patients.[285]46 TCM has outstanding advantages in improving immune function,[286]47 reducing the toxicity and drug resistance rate of antiviral drugs,[287]48,[288]49 reducing the risk of death[289]50 and prolonging the survival rate.[290]23 Therefore, TCM is considered as an effective and safe complementary alternative therapy for the adjuvant treatment of AIDS, however the mechanism is still unclear. In this study, a network pharmacology approach was adopted to describe the relationship between active compounds, targets and signaling pathways and to validate them in combination with in vitro experiments, thus revealing the potential mechanisms of JAKCP. In this study, 147 active compounds and 351 targets of JAKCP were identified by network pharmacology, which indicated that JAKCP exerted its pharmacological effects on AIDS through multiple pathways and multiple targets. Quercetin, Kaempferol, Stigmasterol, Beta-sitosterol and Epigallocatechin gallate were identified as the top 5 important active compounds. Among them, Quercetin demonstrated excellent antioxidant, anticancer, antiviral, antibacterial and anti-inflammatory activities, and quercetin has potential antiviral and immunomodulatory activities against hepatitis B and C, herpes simplex virus types 1 and 2, influenza virus, human coronavirus and HIV.[291]51 Quercetin reactivates HIV-1 expression by inducing nuclear translocation of NF-κB, and is less toxic than other HIV-1 activators.[292]52 Quercetin-3-O-β-d- Glucopyranoside and quercetin-3-O-β-d-galactopyranoside have inhibitory activity against HIV-1 IN inhibitors with IC50 values of 19.39 and 21.80 μM, respectively.[293]53 In addition, Quercetin is a potential HAART adjuvant, and it was found that Quercetin significantly attenuated AZT-induced upregulation of pro-inflammatory cytokines.[294]54 Kaempferol and Kaempferol-7-O-glucoside have been reported to possess not only potent anti-HSV activity but also anti-HIV-1 reverse transcriptase activity, and a 100ug/mL concentration of kaempferol effectively inhibited HIV-1.[295]55 As for sitosterol, the main phytosterol in herbs, it has antiviral and immunomodulatory activities.[296]56 It can be seen that the activity of these single compounds on HIV has been strongly confirmed. In addition to this, there are other compounds with very high biological activity.[297]57,[298]58 We speculate that JAKCP containing the above compounds is likely to play a role in the treatment of AIDS in a multi-channel and multi-target manner. Therefore, it is the focus of this study to verify whether JAKCP can play a therapeutic role through these active compounds. GO analysis indicated that JAKCP was associated with some major biological processes, such as T cell activation, Apoptotic signaling pathway and positive regulation of cytokine production. KEGG pathway analysis showed that JAKCP has therapeutic effects on AIDS by regulating cytokine–cytokine receptor interaction, JAK-STAT signaling pathway, apoptosis and other pathways. This study revolves around “T cell activation”, which is essential for coordinating immune responses. The formation of immune synapses, molecular components and reorganization of membrane proteins and actin cytoskeletons are the main features of T cell activation. Studies have suggested that reduced T cell receptor (TCR) pool diversity and oligoclonal T cell expansion after long-term ART in HIV-infected patients correlate with elevated CD8+ T cell counts, which correlates with systemic level evidence of sustained T cell activation in the genome-wide blood transcriptome.[299]59 In addition, cellular experiments confirmed that upregulated miR-124a silences the expression of the target gene SIRT1, thereby regulating the activation of Th2 CD4 + T cells, and activated Th2 CD4 + T cells could secrete IL-10 and TGF-β cytokines involved in the immune response, which in turn enhances the immunity of patients.[300]60 Another study reported that CD4+ T cell immune activation, IL-2 production and circulating expression during acute HIV infection were associated with a decrease in CD4+ T cells 2 years after infection.[301]61 These all imply that the level of T-cell activation is closely related to the development of AIDS disease. Therefore, we performed cellular experiments to validate this pathway for T-cell activation. As for cytokines, changes in cytokine levels in HIV-infected patients affect the function of the immune system and have the potential to directly influence the course of HIV disease by enhancing or inhibiting HIV replication. In particular, the balance between pro-inflammatory cytokines IL-1, IL-6 and IL-10 may simultaneously play an anti-inflammatory role.[302]62 This is also consistent with the level of inflammatory cytokine expression in our study. Apoptosis is the main mechanism of progressive loss of CD4+ T cells in HIV-1 disease, and HIV-1 proteins associated with the regulation of apoptosis include gp120, Vpr, Nef, and Tat, which have been confirmed to induce apoptosis by upregulation of the apoptosis effector molecule Fas ligand (FasL), inhibition of manganese-dependent superoxide dismutase expression, and cell cycle protein dependent kinase activation.[303]63,[304]64 With regard to the JAK/STAT pathway, this includes phosphorylation of STAT3 and STAT1, as well as activation of RAS/MAPK, transduced through signaling by IL-6ST (another name: sGP130).[305]65 These studies are consistent with the results predicted by our network pharmacology, which further suggest that these pathways may play a role in the process of anti-HIV and regulating immune function. Therefore, we speculate that the effect of JAKCP on AIDS may be related to the above biological process. In this study, IL-1ɑ, IL-1β, IL-2, IL-6, IL-6ST and IL-10 were identified as 6 hub protein targets in the AIDS “T cell activity” pathway. Experimental verification showed that Jianaikang medicated serum promoted the survival of HIV pseudovirus-infected MT-4 cells in a concentration-dependent manner, and effectively down-regulated the mRNA and protein expressions of the predicted targets IL-1ɑ, IL-1β and IL-6, up-regulated IL-2, IL-6ST and IL-10 mRNA and protein expression. The expression of inflammatory cytokines is regulated by the “T cell activation” signaling pathway, which in turn affects the activity of MT-4 cells. We speculate that stimulating the “T cell activation” pathway can increase the activity of MT-4 cells and reduce cell apoptosis, thereby exerting an immunoregulatory effect, which also implies that JAKCP and its active compounds can promote the survival of immune cells and regulate the balance between cytokines, thereby promoting AIDS immune regulation, and thus, the balance between cytokines is of great significance in HIV disease. Multiple studies have demonstrated overproduction of pro-inflammatory cytokines in HIV-infected individuals with elevated levels of IL-1, IL-6 and TNF-α in serum and culture supernatants, and high levels of IL- 1α and IL-1β, the oversecretion of these three cytokines in turn increases HIV replication.[306]66,[307]67 Cellular experiments confirmed that acutely HIV-infected cells released significantly higher levels of TNF-α and IL-1β after stimulation with LPS or a combination of LPS plus IFN-γ compared to chronically infected or uninfected cells.[308]68 These studies are basically consistent with the results of our present study. Our experiments have proved from the cellular level that IL-1 and IL-6 have high expression levels in the AIDS cell model, which is consistent with IL-1α and IL-6 in human HIV-infected patients, and the cytokine decreased after treatment, indicating that Jianaikang medicated serum has a certain regulatory effect on IL-1ɑ and IL-6. Therefore, these pathways could represent therapeutic targets for AIDS. IL-10 is an important anti-inflammatory and immunoregulatory cytokine that exerts transcriptional and post-transcriptional control on IL-1, TNF-α and IL-6 and other inflammatory cytokines to prevent the production of mRNA and protein. Natural terminator of cytokine synthesis by activated monocytes/macrophages.[309]69 According to reports, due to the use of different detection methods, the detection amount of IL-10 in serum or in vivo of HIV/AIDS patients has different degrees of sensitivity and specificity, and may be overproduced in some HIV patients,[310]70 and IL-10 Levels increase as HIV disease progresses, and IL-10 levels in untreated AIDS patients are relatively lower than in treated patients.[311]71,[312]72 IL-6ST (sGP130) is a stabilizer of the IL-6 receptor complex, and sGP130 production is associated with IL-6. sGP130 is associated with AIDS-related Kaposi’s sarcoma AIDS-KS risk was significantly negatively correlated.[313]73 IL-2 is an important control point in controlling the functional balance of regulatory T cells and effector T cells in vivo, and animal experiments have shown that disruption of the IL-2 pathway leads to lymphoid hyperplasia and autoimmunity, rather than immunodeficiency, suggesting that IL-2 is the main. The physiological function is to limit rather than enhance T cell responses.[314]74 On the other hand, cellular experiments confirmed that IL-2 production was significantly reduced and immune function was significantly impaired in latent HIV-1-infected cells compared with uninfected parental cells.[315]59 In addition, cytokines are also involved in the pathogenesis of other immune diseases.[316]75 In summary, the results of this experiment are consistent with these in vitro and in vivo studies, suggesting that IL-1ɑ, IL-1β, IL-2, IL-6, IL-6ST and IL-10 can directly or indirectly participate in the pathogenesis of AIDS, Pathological processes and related treatments, therefore, these inflammatory cytokines are potential targets for the treatment of AIDS, and JAKCP may play an immunomodulatory role by regulating the balance between anti-inflammatory cytokines and anti-inflammatory cytokines. The results of this experiment found that although the expression of most inflammatory cytokines had a certain dose–effect relationship with the concentration of Jianaikang medicated serum, the individual inflammatory cells did not. For example, [317]Figure 10 uses ELISA to detect the expression of IL-6ST in the middle-dose group of Jianaikang medicated serum is lower than that of the low-dose group, [318]Figure 12 uses Western-blot method to detect the IL-1β and low-dose groups in Jianaikang medicated serum, the expression of IL-6ST does not have a certain dose-response relationship. The reason is that, first of all, this phenomenon may be related to the interaction of multiple components of TCM compounds. According to the literature, this phenomenon is relatively common in the research of TCM compounds. Cell experiments by Zeye Zhang and Yanjun Cao on Chinese herbs also yielded similar results to this experiment.[319]76,[320]77 Secondly, compared with a single compound, TCM compounds have multiple components acting at the same time. This effect may be a combination of positive, negative, transformation and superposition. This hypothesis has been proposed by some Chinese scholars.[321]78 There is a coordinated and unified relationship of synergy, addition and antagonism. This relationship may be synergistic promotion, inhibition of rejection or intermittent positive and negative synergy or rejection with the change of drug concentration. Finally, the results of this study showed that there was no significant difference in the expression of IL-6ST between the middle and low dose groups (P = 0.0615), which means that although there was a difference in the expression of IL-6ST between the middle and low dose groups, this difference is moot. It can be seen that there are many mysteries in the research on the mechanism of action of TCM compounds, which need to be further explored. To sum up, the experimental verification preliminarily confirmed that our prediction based on network pharmacology on how JAKCP play an immunomodulatory effect may be correct and credible. However, this study still has some limitations, for example, the diversification of computing software makes the screening, integration and processing of data not systematic; the database of TCM is incomplete and there are deviations, and the accuracy and completeness of the collected data cannot be guaranteed.[322]79 Even so, network pharmacology is still an effective means to study the mechanism of action of TCM compounds. Based on this, our research team carried out a preliminary study on the mechanism of JAKCP regulating AIDS immune function. In the following research work, we will use a combination of mass spectrometry, gas spectrum component detection and network pharmacology prediction to conduct more specific and accurate research on the relevant targets of single Chinese herbs and active compounds in the AIDS cell model. In addition, these and the verification results of TCM compounds can be comprehensively analyzed, so as to clarify how the compound JAKCP exert the therapeutic effect and provide more clues for in-depth mechanism research. Conclusion Based on network pharmacology, this study predicted that JAKCP could regulate AIDS immune function through multiple components, multiple pathways and multiple targets. Consistent with the predicted results, the experimental results showed that JAKCP could down-regulated the mRNA and protein levels of IL-1ɑ, IL-1β and IL-6, and up-regulated IL-2, IL-6ST and IL-10 through the “T cell activation” signaling pathway, regulate the balance between pro-inflammatory cytokines and anti-inflammatory cytokines, increase immune cell activity and play an immunomodulatory role. JAKCP may be considered as a good candidate for the treatment of AIDS. Acknowledgments