Abstract

Purpose

   OP and OA are chronic bone diseases with high incidence in the
   middle-aged and elderly populations. The latest research shows that the
   pathological environment of OP may be involved in the aggravation of
   the pathological process of OA, and the pathological state of OP plays
   an important role in the aggravation of OA pathology. EXD is a
   traditional Chinese medicine decoction that has been used to treat
   osteoporosis. Therefore, we further study whether OA will be aggravated
   in the OP environment and whether EXD can alleviate OA by intervening
   in the OP environment. The purpose of this study was to analyze the
   effect of OP on OA metabolites by using metabolomic methods and to
   explore the intervention mechanism of EXD on osteoporotic OA.

Method

   Thirty-two SD rats were randomly divided into normal group, OA group,
   OP-OA group, and EXD group. EXD was administered by gavage.
   Histopathological evaluation of cartilage tissue was performed using
   Saffron fast green and HE staining. Western blot and qRT-PCR were used
   to detect the expression levels of chondrogenesis genes SOX9, COL2A1,
   and COMP in cartilage tissue. GC-TOFMS and LC-QTRAP-MS/MS metabolomics
   methods were used to analyze the changes of metabolites in serum
   samples of rats in each group.

Result

   The slice results showed that the cartilage damage in the OP-OA group
   was more serious than that in the OA group, which was significantly
   relieved after EXD intervention, indicating that the cartilage damage
   in the OP-OA group was more severe than that in the OA group and
   further reduced the protein and gene expressions of cartilage markers
   SOX9, COL2A1, and COMP. Thirty-seven substances were identified, and
   gentiopicroside, emodin, quercetin, and diosmetin were analyzed as
   possible active components of EXD. EXD treatment significantly reduced
   cartilage damage and reversed the expression of these markers.
   Metabolomics showed that EXD attenuated cartilage destruction by
   modulating the expression of cystine, chenodeoxycholate, and
   D-Turanose, involving glycolysis/gluconeogenesis, pantothenate, and CoA
   biosynthesis metabolic pathways.

Conclusion

   The OP environment may promote the progression of OA through metabolic
   factors. The benign intervention of EXD in osteoporotic OA involves
   cystine, chenodeoxycholate, and D-Turanose, and their associated
   glycolysis/gluconeogenesis, pantothenate, and CoA biosynthesis
   metabolic pathways. Therefore, we have a deep understanding of the
   metabolic-related intervention of EXD in osteoporotic OA and are eager
   to better understand the mechanism of multi-targeted intervention of
   EXD in bone metabolic lesions.

   Keywords: knee osteoarthritis, osteoporosis, metabolomics, erxian
   decoction, osteoporotic osteoarthritis

Introduction

   Osteoporosis (OP) and osteoarthritis (OA) are the two most common
   diseases in the chronic degeneration of the musculoskeletal muscle
   system. The incidence rate is increasing year by year, which seriously
   endangers the physical and mental health of human beings ([41]1–[42]4).
   Previous studies have shown that metabolic disorders are the main
   pathological features of OP, manifested as osteopenia, fracture of
   trabecular bone, and thinning of cortical bone ([43]5), while the main
   pathological features of OA are cartilage loss, osteophyte formation,
   and synovial inflammation in the trochlear joint ([44]6). Therefore, OP
   has been considered to be inversely associated with OA for a long time,
   and low bone mass may even have a protective effect on OA at that time.

   With the deepening of research, scholars have gradually realized that
   OP and OA may also occur in the same patient. Chu L et al. show that
   there are similarities in the pathological changes of subchondral bone
   in OP and OA patients. More ideas in recent years have suggested that
   SB loss may negatively affect osteochondral biomechanics, ultimately
   accelerating OA progression to osteoporotic OA ([45]7). Different from
   the characteristic subchondral bone sclerosis and hyperplasia of OA,
   the disorder of subchondral bone metabolism in the early stage of OA is
   mainly bone resorption, showing decreased bone mass, decreased
   trabecular bone, and stress microfracture, which is similar to OP
   ([46]6). At the same time, the above pathological changes tend to take
   precedence over cartilage degeneration in OA in time, indicating that
   the metabolic disorder of subchondral bone may be an important reason
   for the development of OA, and OP may have a driving effect on the
   progression of OA. Unfortunately, the specific effector targets and
   mechanism of action in this process are still lacking reports.

   The reason why recent studies have focused on the role of metabolic
   abnormalities in Osteoporotic OA is precisely due to the similarities
   in the loss of bone homeostasis between OP and OA ([47]8). Studies have
   shown that under high body mass index, excessive adipokines release and
   metabolic changes lead to the occurrence of OP and increased fracture
   risk ([48]9–[49]11). It is well known that a high body mass index not
   only brings more mechanical load to OA by increasing body weight but
   also increases the risk of OA by inducing sterile inflammation through
   adipokines ([50]12). Therefore, the abnormal lipid metabolism
   accompanying the pathological progress of OP may induce OA at the same
   time, thereby accelerating the progress of OA. Metabolic differences in
   accelerated OA progression provide the possibility and a rationale for
   the treatment of Osteoporotic OA.

   From a therapeutic point of view, some drugs for the treatment of OP,
   such as Pueraria lobata and Rhizoma Drynariae, etc., can also benefit
   OA in the clinical treatment of OA, that is, cutting off the progress
   of OP may alleviate OA ([51]13–[52]16). The traditional Chinese
   medicine decoction ErXian Decoction (EXD) is included in the famous
   medical book “Wenbing Tiaobian” in the Ming Dynasty ([53]17). It
   consists of epimedium, curcuma, Morinda officinalis, angelica,
   phellodendron, and anemarrhena. It was widely used in the treatment of
   osteoporosis with a definite curative effect ([54]18–[55]21).
   Interestingly, EXD also achieved satisfactory results in the treatment
   of OA. Combined with the above, it is reasonable to speculate that the
   benefit of EXD in the treatment of OP and OA may be aimed at the
   metabolic disorders shared by the two diseases and may be used for the
   treatment of Osteoporotic OA. Therefore, in this study, an experimental
   animal OA model and Osteoporotic OA model were constructed by surgery,
   and gas chromatography-time of flight mass spectrometry (GC-TOFMS) and
   liquid chromatography quadrupole ion trap tandem mass spectrometry
   (LC-QTRAP-MS/MS) techniques were combined to study the metabolomic
   differences and related mechanisms under the action of OA, Osteoporotic
   OA and EXD.

Material and Methods

Preparation for EXD

   The herbs that make up EXD include 9 g epimedium, 9 g curcuma, 9 g
   Morinda officinalis, 9 g angelica, 6 g phellodendron, 6 g anemarrhena
   (all herbs were obtained from Jiangsu Provincial Hospital of
   Traditional Chinese Medicine) ([56]22), and all herbal materials used
   in our study were approved by Nanjing University of Traditional Chinese
   Medicine. It met the quality requirements of the 2015 edition of the
   Chinese Pharmacopoeia. Four times the mass of water for each
   sub-medicine were added, mixed and soaked, boiled two times, 30 min
   each time, and extracted three times.

Experimental Design

   Thirty-two 2-month-old Sprague-Dawley (SD) female rats used in the
   experiment were provided by Nanjing Qinglongshan Animal Farm (ethical
   number: ACU211204), weighing 180-220 g. Rats were maintained in a
   specific pathogen-free laminar flow environment at a temperature of 25
   ± 2°C, humidity 55%, and a 12-h light/dark regimen. The animal care and
   use protocol followed the National Institutes of Health Guide for the
   Care and Use of Laboratory Animals and was approved by the Animal Care
   and Use Committee of Nanjing University of Chinese Medicine.

   The rats were randomly divided into four groups: Normal group (n=8), OA
   group (n=8), Osteoporotic-osteoarthritis (OP-OA) group (n=8), and EXD
   group (n=8). The model of OA was obtained by amputating the anterior
   cruciate ligament of the rat knee joint by ACLT method, and the ACLT
   model was determined by the anterior drawer test ([57]23). The OP model
   was obtained by removing both ovaries from rats using bilateral
   oophorectomy according to the references ([58]24). In the OP-OA group