network pharmacology-based study of the potential mechanism of BuShenHuoXue formula in treating osteoarthritis

Background: To investigate the potential mechanism underlying the efficacy of BuShenHuoXue (BSHX) formula on Osteoarthritis (OA) and its molecular mechanism. Materials and Methods: Data as for bioactive chemicals of individual herb in BSHX formula and their targets were collected from Traditional Chinese Medicine Systems Pharmacology database and OA-associated targets from Gene Expression Omnibus database, compound-disease target network and protein-protein interactions network were built, picturized and analyzed by Cytoscape. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment of key targets were carried out and analyzed to probe into the core pathway and their main functions further. The chondrocytes of SD rats were cultured in vitro, and 50 μ g/ml IL-1 β was added to the chondrocytes to induce apoptosis. Different concentrations of quercetin were added to the experimental group and the apoptosis rate of chondrocytes, the difference of the expression of SELE, MMP2, and COL1 genes and their protein expression level were further detected. Results: A total of 104 candidate chemicals and 42 crossing targets were screened out. Leading target genes are PTGS2, NCOA2 and HSP90AA1, whereas quercetin and luteolin are principal ingredients. Potential pathways against OA are AGE-RAGE signaling pathway in diabetic complications, Relaxin signaling pathway, IL-17 signaling pathway, Tyrosine metabolism and Endocrine resistance. Our study showed that quercetin could inhibit the apoptosis of chondrocytes induced by IL-1 β , decrease SELE, MMP2 and COL1 mRNA expression, likewise decrease the expression of SELE, MMP2 and COL1 protein. Conclusion: This study investigated the bioactive chemicals, crossing targets and possible mechanisms of BSHX formula against OA by network pharmacology strategy, results suggests that quercetin in BSHX formula may target on SELE, MMP2, and COL1 genes and then inhibit the progression of OA through the AGE-RAGE signaling pathway in diabetic complications. By the mechanism of reducing the apoptosis rate of SD rat chondrocytes and down-regulation the expression of genes involved in inflammation, we made sure that quercetin as principal ingredient can protect the cartilage. In addition, the conclusion of this study still need to be confirmed by in vivo and vitro experiments.

2 effective pharmacological therapies, it is imperative to discover potential chemicals with ameliorative effects.
Traditional Chinese Medicine (TCM) is a general term for traditional medicines mainly derived from different natural medicines and herb products. It dates back from ancient China and has made significant contributions to human health [5,6]. With minimal side effects, it shows better prospect as complementary or alternative medicine for osteoarthritis. Furthermore, it shows remarkable effects in protecting articular cartilage [7,8]. In recent years, the advancement of analytical technologies and methodologies has greatly accelerated the research of TCM. In TCM theory, sustainable homeostasis maintenance and regeneration of bone and cartilage depend on sufficient essence of kidney and liver [9]. Deficient essence of kidney and liver together with blood stasis can ultimately lead to cartilage damage. BSHX formula, which consist of 10 main herbs (Glycyrrhiza uralensis, Rehmannia glutinosa, Eucommia ulmoides, Aconitum carmichaeli, Lycium barbarum, Cinnamomum cassia, Cornus officinalis, Prunus persica, Carthamus tinctorius, Dioscoreae opposite), has been used in practice for many years in OA treatment and yielded positive outcome [10,11]. Besides, Wang [12] testified BSHX formula and proved that it could prohibit cartilage degradation in mouse OA model. While the unclear mechanism of TCM has hampered its clinical practice in the world, the therapeutically effect of BSHX on osteoarthritis remains to be further elucidated.
Network pharmacology(NP) is an burgeoning drug research strategy that studies the effects of drugs on diseases in a complex biological network and uses related gene databases to explore the mechanism [13]. NP accelerate the development of pharmacology and elucidates mechanisms of drug action by establishing a multi-layer network of disease-phenotype-gene-drug, which is of the same to TCM research [14].
In this study, a compound-disease target network was constructed with the methods of NP prediction. We tried to investigate the bioactive chemicals, crossing targets and possible mechanisms of BSHX against osteoarthritis by NP strategy involving multiple drug target prediction, protein-2 protein interaction network and enrichment analysis. A following experiment was conducted which validated the effects of bioactive ingredients for BSHX in the treatment of OA.

Searching and screening for potential Osteoarthritis targets
OA-associated targets were acquired from Gene Expression Omnibus(GEO: https://www.ncbi.nlm.nih.gov), an international public gene expression database affiliated with NCBI and covers sufficient disease-differential genes profiling by array [16]. Selecting gene expression profile of homo sapiens in 40 patients(Female/Male:22/18) with knee OA which differ from 10 normal ones(Female/Male:6/4) with the key word "osteoarthritis" and "normal" (GEO accession: GSE51588). Download the sample series matrix file and the platform file (GPL13497) required by our study. Differentially expressed genes (DEGs) were obtained based on two criteria (| log FC |>1 and adj. P Value<0.05) by limma package in R software (https://www.r-project.org, ver 3.6.2).

Compound-disease target network
Intersect BSHX bioactive drug targets and OA targets so as to obtain the crossing targets for compound network by Strawberry-perl-5.30.1.1 software. The compound-disease target network was built, visualized and analyzed by Cytoscape software (https://cytoscape.org, ver3.7.2). In the network, nodes indicate OA target genes and BSHX bioactive chemicals, while edges indicate interactions between them.

Protein-protein interactions (PPI) network
The protein-protein interactions (PPI) network was constructed by Cytoscape software based on target genes which aimed at investigating the interactions between gene regulatory proteins. Select BIP, BIOGRID, HPRD, INTACT, MINT and BIND as tool databases to construct the PPI network.
To simplify the network, the topological analysis of the network was performed twice using the CytoNCA plug-in according to the screening conditions DC degree≥61 and BC degree≥100.

GO and KEGG pathway enrichment analysis
In order to clarify the biological functions and pathways of BSHX chemicals for treating OA, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of target genes were carried out. GO enrichment analysis was executed by Cytoscape plugin ClueGO (P≤0.05) and consists of three major categories: molecular functions (MF), biological process (BP) and cell chemicals (CC). Then accomplished KEGG pathway enrichment analysis (P≤0.05, Q≤0.05) using bioconductor package in R software, set output barplot to show top 20 entries.

Experimental validation
A series of experiments were conducted to explore the protective effect of quercetin on interleukin-1β-induced apoptosis of rat chondrocytes: Chondrocytes isolated from the joints of normal rats were cultured in vitro, randomly divided into 5 groups: control group (no intervention), IL-1β (50ng /ml), and 3 groups of quercetin (100μM, 200μM, 400μM-quercetin + IL-1β). Alcian blue staining and type II collagen staining were used for observation and determination. The TUNEL 2 staining method was used to observe the apoptosis of chondrocytes under a fluorescence microscope.

Bioactive chemicals and targets in BuShenHuoXue
A total of 1408 chemicals and 10987 targets in BSHX were collected from TCMSP. After screening by OB≥30% and DL≥0.18, we obtained 269 bioactive chemicals and 3591 targets in all.
These chemicals were identified as potential bioactive molecules for further study. The results of 269 chemicals and screening parameters of BSHX are presented in Table 2.

Osteoarthritis-associated targets
The dataset (GSE51588) we selected has a total of 50 samples including 10 control group(normal) samples and 40 test group (OA) samples and obtained altogether 21752 targets related to OA. We got 1591 differential genes including 950 up-regulated genes and 641 down-regulated genes after differential analysis. The top 40 differential expression genes are shown in heat map ( Figure 1).

Analysis of compound-disease target Network
42 target genes were obtained through taking intersection of BSHX targets and 1591 OA differential genes. Combined with 104 corresponding bioactive chemicals we built a visualized compound-disease target network ( Figure 2). There were 146 nodes and 208 edges shown in the network. Based on the analysis result, each compound was related to at least one gene, chemicals with the most OA targets were quercetin (MOL000098) with 22 edges and luteolin (MOL000006) with 9 edges, indicating that these chemicals might be bioactive ingredients for BSHX in the treatment of OA. 2 The protein interactions of compound-disease target obtained from taking intersection of BSHX targets and OA differential genes were shown in PPI network. The overall network included 1229 nodes and 15419 edges, after topological analysis, we got the preliminary screening network with 124 nodes and 2358 edges, and eventually the core network with 31 nodes and 287 edges ( Figure   3). Nodes in the core protein network are TP53, ESR1, HSPA8, NPM1 and VCP.

GO and KEGG pathway enrichment analysis
In GO enrichment analysis of BP, CC and MF, term connections were received according to P value≤0.05. GO enrichment analysis of target proteins is shown in Figure 4. Biological process with most significant enrichment are extracellular structure organization, muscle cell proliferation, extracellular matrix disassembly. Enriched KEGG pathways are shown in Table 3 and Figure 5,6. The major pathways for herbal intervention in OA are AGE-RAGE signaling pathway in diabetic complications (hsa04933), Relaxin signaling pathway (hsa04926), IL-17 signaling pathway (hsa04657), Tyrosine metabolism (hsa00350) and Endocrine resistance (hsa01522).Typically, figure   7 shows distribution of BSHX targets in AGE-RAGE signaling pathway in diabetic complications in KEGG.

Experimental validation
3.6.1 Observation of the culture results of rat chondrocytes After digestion and isolation of primary cells, 80-90% of the cells were fused after 10-14 days of culture. Alcian blue staining and type II collagen staining were used for observation (Figure 8a,b ).

Quercetin can reduce the apoptosis rate of SD rat chondrocytes
The results were observed by flow cytometry that in the control group the apoptosis rate was 1.73%, and the experimental group (IL-1βgroup, the 100μM quercetin group, the 200μM quercetin group and 400 μ M quercetin group) of chondrocytes apoptosis rate 12.09%, 8.78%, 6.31 %, 4.31 % ( Figure 9a,b,c,d,e). 2 The TUNEL staining method was used to observe the apoptosis of chondrocytes under a fluorescence microscope, which could be further verified more intuitively, indicating that compared with the control group, IL-1β can significantly increase the apoptosis of chondrocytes, while quercetin can effectively reduce the apoptosis induced by IL-1β( Figure 10,11 a,b,c,d,e).
3.6.3 Quercetin decreased SELE, MMP2 and COL1 mRNA expression PCR results revealed the expression levels of SELE, MMP2 and COL1 genes mRNA. By taking GAPDH as internal reference, we set the relative expression rate of GAPDH as 1, and obtained the relative expression rates of the three genes in each group (Figure 12).

Quercetin decreased the expression of SELE, MMP2 and COL1 protein
By Western blotting results, it is found that the expression of the protein mediated by the genes SELE, MMP2 and COL1 are similar in trend in PCR. Also using GAPDH as internal reference, we obtained the relative expression rates of the three proteins (Figure 13，Figure 14). Western blot analysis showed that the expression of SELE, MMP2 and COL1 protein decreased significantly.
The results showed that quercetin could inhibit IL-1β-mediated chondrocyte apoptosis when it reached a certain concentration. By inhibiting the expression of SELE, MMP2 and COL1 protein quercetin can protect the cartilage.

4.Discussions
OA is a highly prevalent degenerative joint disease of unknown etiology that involves degeneration of articular cartilage, limited intra-articular inflammation manifested by synovitis, and changes in the subchondral bone [17][18][19][20][21]. Due to the prevalence of obesity and an aging population, the public-health consequences of OA and OA-related disability have developed into a pressing issue, and novel therapeutic strategies are urgently required [22].
Osteoarthritis belongs to the category of "Bi Zheng" in terms of traditional Chinese medicine. It was first recorded in the book "Huangdi Neijing" in which the etiology and pathogenesis were described in detail. The basic pathogenesis of osteoarthritis is local qi stagnation and blood stasis in the theory of traditional Chinese medicine. TCM chemicals regulate the progression of OA by interfering with some targets in the certain signaling pathway [23]. Currently, the main etiological treatment is kidney tonifying and blood activating.
TCM preparation is to arrange the functional relationship of various herbs according to combinatorial rules of "Jun-Chen-Zuo-Shi" based on patients' syndrome, which indicates a major therapeutic chemicals combined with other agents used to restore balance of body functions and reduce toxicity such as insomnia, palpitation, infection and fever. Traditional Chinese medicine acts as a promising therapeutic option for OA. Bushenhuoxue formula is a traditional Chinese remedy that proved to be efficient for quite a long time in the first affiliated hospital of Zhejiang Chinese Medical University. Their previous researches have shown that BSHX formula could prohibit articular cartilage degradation in vivo. It was found that after 8 weeks of treatment with kidney-tonifying and bloodactivating prescription, Il-6 and MMP-13 in knee joint fluid of rabbits with knee arthritis were reduced.
From the perspective of system biology, network pharmacology has been advocated to reinforce the established methodology for pharmacologic approach [24]. As a high throughput method, drugtarget prediction technology is cost-effective, can promptly forecast the component targets and afford basis for the application of network pharmacology in Traditional Chinese Medicine [25]. As a system biology-based methodology, it offers an applicable approach to evaluate pharmacological effects of TCM at molecular level. Moreover, targeted drugs for OA have been a great challenge for the development of pharmaceutical formulations [26]. Therefore, it represents a highly attractive tool owing to its potential to investigate complex relationships between chemicals and target proteins [27]. Naiqiang Zhu undertook a network pharmacology analysis of the mechanism of the effects of Shaoyao Gancao Decoction in OA, and the results suggested that SGD exerted its pharmacological effects in OA by modulating multiple pathways, including cell cycle, cell apoptosis, drug metabolism, inflammation, and immune modulation [23]. Laigen Zhang used a network pharmacology approach to elucidate the molecular mechanism governing the effect of Radix Achyranthis Bidentatae (RAB) on osteoarthritis (OA), and the results showed possible mechanisms of action for RAB include regulating the immune and inflammatory responses, reducing chondrocyte apoptosis, and protecting the joint synovial membrane and cartilage to control disease development [28]. However, no further research was conducted to verify the prediction in both two researches then reduces their persuasiveness and credibility.
In this research, a total of 104 chemicals and 42 target genes were sorted out by our screening condition, each bioactive compound acts on at least 1 OA differential genes. Great majority of chemicals derive from licorice which indicated that licorice might be the principal effective herb of BSHX in treating OA, other herbs could play a supporting role. Quercetin and luteolin are bioactive ingredients with the most targets and they exist in multi-herb (quercetin from licorice, Lycii Fructus, Carthami Flos and Eucommiae Cortex, luteolin from Carthami Flos).
Osteoarthritis (OA) is featured by unusual extracellular matrix content (ECM) combined with articular surface erosion [29]. The precisely regulated balance of ECM synthesis, metabolism and repairment is disrupted, leading to progressive degradation of articular cartilage. Enrichment analysis of Biological Process revealed potential functions of these chemicals against OA are extracellular structure organization; muscle cell proliferation and extracellular matrix disassembly. Potential pathways are AGE-RAGE signaling pathway in diabetic complications, Relaxin signaling pathway and IL-17 signaling pathway. It is noteworthy that among the 6 target genes 2 (SELE/MAPK14/MMP2/F3/COL1A1/COL3A1) in the AGE-RAGE signaling pathway in diabetic complications pathway, all of them can be interfered by quercetin, indicating that quercetin is likely to be the most promising OA treatment compound in BSHX. Quercetin was reported to exert painrelieving influence in various pain models [30] and, specially, can reduce: (i) CAR-induced mechanical hyperalgesia [30], (ii) chemotherapy-induced neuropathic pain [31], (iii) diabetic neuropathic pain [32], and (iv) muscle mechanical hyperalgesia [30]. Showing a collaborative effect and the capacity to target the oxidative stress cascade as an antioxidant, Quercetin has been employed successfully as an supplementary in vivo and experimental arthritic models [33,34]. Beyond this, other pathways remain to be tested to promote the treatment of OA.

5.Conclusions
In conclusion, TCM compound has the characteristics of multiple chemicals, multiple targets and multiple pathways in disease prevention and treatment, which are exactly what network pharmacology focus on. Our study uses network pharmacology to predict the targets and potential mechanisms of BSHX formula for OA treatment, and ultimately focuses on quercetin targeting on SELE, MMP2, and COL1 genes in BSHX formula and then inhibit the progression of OA through the AGE-RAGE signaling pathway in diabetic complications. Further experiments showed that quercetin could inhibit the apoptosis of chondrocytes induced by IL-1β. By the mechanism of down-regulating the expression of genes involved in inflammation such as SELE, MMP2 and COL1, we made sure that quercetin can protect the cartilage. In addition, more studies are needed to reduce the toxicity of TCM and prove the rationality and compatibility of it.
In this work, we applied a novel and efficient analysis method for identification of the bioactive chemicals, crossing targets and possible molecular mechanisms of traditional Chinese herbal formula-BSHX formula. Still, there are some limitations in the present method. First, the omission of synergistic effects among ingredients in herbal formula may produce bias and incomplete results.
Second, our method could not discriminate whether an ingredient could directly bind a target or just  Gancao, pink from Duzhong, orange from Honghua, purple from Fuzi, gray from Shanyao, yellow from Gouqizi, brown from Shanzhuyu, gold from Taoren, and red from multi-herb. In the right circle, blue elliptic nodes derive from genes.