Network Pharmacology and Experimental Verification Strategies to Illustrate the Mechanism of Jian-Pi-Yi-Shen Formula in Suppressing Epithelial-Mesenchymal Transition.

Jian-Pi-Yi-Shen formula (JPYSF), a traditional Chinese medicine, has been recommended to treat renal fibrosis for decades. Previous studies had shown that JPYSF could inhibit epithelial-mesenchymal transition (EMT), an important regulatory role in renal fibrosis. However, the mechanism of JPYSF action is largely unknown. In this study, network pharmacology and experimental verification were combined to elucidate and identify the potential mechanism of JPYSF against renal fibrosis by suppressing EMT at molecular and pathway levels. Network pharmacology was first performed to explore the mechanism of JPYSF against renal fibrosis targeting EMT, and then a 5/6 nephrectomy (5/6 Nx)-induced rat model of renal fibrosis was selected to verify the predictive results by Masson's trichrome stains and western blot analysis. Two hundred and thirty-two compounds in JPYSF were selected for the network approach analysis, which identified 137 candidate targets of JPYSF and 4,796 known therapeutic targets of EMT. The results of the Gene Ontology (GO) function enrichment analysis included 2098, 88, and 133 GO terms for biological processes (BPs), molecular functions (MFs), and cell component entries, respectively. The top 10 enrichment items of BP annotations included a response to a steroid hormone, a metal ion, oxygen levels, and so on. Cellular composition (CC) is mainly enriched in membrane raft, membrane microdomain, membrane region, etc. The MF of JPYSF analysis on EMT was predominately involved in proximal promoter sequence-specific DNA binding, protein heterodimerization activity, RNA polymerase II proximal promoter sequence-specific DNA binding, and so on. The involvement signaling pathway of JPYSF in the treatment of renal fibrosis targeting EMT was associated with anti-fibrosis, anti-inflammation, podocyte protection, and metabolism regulation. Furthermore, the in vivo experiments confirmed that JPYSF effectively ameliorated interstitial fibrosis and inhibited the overexpression of α-SMA, Wnt3a, and ß-catenin, and increased the expression of E-cadherin by wnt3a/ß-catenin signaling pathway in 5/6 Nx-induced renal fibrosis rats. Using an integrative network pharmacology-based approach and experimental verification, the study showed that JPYSF had therapeutic effects on EMT by regulating multi-pathway, among which one proven pathway was the Wnt3a/ß-catenin signaling pathway. These findings provide insights into the renoprotective effects of JPYSF against EMT, which could suggest directions for further research of JPYSF in attenuating renal fibrosis by suppressing EMT.

Wen Luo Yin inhibits angiogenesis in collagen-induced arthritis rat model and in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE AND AIM OF THE STUDY: Wen Luo Yin (WLY) is a traditional Chinese formula, which has the traditional use of scattering cold pathogen, draining dampness, freeing the flow of network vessels and relieving pains. It is extensively used in the treatment of rheumatoid arthritis (RA) patients for more than 2000 years, but its actions on angiogenesis of RA have not been clarified. The present study aims to determine the anti-angiogenic activity of WLY on collagen-induced arthritis (CIA) rat model and in human fibroblast-like synoviocytes of RA (HFLS-RA) and human umbilical vein endothelial cells (HUVEC). MATERIALS AND METHODS: For in vivo experiment, arthritis was induced by immunization with bovine II collagen in DA rats. Treatment with WLY (3.45, 6.9, 13.8 g/kg, p.o., daily), or vehicle began from day 1 to day 28 of first immunization. The arthritis score, arthritis incidence, microfocal computed tomography analysis and histopathology evaluation of inflamed joints were assessed. Angiogenesis was measured by synovial vessel density with immunohistochemistry and histomorphometric analysis in synovial membrane tissues of joints. For in vitro experiments, HFLS-RA and HUVEC were used. Assays to determine HFLS-RA migration and adhesion were performed in the presence of vascular endothelial growth factor (VEGF)165 or interleukin (IL)-1ß and/or the WLY (8, 16, 32 mg/ml). Angiogenesis was assessed by measuring the migration, adhesion, and tube formation of HUVEC. Further the effect of treatment with WLY on expression levels of angiogenic activators in sera of CIA rats and in IL-1ß-induced HFLS-RA were evaluated by enzyme linked immunosorbent assay. RESULTS: WLY significantly decreased the arthritis score and arthritis incidence, and inhibited inflammation, pannus formation, cartilage and bone destruction of inflamed joints in CIA rats. More interestingly, doses of 3.45-13.8 g/kg WLY could markedly reduce the capillaries, small, medium and large vessel density in synovial membrane tissues of inflamed joints. Moreover, WLY suppressed the VEGF-induced chemotactic migration of HFLS-RA and HUVEC, and inhibited matrigel-induced cell adhesion of them. It also disrupted tube formation of HUVEC on matrigel. Furthermore, WLY significantly reduced the expression of angiogenic activators including tumor necrosis factor-α, IL-1ß, IL-17, VEGF, VEGFR, angiopoietin (Ang)-1, Ang-2 and Ang-2 receptor in sera of CIA rats and/or in IL-1ß-induced HFLS-RA/HUVEC. CONCLUSIONS: Our data suggest for the first time that WLY posses the anti-angiogenic effect in RA both in vivo and in vitro by downregulating angiogenic activators.

[Comparative study of effect of Atractylodes lancea between geo-authentic and non-authentic producing areas on collagen-induced arthritis in rats].

OBJECTIVE: To compare the effects of Atractylodes lancea from different producing area on collagen-induced arthritis (CIA) in rats. METHOD: Wistar rats were induced by type II bovine collagen. CIA rats were treated daily with oral administration of A. lancea from the geo-authentic and non-authentic producing area of Maoshan, Jiangsu province, and non-geo-authentic and non-authentic producing areas of Yingshan, Hubei province and Huayin, Shanxi province from day 7 after the day of the first immunization to day 35. Clinical symptoms as well as clinical scores and incidence were observed. All rats were sacrificed on day 35 after immunization to observe histopathologic and radiologic changes. Antibody to type II collagen in sera was measured by enzyme-linked immunosorbent assay (ELISA), the levels of pro-inflammatory cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-6 (IL-6) in sera and article-homogenated supernants by radiommunoassay, and inflammatory mediator of PGE2 in sera using ELISA. RESULT: A. lancea from Jiangsu province can ameliorate clinical symptom, reduce arthritis index and arthropathy of inflammatory joints, inhibit the production of IgG and IgM in sera, directly suppress the production of exogenous and endogenous cytokines of IL-1beta, TNFalpha and IL-6 and PGE2. A. lances from Hubei and Shanxi provinces can inhibit the production of IgM in sera, and A. lanceas from Hubei province also depress the production of IL-1beta in sera and IL-6 in supernants. CONCLUSION: A. lancea from Jiangsu province is effective in CIA rats through inhibiting the production of pro-inflammatory cytokines and the inflammatory mediators.