Wenyang Huazhuo Tongluo formula alleviates pulmonary vascular injury and downregulates HIF-1α in bleomycin-induced systemic sclerosis mouse model.

BACKGROUND: Vascular damage, autoimmune abnormalities, and fibrosis are the three pathological features of systemic sclerosis (SSc).However, pulmonary vascular damage is the main factor affecting the progression and prognosis of SSc. The main purpose of this study was to explore the molecular mechanism of Wenyang Huazhuo Tongluo Formula in alleviating pulmonary vascular injury in bleomycin-induced SSc mouse model. METHODS: Masson staining and H&E staining were used to analyze the degree of pulmonary vascular fibrosis and the infiltration of leukocyte cells in lung tissue ofbleomycin-induced SSc mouse models treated with saline (BLM group), Wenyang Huazhuo Tongluo Formula (WYHZTL group) and HIF-1α inhibitor KC7F2 (KC7F2 group). Blood vessel exudation was determined by analyzing the cell number and albumin concentration in bronchoalveolar lavage fluid using a cell counter and ELISA assay, respectively. The degree of vascular injury was assessed by measuring the expression levels of vWF, E-selectin, ICAM-1, VCAM-1, VE-cadherin and claudin-5 in serum and pulmonary vascular endothelial cells using ELISA and immunofluorescence staining. Finally, the effect of Wenyang Huazhuo Tongluo Formula on the expression of HIF-1α was detected using immunofluorescence staining. RESULTS: Wenyang Huazhuo Tongluo Formula and KC7F2 significantly inhibited bleomycin-induced pulmonary vascular fibrosis and the level of perivascular inflammatory cell infiltration. The number of cells and the concentration of albumin were significantly reduced in the bronchoalveolar lavage fluid of the WYHZTL group and KC7F2 group compared with the BLM group. In addition, treatment with Wenyang Huazhuo Tongluo Formula and KC7F2 significantly downregulated the expression levels of vWF, E-selectin, ICAM-1, VCAM-1 and HIF-1α, but upregulated the expression of VE-cadherin and claudin-5 in serum and pulmonary vascular endothelial cells, compared with treatment with saline. CONCLUSIONS: This study reveals that Wenyang Huazhuo Tongluo Formula plays a new role in the treatment of SSc by alleviating pulmonary vascular damage. Furthermore, we found that Wenyang Huazhuo Tongluo Formula alleviates pulmonary vascular injury and inhibits HIF-1α expression.

Effect of Wenyang Huazhuo Tongluo formula on Sema3A/Nrpl signaling pathway in systemic sclerosis mouse model / 中国药理学通报

Aim To study the effeet of Wenyang Hua- Nrpl signaling pathway in systemic sclerosis ( SSc ) zhuo Tongluo formula ( WYHZTLF) on the Sema3A/ mouse model, and to explore its mechanism in the treatment of SSe.Methods The systemic sclerosis mouse model was constructed and divided into control group, model group, WYHZTLF low (21 g • kg 1 ) , medium (42 g • kg 1 ) , high (84 g • kg 1 ) dose group and the Sema3A inhibitor epigallocatechol gallate (EGCG) group.All groups were given intragastric administration for four weeks, and the control and model groups were treated with saline.Histopathology and dermal thickness were detected by HE, VEGFA pro-tein expression levels were detected by immunohisto- chemistry; the protein expression levels of Sema3A, Nrpl and VEGFA were determined by Western blot; Sema3A expression levels in mouse serum were detec-terl by ELISA.Results Comparer] with model group, WYHZTLF significantly alleviated the skin lesions in systemic sclerosis mouse model, significantly inhibited the dermal thickness, inhibited the protein expression levels of VEGFA, Sema3A and Nrpl , and reduced the expression levels of serum Sema3A.Conclusion WYHZTLF can improve the vascular injury of SSc, and its mechanism may be related to the inhibition of VEGFA and Sema3A/Nrpl signaling pathway.

Study on the mechanism of Wenyang huazhuo tongluo formula contained serum inhibiting the proliferation of Th17 cells in scleroderma patients / 中国药房

OBJECTIVE To study the effects of Wenyang huazhuo tongluo formula conta ined serum on the expression and methylation of retinoic acid related nuclear orphan receptor （RORγt），and to explore the mechanism of its regulation of Th 17 cell proliferation in the treatment of scleroderma. METHODS Wistar rats were given 15，30，60 g/（kg·d）Wenyang huazhuo tongluo formula intragastrically to prepare different doses of drug-contained serum ，and peripheral blood of scleroderma patients were collected to sort Th 17 cells. Using blank serum as blank control ，methyltransferase inhibitor decitabine （DCA）as positive control ， Th17 cells were treated with different concentrations of drug-contained serum ，and then the proliferation of Th 17 cells was detected by CCK- 8；mRNA expressions of RORγt and IL-17 were detected by qRT-PCR ，and the protein expression of RORγt was detected by Western blot assay ；the protein expression of IL- 17 in the cell supernatant was detected by ELISA ，and the methylation level of RORγt gene promoter was detected by methylation-specific PCR. The transcriptional activity of RORγt gene promoter was detected by dual-luciferase assay. RESULTS Compared with blank control ，Wenyang huazhuo tongluo formula contained serum and DCA could inhibit the proliferation of Th 17 cells（P＜0.05），reduced the mRNA and protein expressions of RORγt and IL-17，enhanced the methylation level of RORγt gene promoter and attenuated ， the transcription activity of RORγt gene promoter. Except for mRNA expression of Th 17 and the methylation level of RORγt gene promoter in drug-contained serum low-dose group ，there were statistical significance in above indexes of other groups（P＜0.05）. CONCLUSIONS Wenyang huazhuo tongluo formula can promote the methylation lev el of RORγt gene，and inhibit the expression of RORγt and the secretion of IL-17，so as to inhibit the proliferation of Th 17 cells.

Wenyang Huazhuo Tongluo formula inhibits fibrosis via suppressing Wnt/ß-catenin signaling pathway in a Bleomycin-induced systemic sclerosis mouse model.

BACKGROUND: Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin and internal organs. So far, no Western medicine treatment can completely inhibit or reverse the progress of SSc, while at the same time, our previous series of studies have shown that the treatment of SSc by the Wenyang Huazhuo Tongluo formula (WYHZTL), a Chinese herbal decoction, shows a delightful prospect. The aim of this study is to further investigate the mechanism of anti-fibrosis of WYHZTL formula in SSc mouse model. METHODS: The Bleomycin-induced SSc mouse model was treated with saline (BLM), high-dosage of WYHZTL formula (WYHZTL-H), medium-dosage of WYHZTL formula (WYHZTL-M), low-dosage of WYHZTL formula (WYHZTL-L) and XAV-939, a small molecule inhibitor of Wnt/ß-catenin signaling pathway, by the intragastric administration and intraperitoneal injection, respectively. The mRNA and protein levels of Wnt/ß-catenin signaling pathway associated genes, fibrosis markers and histopathology were detected by reverse transcription-quantitative polymerase chain reaction, Western blotting and hematoxylin/eosin-staining. The levels of Wnt1, CTGF and DKK1 protein in serum were detected by enzyme-linked immunosorbent assay. RESULTS: Compared with BLM group, the WYHZTL formula and XAV-939 could significantly inhibit the thickness of the skin tissue of the SSc mouse model. The mRNA expression levels of GSK3ß and DKK1 in the WYHZTL formula and XAV-939-treated group were significantly higher than those in the BLM group, while Wnt1, ß-catenin, TCF4, cyclin D1, survivin, VEGF, CTGF, FN1, collagen I/III were decreased. Compared with BLM group, the protein expression levels of GSK3ß and DKK1 in the WYHZTL formula and XAV-939-treated group were upregulated, while Wnt1, ß-catenin, cyclin D1, survivin, CTGF, FN1, collagen I/III were downregulated. WYHZTL formula and XAV-939 could inhibit expression of Wnt1 and CTGF, but promoted DKK1 in serum. Furthermore, WYHZTL-H seemed more effective than WYHZTL-M and/or XAV-939 on regulating Wnt1, ß-catenin, TCF4, GSK3ß, DKK1, cyclin D1, survivin, VEGF, CTGF, FN1 and collagen I/III. CONCLUSION: This present study demonstrates that WYHZTL formula has anti-fibrosis effect in Bleomycin-induced SSc mouse model in a dosage-dependent manner, and the molecular mechanism may be related to the inhibition of Wnt/ß-catenin signaling pathway.