Mechanism Of Total Alkaloids From Capparis Spinosa In Improving Tissue Fibrosis In Systemic Scleroderma Based On Toll-like Receptor Pathway

Objective: To investigate the specific mechanism of Capparis spinosa in the treatment of systemic scleroderma(SSc)by observing the expression of some genes or proteins in Toll-like signaling pathway in Capparis spinosa total alkaloids treated mice with systemic scleroderma(SSc).Methods: 1.90 BALB / c mice were divided into 6groups: blank group,SSc model group,low-dose,medium-dose,and high-dose total alkaloids of Capparis spinosa group,and positive drug penicillamine group.In addition to the blank group,the other groups were subcutaneously injected with bleomycin 28 days to create SSc mice model.After 60 days of modeling,each test drug group applied the low,medium and high doses of total alkaloids of spinosa on the back of the mice at a certain dosage,and the mice in the positive group were gavaged with penicillamine.2.The mice were killed 4 hours after the last administration.The skin of mice was taken and stained with HE to observe the histopathological improvement of the mice in each group.3.Masson staining was used to observe the effect of total alkaloids of Capparis spinosa on collagen expression in skin tissue of mice with systemic scleroderma.4.The expressions of TLR3,TLR4,MD2,RP105,TRAF6,IRAK-1,TRIF and My D88 were detected by immunohistochemistry.5.Western blotting was used to detect the relative expression of TLR3,TLR4,MD2,RP105,TRAF6,IRAK-1,TRIF,My D88,NF-κB,TAK1,TNFAIP3 proteins in the skin of each group of mice.6.q RT PCR was performed to detect the relative expression of TLR3、TLR4、IL-6、IL-13、TGF-β m RNA in the skin of mice in each group.Results: 1.HE staining results showed that mice in the SSc model group showed significant inflammatory and fibrotic lesions compared with the blank group.2.Masson staining results showed that: compared with the blank group,the collagen deposition of SSc model group was significantly increased;compared with the model group,the collagen deposition of Capparis spinosa total alkaloids middle and high dose groups and positive drug penicillamine group was significantly reduced.3.Immunohistochemical results showed that: compared with the blank group,the relative expression of TLR3,TLR4,MD2,RP105,TRAF6,IRAK-1,TRAIF,My D88 protein expression in the model group was significantly up-regulated(P < 0.01);compared with the model group,the relative expression of TLR3,TLR4,MD2,RP105,TRAF6,IRAK-1,TRIF and My D88 protein in each dose group and penicillamine group was significantly down regulated(P < 0.05,P < 0.01).4.The results of Western blotting showed that:compared with the blank group,the relative expression levels of TLR3,TLR4,MD2,RP105,TRAF6,IRAK-1,TRAF,My D88,NF-κB,and TAK1 protein in the model group were significantly up-regulated(P < 0.05,P < 0.01);and TNFAIP3 expression was downregulated(P <0.05);compared with the model group,the relative expression levels of TLR3,TLR4,MD2,RP105,TRAF6,My D88 protein in each dose group and penicillamine group were significantly down regulated(P < 0.05,P < 0.01),and the relative expression levels of IRAK-1 protein in the low and high dose groups and penicillamine group were significantly down regulated(P < 0.05);and the relative expression levels of TRIF and TNFAIP3 were significantly down regulated in the lowdose group(P < 0.05).5.q RT PCR results showed that: TLR4,IL-6,IL-13,TGF-β,TLR3 m RNA expression was significantly up-regulated in the model group compared with the blank group(P < 0.01);LR4,IL-6,IL-13,TGF-β,TLR3 m RNA expression was significantly down-regulated in each dose administration group and penicillamine group compared with the model group(P<0.01).Conclusion: According to the results of the study,the total alkaloids of Capparis spinosa can improve the skin fibrosis of systemic scleroderma to a certain extent by down regulating the abnormal expression of TLR3,TLR4,MD2,RP105,TRAF6,IRAK-1,TRIF,My D88,NF-κB and TAK1 and other genes or proteins.Suggesting that the improvement of total alkaloids of Capparis spinosa on skin fibrosis of systemic scleroderma may be achieved through TLR4 and TLR3 signaling pathway in Toll-like receptor pathway,and this study provides a reference for further study on the mechanism of total alkaloids of Capparis spinosa in the treatment of SSc in the future.