白术转腰汤对大鼠腰椎黄韧带纤维化及M2巨噬细胞TGF-β1介导炎症反应的影响OA北大核心CSTPCD

Objective Zhuanyao decoction is a traditional Chinese herbal compound prescription orignated from Bianzhenglu in Ming Dynasty;Baizhu Zhuanyao decoction(BZZYD),adding spatholobus suberectus,sappanwood,and cyathula root,is intended to enhance the therapeutic effects on the waist and hips.We aimed to investigate the effects of BZZYD on lumbar ligamentum flavum fibrosis and its possible immune regulation mechanism.Methods(i)By using a random number table method,twenty-four male SD rats were divided into the normal,model,chlorophosphate,and BZZYD groups,with six rats per group.The rats in all groups,except for the normal group,were used to establish a rat model of lumbar ligamentum flavum fibrosis using lumbar instability method.The rats in the BZZYD group received Baizhu Zhuanyao decoction through gavage(13.6 g/kg),and the other groups were administered the same amount of saline by gavage once a day for 30 days.The rats in the chlorophosphate group were subcutaneously injected with disodium chlorophosphate liposome(5 g/L,0.5 mL)at the original incision immediately after surgery and at Day 9,18,and 27.After 30 days,the rats were sarcrificed through excessive anesthesia,and the ligamentum flavum was histologically evaluated using HE and Masson staining.The collagen volume fraction(CVF)was calculated.Transforming growth factor-β1(TGF-β1),tumor necrosis factor-α(TNF-α),and interleukin-1β(IL-1β)protein expressions in the ligamentum flavum were detected using immunohistochemical staining.CD163 and TGF-β1 protein expression in M2 macrophages were detected using the immunofluorescence double-labeling method.(ⅱ)M2 macrophage and fibroblast were cultured in the three ways:separately,co-culture,and co-culture of pre-treatment of BZZYD containing serum on M2 macrophage and fibroblast.TGF-β1,TNF-α,and IL-1β mRNA expressions in M2 macrophage and fibroblast were compared using RT-qPCR.Results(ⅰ)Compared with those in the normal group,the ligamentum flavum fibers were dense and twisted,immune cells infiltrated,and the CVF was increased in the model group.TGF-β1,TNF-α,and IL-1β protein expression in the ligamentum flavum tissues of the model group were increased,and TGF-β1 and TNF-α protein expression in the BZZYD group were increased(P<0.05).Compared with that of the model group,extracellular matrix accumulation decreased in the chlorophosphate and BZZYD groups,the CVF was decreased,and TGF-β1,TNF-α,and IL-1β protein expressions in ligamentum flavum tissue were decreased(P<0.05).Compared with that of the chlorophosphate group,extracellular matrix accumulation increased,the CVF was increased,TGF-β1 and IL-1β protein expressions in ligamentum flavum tissue were increased(P<0.05).CD163 and TGF-β1 proteins were not expressed in the normal and chlorophosphate groups.CD163 and TGF-β1 proteins were expressed and co-localized in the model group,and CD163 protein expression was observed in the BZZYD group,however,TGF-β1 expression was low.(ⅱ)The co-culture system increased mRNA expressions of TGF-β1 and IL-1β in M2 macrophages,TNF-α and IL-1β in fibroblasts,compared to the two kinds of cells cultured separately.And after pre-treatment on M2 macrophages,the mRNA expressions all decreased compared co-culture system(P<0.05).Conclusion BZZYD can significantly inhibit lumbar ligamentum flavum fibrosis,reduce TGF-β1 and IL-1β expression in M2 macrophages,affect TNF-α and IL-1β expression in fibroblasts,and inhibit the positive feedback of ligamentum flavum inflammation-fibrosis.