基于国医大师张大宁补肾活血理论探讨肾衰排毒胶囊调控ERK2/HIF-1α/VEGF信号通路抑制腹膜间皮细胞炎症的作用机制OA

Objective To investigate the improving effect of Shenshuai Paidu Capsules on peritoneal mesothelial cells stimulated by inflammation and to explore the underlying mechanisms.Methods Forty SPF/VAF grade male SD rats were divided into four groups using the random stratification method:a blank group,along with low-,medium-,and high-dose traditional Chinese medicine(TCM)groups.The low-,medium-,and high-dose TCM groups were administered Shenshuai Paidu Capsules via gavage at 0.1575 g/100 g,0.315 g/100 g,and 0.63 g/100 g,respectively,while the blank group was administered normal saline via gavage at 1 ml/100 g.After 7 consecutive days of intervention,blood samples were collected from the abdominal aorta to prepare serum,based on which peritoneal mesothelial cells were cultured in separate groups.The blank group consisted of normal serum only.The model group consisted of normal serum and lipopolysaccharide(LPS).The low-,medium-,and high-dose groups consisted of LPS and serum from the low-,medium-,and high-dose TCM groups,respectively.Then,methyl thiazolyl tetrazolium(MTT)reaction was conducted to observe cell viability.Immunofluorescence was performed to determine phosphorylated p65 protein expression.Western blot was employed to detect extracellular signal-regulated kinase 2(ERK2),hypoxia-inducible factor-1 alpha(HIF-1α),and vascular endothelial growth factor(VEGF)protein expression.Furthermore,real-time quantitative polymerase chain reaction(RT-qPCR)was applied to measure ERK2,HIF-1α,VEGF,Tumor Necrosis Factor-alpha(TNF-α),Interleukin-6(IL-6),and Interleukin-10(IL-10)mRNA expression levels in each group.Results Compared with the blank group,the model group exhibited decreased cell viability,increased phosphorylated p65 protein expression,elevated VEGF,HIF-1α,and ERK2 protein expression levels,up-regulated ERK2,HIF-1α,VEGF,TNF-α,and IL-6 mRNA expression levels,alongside decreased IL-10 mRNA expression level(P＜0.05).Compared with the model group,the low-,medium-,and high-dose groups showed increased cell viability,decreased phosphorylated p65 protein expression,reduced ERK2,HIF-1α,VEGF,TNF-α,and IL-6 mRNA expression levels,alongside elevated IL-10 mRNA expression level(P＜0.05).Specifically,VEGF protein expression level was decreased in the low-dose group(P＜0.05).ERK2 protein expression level was decreased in the medium-dose group(P＜0.05).VEGF,ERK2,and HIF-1α protein expression levels were decreased in the high-dose group(P＜0.05).Compared with the low-dose group,the high-dose group demonstrated lower VEGF and ERK2 protein expression levels(P＜0.05),while the medium-,and high-dose group displayed lower ERK2,HIF-1α,VEGF,and TNF-α mRNA expression levels(P＜0.05).IL-10 mRNA expression level was increased in the high-dose group(P＜0.05).Compared with the medium-dose group,the high-dose group exhibited lower ERK2,HIF-1α,VEGF,TNF-α,and IL-6 mRNA expression levels,alongside higher IL-10 mRNA expression levels(P＜0.05).Conclusion The ERK2/HIF-1 α/VEGF pathway is involved in inflammatory response regulation in peritoneal mesothelial cells.Shenshuai Paidu Capsules alleviate peritoneal mesothelial cell inflammation via down-regulating HIF-1α,ERK2,and VEGF expression levels,while reduce phosphorylated p65,IL-6,IL-10,and TNF-α expressions.