摘要
目的观察盐酸青藤碱对膝关节黏连强直的家兔成纤维细胞增殖和相关基因表达的影响,并进一步尝试探讨其对抗膝关节黏连强直的作用机制。方法以体外培养法培养成纤维细胞,并设对照组、盐酸青藤碱低中高浓度实验组。CCK-8法检测成纤维细胞增殖的情况;实时荧光定量聚合酶链反应(real-time quantitative PCR,RT-qPCR)法检测经过盐酸青藤碱处理后,成纤维细胞相关基因mRNA表达的改变,用ELISA法检测药物的作用对血清中炎症因子等水平的影响,蛋白质印迹法(Western blot)检测相关蛋白质的表达。结果盐酸青藤碱能降低成纤维细胞存活率,且随浓度升高存活率逐渐降低。盐酸青藤碱中各个组的效果均十分明显(P<0.05)。在相关基因的mRNA表达层面,与对照组比较,盐酸青藤碱各组炎症因子均显著下调(P<0.05),凋亡蛋白的表达量显著上升、Bcl-2的mRNA表达量下降(P<0.05),而PI3K/mTOR/AKT3信号通路分子的mRNA表达量均下降(P<0.05)。在蛋白质表达层面,与对照组相比较,中、高剂量盐酸青藤碱组血清中炎症因子IL-6、IL-8、IL-1β、TGF-β的水平均明显下调(P<0.05),凋亡蛋白cleaved-PARP、cleaved caspase-3/7及Bax的表达量均上调,并且与给药剂量成正相关,而抗凋亡蛋白Bcl-2、PI3K/AKT3/mTOR信号通路的表达量则与给药剂量成负相关。盐酸青藤碱对家兔膝关节成纤维细胞的存活表现为显著的抑制作用,作用机制或与下调炎症因子IL-6、IL-8、IL-1β的表达,并促进凋亡蛋白cleaved-PARP、cleaved caspase-3/7及Bax的表达,抑制Bcl-2的表达,抑制其下游PI3K/AKT3/mTOR信号通路的基因表达有关。结论盐酸青藤碱可抑制黏连性膝关节强直家兔膝关节成纤维细胞的炎症反应和加速成纤维细胞的凋亡,或可通过该机制为改善和治疗黏连性膝关节强直提供新的方法。
Objective This study aimed to observe the impact of sinomenine hydrochloride on the proliferation of fibroblasts and the mRNA expression of related genes in knee joint adhesion and contracture in rabbits.Additionally,we sought to explore its potential mechanisms in combating knee joint adhesion and contracture.Methods Fibroblasts were cultured in vitro,and experimental groups with varying concentrations of sinomenine hydrochloride were established alongside a control group.Cell proliferation was assessed using the CCK-8 assay.Changes in the mRNA expression of fibroblast-related genes following sinomenine hydrochloride treatment were evaluated using RT-qPCR.The impact of the drug on serum levels of inflammatory cytokines was determined using the ELISA method,and the expression of related proteins was assessed using Western blot.Results Sinomenine hydrochloride was found to inhibit fibroblast viability,with viability decreasing as the concentration of sinomenine hydrochloride increased.The effects of sinomenine hydrochloride in all experimental groups were highly significant(P<0.05).At the mRNA expression level,compared to the control group,sinomenine hydrochloride led to a significant downregulation of inflammatory cytokines in all groups(P<0.05).Additionally,the expression levels of apoptosis-related proteins significantly increased,while Bcl-2 mRNA expression decreased(P<0.05).The mRNA expression levels of the PI3K/mTOR/AKT3 signaling pathway also decreased(P<0.05).At the protein expression level,in comparison to the control group,the levels of inflammatory cytokines IL-6,IL-8,IL-1β,and TGF-βwere significantly downregulated in the middle and highdose sinomenine hydrochloride groups(P<0.05).The expression levels of cleaved-PARP,cleaved caspase-3/7,and Bax increased and were positively correlated with the dose,while the expression levels of the anti-apoptotic protein Bcl-2 and the PI3K/AKT3/mTOR signaling pathway were negatively correlated with the dose.Sinomenine hydrochloride exhibited a significant inhibitory effect on the viability of rabbit knee joint fibroblasts,which may be associated with the downregulation of inflammatory cytokines IL-6,IL-8,and IL-1β,promotion of apoptosis-related proteins cleaved-PARP,cleaved caspase-3/7,and Bax,suppression of Bcl-2 expression,and inhibition of gene expression in the downstream PI3K/AKT3/mTOR signaling pathway.Conclusion Sinomenine hydrochloride can inhibit the inflammatory response of fibroblasts in adhesive knee joints and accelerate fibroblast apoptosis.This mechanism may offer a novel approach to improving and treating knee joint adhesion.
作者
侯新聚
雷洪峰
陈勇
黎芙希
孙敬宁
刘佳铭
马红梅
HOU Xin-Ju;LEI Hong-Feng;CHEN Yong;LI Fu-Xi;SUN Jing-Ning;LIU Jia-Ming;MA Hong-Mei(Nanchang Hongdu Hospital of Traditional Chinese Medicine,Nanchang 330008,China;The Fourth Clinical Medical College,Guangzhou University of Chinese Medicine,Guangzhou 510006,China;Guangzhou University of Chinese Medicine,Second Clinical Medical College,Guangzhou 510006,China)
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2024年第4期959-968,共10页
Progress In Biochemistry and Biophysics
基金
江西省卫生健康委科技计划(202140175)
江西省中医药管理局科技计划(2021A196)资助项目。
关键词
盐酸青藤碱
黏连性膝关节强直
家兔
成纤维细胞
sinomenine hydrochloride
adhesion knee rigidity
domestic rabbits
fibroblasts
