独活寄生汤调控miR-214-3p抑制软骨细胞铁死亡

Duhuo Jisheng Decoction Inhibiting Ferroptosis of Hondrocytes by Regulating miR-214-3p

目的:基于miR-214-3p探讨独活寄生汤(Duhuo Jisheng decoction,DHJSD)对软骨细胞铁死亡的影响。方法:采用机械-Ⅱ型胶原酶消化法提取原代软骨细胞,免疫组织化学法(immunohistochemistry,IHC)检测Ⅱ型胶原进行细胞鉴定。慢病毒转染软骨细胞,分为NC组(转染si-NC)、miR-214-3p组(转染si-miR-214-3p),qRT-PCR法检测miR-214-3p mRNA表达水平。随后,将软骨细胞分为空白组、模型组(Erastin 1μmol·L^(-1))、DHJSD组(Erastin 1μmol·L^(-1)+DHJSD 300 mg·L^(-1))、si-NC组(Erastin 1μmol·L^(-1)+DHJSD 300 mg·L^(-1)+转染si-NC)、si-miR-214-3p组(Erastin 1μmol·L^(-1)+DHJSD 300 mg·L^(-1)+转染si-miR-214-3p)。采用qRT-PCR法检测细胞miR-214-3p mRNA表达水平,Western blot法检测细胞谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)、酰基辅酶A合成酶长链家族成员4(recombinant acyl coenzyme A synthetase long chain family member 4,ACSL4)、Ⅱ型胶原蛋白(CollagenⅡ)、基质金属蛋白酶13(matrix metalloproteinase 13,MMP-13)表达水平。结果:IHC显示,阳性组软骨细胞胞质呈棕黄色,细胞核呈蓝色。qRT-PCR显示,与NC组比较,miR-214-3p组细胞miR-214-3p mRNA水平降低(P<0.05)。模型组细胞miR-214-3p mRNA水平较空白组降低,经DHJSD治疗后升高(P<0.01)。与si-NC组比较,si-miR-214-3p组细胞miR-214-3p mRNA水平降低(P<0.05)。Western blot显示,与空白组比较,模型组细胞GPX4、CollagenⅡ表达水平降低,ACSL4、MMP-13表达水平升高(P<0.05),经DHJSD治疗后得到逆转(P<0.05)。与si-miR-214-3p组比较,si-NC组、DHJSD组细胞GPX4表达水平升高,MMP-13表达水平降低(P<0.05)。结论:DHJSD可抑制软骨细胞铁死亡,其机制可能与上调miR-214-3p基因表达水平有关。

Objective:To explore the effect of Duhuo Jisheng Decoction(DHJSD)on ferroptosis chondrocyte by regulating miR-214-3p.Methods:Primary chondrocytes were extracted by mechanical-typeⅡcollagenase digestion,and typeⅡcollagen was detected by immunohistochemistry(IHC)for identification.Chondrocytes were lentivirally transfected and divided into NC group(transfected with si-NC),miR-214-3p group(transfected with si-miR-214-3p),and miR-214-3p expression level was detected by qRT-PCR.Subsequently,chondrocytes were divided into blank group,model group(Erastin 1μmol·L^(-1)),DHJSD group(Erastin 1μmol·L^(-1)+DHJSD 300 mg·L^(-1)),si-NC group(Erastin 1μmol·L^(-1)+DHJSD 300 mg·L^(-1)+transfected si-NC),si-miR-214-3p group(Erastin 1μmol·L^(-1)+DHJSD 300 mg·L^(-1)+transfected with si-miR-214-3p).The expression level of miR-214-3p was detected by qRT-PCR,and the expression level of glutathione peroxidase 4(GPX4),recombinant acyl coenzyme A long chain family member 4(recombinant acyl coenzyme A)was detected by Western blot.synthetase long chain family member 4(ACSL4),collagen typeⅡ(CollagenⅡ),and matrix metalloproteinase 13(MMP-13)expression levels.Results:With IHC,it was showed that the cytoplasm of chondrocytes in the positive group was brownish-yellow,and the nuclei were blue.With qRT-PCR,it was showed that the miR-214-3p mRNA level of the cells in the miR-214-3p group was reduced compared with that of the NC group(P<0.05).The miR-214-3p mRNA level of cells in the model group was reduced compared with the blank group and was elevated by DHJSD treatment(P<0.01).The miR-214-3p mRNA level of cells in the si-miR-214-3p group was reduced compared with that of the si-NC group(P<0.05).With western blot,it was showed that the expression level of GPX4 and CollagenⅡin cells of the model group was reduced compared with that of the blank group,and the expression level of ACSL4 and MMP-13 was elevated(P<0.05),and it was reversed with DHJSD treatment(P<0.05).Compared with the si-miR-214-3p group,the expression level of GPX4 in cells of the si-NC group and DHJSD group was increased,and the expression level of MMP-13 was decreased(P<0.05).Conclusion:DHJSD can inhibit chondrocyte ferroptosis,and its mechanism may be related to the up-regulation of miR-214-3p expression.