白脉软膏含药血浆对大鼠退变滑膜细胞和软骨细胞共培养体系的作用及机制研究

Effects of Plasma Containing Baimai Ointment on Co-cultivation of Rat Degenerative Synoviocytes and Chondrocytes and Its Mechanism

【目的】观察白脉软膏含药血浆对大鼠退变滑膜细胞和软骨细胞共培养体系的干预作用,并从"滑膜—软骨"轴角度探讨骨关节炎(OA)的发病机制。【方法】取新生SD大鼠膝关节滑膜,经消化酶消化后获得原代滑膜细胞,取第1代(P1)细胞进行实验。实验设空白组、模型组、西药组、白脉组。模型组加入脂多糖(LPS)进行诱导,西药组与白脉组在模型组LPS诱导的基础上分别加入扶他林软膏、白脉软膏含药血浆干预。LPS诱导24 h后观察滑膜细胞形态变化,酶联免疫吸附分析(ELISA)检测滑膜细胞上清中白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)含量。此后,各组皆与正常软骨细胞进行共培养。共培养24 h后观察软骨细胞形态,实时定量PCR(qPCR)检测软骨细胞基质金属蛋白酶3(MMP3)、带有血小板凝血酶敏感蛋白样模体的解整链蛋白金属蛋白酶4(ADAMTS4)、TNF-α、性别决定区Y框蛋白9(Sox9)mRNA表达情况,Westernblot法检测Ⅱ型胶原蛋白(COL2A1)、基质金属蛋白酶13(MMP13)、磷酸化核因子κBp65(p-NF-κBp65)表达情况。【结果】LPS诱导后滑膜细胞形态由原来的多角形变成长梭形。ELISA结果显示,模型组滑膜细胞上清中IL-1β、TNF-α含量较空白组升高(P <0.05),而白脉组IL-1β、TNF-α含量较模型组下降。软骨细胞与退变滑膜细胞培养24 h后由原来的多角形变为长梭形。qPCR结果显示:与空白组比较,模型组软骨细胞MMP3、ADAMTS4、TNF-αmRNA表达升高(P <0.05),Sox9mRNA表达降低(P <0.05);与模型组比较,白脉组软骨细胞MMP3、TNF-α、ADAMTS4 mRNA表达明显降低(P <0.05),Sox9 mRNA表达水平升高(P <0.05)。Western blot结果显示:与空白组比较,模型组软骨细胞MMP13、p-NF-κBp65表达升高(P <0.05),COL2A1表达明显降低(P <0.05);与模型组比较,白脉组软骨细胞MMP13、p-NF-κBp65表达降低(P <0.05),COL2A1表达升高(P <0.05)。【结论】退变滑膜细胞可导致软骨细胞退变并促进骨关节炎发展,白脉软膏对此病理过程有明显的抑制作用。

Objective To investigate the intervention effects of plasma containing Baimai Ointment on the co-cultivation of rat degenerative synoviocytes and chondrocytes,and to discuss the pathogenesis of osteoarthritis(OA)in terms of synovium-cartilage axis.Methods Synovium was taken out from articular synovium of the newly born SD rats and then was digested with enzymes to obtain the primary synoviocytes.Synoviocytes at passage one were used for the experiment.The blank group,model group,western medicine group,and Baimai group were set up.Synoviocytes in the model group were cultured with lipopolysaccharide(LPS),and the western medicine group and Baimai group were given respectively Diclofenac Diethylamine Emulgel(DDE)-containing plasma and Baimai Ointment-containing plasma together with LPS induction.After 24-hour LPS induction,the morphological changes of the synoviocytes were observed,and the contents of interleukin 1β(IL-1β)and tumor necrosis factorα(TNF-α)in the supernatant were detected by enzyme-linked immunosorbent assay(ELISA).And then the normal cartilage cells were added into every group for co-cultivation with synoviocytes.After 24-hour co-culture,the morphological changes of the chondrocytes were observed,the mRNA levels of matrix metalloproteinases 3(MMP3),A disintegrin and metalloproteinase with thrombospondin motif 4(ADAMTS4),TNF-α,and sex determining region Y-box 9(Sox9)were detected by real time quantitative polymerase chain reaction(qPCR),and the protein expression levels of typeⅡcollagen(COL2 A1),phosphorylated nuclear factor kappaBp65(p-NF-κBp65)and matrix metalloproteinases 13(MMP13)were determined by Western blotting method.Results After LPS induction,the shape of synoviocytes was changed from polygon to long spindle-shape.ELISA results showed that the contents of IL-1βand TNF-αin the synoviocyte supernatant of the model group were increased as compared with those of the blank group(P<0.05),and the contents of IL-1βand TNF-αin the synoviocyte supernatant of Baimai group were decreased as compared with those of the model group(P<0.05).After 24-hour co-cultivation of chondrocytes together with degenerated synoviocytes,the shape of chondrocytes was changed from polygon to long spindle-shape.QPCR results showed that the mRNA expression levels of MMP3,ADAMTS4,and TNF-αwere increased,and Sox9 mRNA expression level was decreased in the model group compared with those in the blank group(P<0.05);the mRNA expression levels of MMP3,ADAMTS4,and TNF-αwere decreased,and Sox9 mRNA expression level was increased in the Baimai group compared with those in the model group(P<0.05).Western blotting results showed that the expression levels of MMP13 and p-NF-κBp65 were increased while the expression level of COL2 A1 was decreased in the model group compared with those in the blank group(P<0.05);the expression levels of MMP13 and p-NF-κBp65 were decreased,and the expression level of COL2 A1 was increased in Baimai group compared with those in the model group(P<0.05).Conclusion The degenerated synoviocytes can cause the degeneration of chondrocytes thus to aggravate OA development,while Baimai Ointment has obvious effects on inhibiting the pathological process.