山姜素调节VEGF/SphK1/S1P信号通路对膝骨关节炎大鼠血管生成的影响

Impacts of alpinetin on angiogenesis in knee osteoarthritis rats by regulating the VEGF/SphK1/S1P signaling pathway

目的探讨山姜素(APT)调节血管内皮生长因子/鞘氨醇激酶1/1磷酸鞘氨醇(VEGF/SphK1/S1P)信号通路对膝骨关节炎(KOA)大鼠血管生成的影响。方法采用改良的Videman法构建KOA大鼠模型,将90只大鼠分为对照组(Control组)、模型组(Model组)、山姜素低剂量组(L-APT组)、山姜素高剂量组(H-APT组)、山姜素高剂量组+慢病毒阴性对照组(APT+NC组)、山姜素高剂量组+过表达SphK1慢病毒组(APT+SphK1组),每组15只。HE染色观察大鼠软骨组织病理变化;酶联免疫吸附试验测定软骨组织白细胞介素(IL)-1β、肿瘤坏死因子α(TNF-α)、IL-6、基质金属蛋白酶-13(MMP-13)水平;TUNEL检测软骨组织细胞凋亡情况;免疫组化检测血管内皮生长因子(VEGF)、CD31蛋白表达情况;Western blot检测血管内皮生长因子受体2(VEGFR2)、磷酸化VEGFR2(p-VEGFR2)、SphK1、S1P蛋白水平。结果与Control组比较,Model组大鼠出现病理损伤,细胞凋亡率、IL-1β、TNF-α、IL-6、MMP-13、VEGF阳性表达、CD31阳性表达和p-VEGFR2、SphK1、S1P蛋白表达水平增加(P<0.05);与Model组比较,L-APT组、H-APT组病理损伤明显减轻,细胞凋亡率、IL-1β、TNF-α、IL-6、MMP-13、VEGF阳性表达、CD31阳性表达和pVEGFR2、SphK1、S1P蛋白表达水平降低(P<0.05);与APT+NC组比较,APT+SphK1组软骨组织病理损伤加重,细胞凋亡率、IL-1β、TNF-α、IL-6、MMP-13、VEGF阳性表达、CD31阳性表达和p-VEGFR2、SphK1、S1P蛋白表达水平增加(P<0.05)。结论APT通过抑制VEGF/SphK1/S1P信号通路抑制KOA大鼠血管生成。

Objective To investigate effects of alpinetin(APT)on angiogenesis in knee osteoarthritis(KOA)rats by regulating vascular endothelial growth factor/sphingosine kinase 1/sphingosine 1 phosphate(VEGF/SphK1/S1P)signaling pathway.Methods The KOA rat model was established by Videman method.Ninety rats were grouped into the control group,the model group,the low-dose kaempferol group(L-APT group),the high-dose kaempferol group(H-APT group),the high-dose kaempferol group+lentivirus negative control group(APT+NC group)and high-dose kaempferol+overexpression of SphK1 lentivirus group(APT+SphK1 group),with 15 rats in each group.Pathological changes of cartilage tissue in rats were observed by HE staining.Contents of IL-1β,TNF-α,IL-6 and MMP-13 in cartilage tissue were measured by enzyme linked immunosorbent assay.Chondrocyte apoptosis of cartilage tissue cells was detected by TUNEL.VEGF and CD31 protein positive expression levels were detected by immunohistochemistry assay.The p-VEGFR2,VEGFR2,SphK1 and S1P protein levels were detected by Western blot assay.Results Rats in the model group showed pathological damage.Compared with the control group,the apoptosis rate,IL-1b,TNF-a,IL-6,MMP-13 levels,VEGF positive expression,CD31 positive expression,p-VEGFR2,SphK1 and S1P protein expression levels were increased in the model group(P<0.05).Compared with the model group,the pathological damage was obviously reduced in the L-APT group and the M-APT group,and cell apoptosis rate,IL-1β,TNF-α,IL-6,MMP-13 levels,VEGF positive expression,CD31 positive expression,p-VEGFR2,SphK1 and S1P protein expression levels were obviously reduced(P<0.05).Compared with the APT+NC group,the pathological injury of cartilage tissue increased in the APT+SphK1 group,cell apoptosis rate,IL-1β,TNF-α,IL6,MMP-13 levels,VEGF positive expression,CD31 positive expression,p-VEGFR2,SphK1 and S1P protein expression levels were obviously increased(P<0.05).Conclusion APT inhibits angiogenesis in knee osteoarthritis rats by inhibiting the VEGF/SphK1/S1P signaling pathway.