耳穴贴压联合电针对膝骨关节炎模型大鼠滑膜纤维化及内质网应激的抑制作用研究

Study of the inhibiting effects of ear point sticking plus electroacupuncture on synovial fibrosis and endoplasmic reticulum stress in knee osteoarthritis model rats

目的观察耳穴贴压联合电针对膝骨关节炎模型大鼠滑膜纤维化及内质网应激的抑制作用。方法将60只Sprague-Dawley(SD)大鼠随机分为正常组(10只)和其余组(50只),其余组造模成功后再次随机分为模型组、耳穴组、电针组、联合组及药物组,每组10只。耳穴组使用磁珠贴于大鼠两侧肾耳穴进行干预,电针组予电针干预,联合组予耳穴磁珠贴压联合电针干预,药物组予塞来昔布水溶液灌胃干预。观察各组大鼠膝关节肿胀度的变化。观察各组大鼠血清白介素-1(interleukin-1,IL-1)、白介素-6(interleukin-6,IL-6)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、锰超氧化物歧化酶(manganese superoxide dismutase,Mn-SOD)、过氧化氢酶(catalase,CAT)和丙二醛(malondialdehyde,MDA)的水平,观察关节液中环氧合酶-2(cyclooxygenase-2,COX-2)、前列腺素E2(prostaglandin E2,PGE2)和一氧化氮(nitric oxide,NO)的水平,观察各组大鼠滑膜组织中白介素-1β(interleukin-1β,IL-1β)及TNF-α的水平。观察大鼠软骨组织病理学变化,比较各组滑膜组织中葡萄糖调节蛋白78(glucose regulated protein 78,GRP78)、C/EBP同源蛋白(C/EBP-homologous protein,CHOP)、基质金属蛋白酶13(matrix metalloproteinase 13,MMP13)、α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、金属蛋白酶组织抑制剂1(tissue inhibitor of metalloproteinase 1,TIMP1)、胶原蛋白Ⅰ(collagenⅠ,Col-Ⅰ)、胶原蛋白Ⅱ(collagenⅡ,Col-Ⅱ)mRNA水平和蛋白水平。结果与正常组比较,模型组大鼠关节肿胀度,血清IL-1、IL-6、TNF-α和MDA水平,关节液COX-2、PGE2和NO水平,滑膜组织IL-1β和TNF-α水平,滑膜组织GRP78、CHOP、MMP-13、α-SMA、TIMP1、Col-Ⅰ、Col-ⅡmRNA及蛋白水平,均显著升高(P<0.05);模型组大鼠血清Mn-SOD和CAT水平显著降低(P<0.05)。与模型组比较,耳穴组、电针组、联合组和药物组大鼠关节肿胀度,血清IL-1、IL-6、TNF-α和MDA水平,关节液COX-2、PGE2和NO水平,滑膜组织IL-1β和TNF-α水平,滑膜组织GRP78、CHOP、MMP-13、α-SMA、TIMP1、Col-Ⅰ、Col-ⅡmRNA及蛋白水平,均显著降低(P<0.05);血清Mn-SOD和CAT水平显著升高(P<0.05);大鼠关节软骨组织病理学明显改善。与耳穴组和电针组比较,联合组大鼠关节肿胀度,血清IL-1、IL-6、TNF-α和MDA水平,关节液COX-2、PGE2和NO水平,滑膜组织IL-1β和TNF-α水平,滑膜组织GRP78、CHOP、MMP-13、α-SMA、TIMP1、Col-Ⅰ、Col-ⅡmRNA及蛋白水平,均显著降低(P<0.05);血清Mn-SOD和CAT水平均显著升高(P<0.05)。结论耳穴贴压联合电针能显著改善膝骨关节炎大鼠关节肿胀程度,抑制病理进展,缓解大鼠滑膜组织炎症反应,修复氧化应激损伤,进而修复膝骨关节炎大鼠滑膜组织纤维化及内质网应激反应。

Objective To observe the inhibiting effects of ear point sticking plus electroacupuncture on synovial fibrosis and endoplasmic reticulum stress in knee osteoarthritis model rats.Method Sixty Sprague-Dawley(SD)rats were randomized into a normal group(10 rats)and another group(50 rats).After successful modeling,rats in the another group were re-randomized into a model group,an ear point group,an electroacupuncture group,a combined group,and a medication group,each having 10 rats.Rats in the ear point group received magnetic pearl plasters applied to bilateral ear Kidney point(CO10),the electroacupuncture group received electroacupuncture intervention,and the medication group received Celecoxib solution via gavage.The rat knee joint swelling severity was observed in each group.The serum levels of interleukin(IL)-1,IL-6,tumor necrosis factor-α(TNF-α),manganese superoxide dismutase(Mn-SOD),catalase(CAT),and malondialdehyde(MDA)were detected.The levels of cyclooxygenase-2(COX-2),prostaglandin E2(PGE2),and nitric oxide(NO)in the joint fluid were determined.The IL-1βand TNF-αlevels in the synovial tissues were also checked.The pathological changes in the cartilage tissues were observed,and the mRNA and protein expression levels of glucose regulated protein 78(GRP78),C/EBP-homologous protein(CHOP),matrix metalloproteinase 13(MMP-13),α-smooth muscle actin(α-SMA),tissue inhibitor of metallo-proteinase 1(TIMP1),collagen I(Col-Ⅰ),and collagenⅡ(Col-Ⅱ)in the synovial tissues were compared among the groups.Result Compared with the normal group,the rat joint swelling degree,the serum levels of IL-1,IL-6,TNF-α,and MDA,the levels of COX-2,PGE2,and NO in the joint fluid,and the IL-1βand TNF-αlevels in the synovial tissues,as well as the mRNA and protein expression levels of GRP78,CHOP,MMP-13,α-SMA,TIMP1,Col-Ⅰ,and Col-Ⅱin the synovial tissues all increased significantly in the model group(P<0.05);the serum levels of Mn-SOD and CAT dropped significantly in the model group(P<0.05).Compared with the model group,the rat joint swelling degree,the serum levels of IL-1,IL-6,TNF-α,and MDA,the levels of COX-2,PGE2,and NO in the joint fluid,the IL-1βand TNF-αlevels in the synovial tissues,and the mRNA and protein expression levels of GRP78,CHOP,MMP-13,α-SMA,TIMP1,Col-Ⅰ,and Col-Ⅱin the synovial tissues all decreased significantly in the ear point,electroacupuncture,combined,and medication groups(P<0.05);the serum levels of Mn-SOD and CAT increased significantly(P<0.05);pathological changes in the articular cartilage tissues improved notably.Compared with the ear point and electroacupuncture groups,the joint swelling degree,the serum levels of IL-1,IL-6,TNF-α,and MDA,the levels of COX-2,PGE2,and NO in the joint fluid,the levels of IL-1βand TNF-αin the synovial tissues,and the mRNA and protein expression levels of GRP78,CHOP,MMP-13,α-SMA,TIMP1,Col-Ⅰ,and Col-Ⅱin the synovial tissues all dropped significantly in the combined group(P<0.05);the serum levels of Mn-SOD and CAT increased markedly(P<0.05).Conclusion Ear point sticking plus electroacupuncture can improve joint swelling degree,inhibit pathological progression,mitigate inflammatory responses in the synovial tissues,and repair oxidative stress damage to repair synovial tissue fibrosis and endoplasmic reticulum stress responses in knee osteoarthritis rats.