目的:探讨黄芩苷干预膝骨关节炎(knee osteoarthritis，KOA)大鼠的疗效和作用机制。方法:将50只大鼠随机分为假手术组、模型组、低剂量黄芩苷组、高剂量黄芩苷组、高剂量黄芩苷联合抑制剂组，每组10只。将假手术组以外的大鼠采用切断前交叉韧带和切除内侧半月板的方法建立右膝关节KOA模型，假手术组大鼠于右膝关节内侧做一切口后缝合。造模成功后，低剂量黄芩苷组、高剂量黄芩苷组大鼠分别按照50 mg·kg-1、100 mg·kg-1的剂量给予黄芩苷生理盐水溶液灌胃;高剂量黄芩苷联合抑制剂组大鼠按照100 mg·kg-1的剂量给予黄芩苷生理盐水溶液灌胃，并按照1 mg·kg-1的剂量给予XMU-MP-1溶液腹腔注射;假手术组和模型组大鼠均给予等量生理盐水灌胃。每天给药1次，连续给药30 d。分别于给药前、给药15 d时、给药30 d时测量大鼠双侧膝关节肿胀程度。给药结束后处死大鼠，取大鼠右侧膝关节软骨组织，采用苏木精-伊红染色和番红O-固绿染色观察膝关节软骨组织病理变化，采用Mankin评分标准评价软骨退变情况，采用ELISA试剂盒检测肿瘤坏死因子-α(tumor necrosis factor-α，TNF-α)、白细胞介素(interleukin，IL)-1β、IL-10、基质金属蛋白酶(matrix metalloproteinase，MMP)-13 表达水平，采用 Western blotting 检测滑膜组织中切割活化的半胱氨酸天冬氨酸蛋白酶(Cleaved-cysteine aspartic acid specific protease，Cleaved-Caspase)-3、B淋巴细胞瘤-2 相关 X 蛋白(Bcl2 associated X protein，Bax)、B 淋巴细胞瘤-2(B-cell lymphoma-2，Bcl-2)蛋白、Yes 相关蛋白(Yes-associated protein，YAP)、Tafazzin(TAZ)蛋白的表达水平。结果:①大鼠右膝关节肿胀程度。给药15 d时和给药30 d时，高剂量黄芩苷组大鼠右膝关节肿胀程度均低于模型组、低剂量黄芩苷组和高剂量黄岑苷联合抑制剂组(P=0。000，P=0。000，P=0。000;P=0。000，P=0。000，P=0。000);模型组、低剂量黄芩苷组、高剂量黄芩苷联合抑制剂组大鼠右膝关节肿胀程度两两比较，差异均无统计学意义(P=0。063，P=0。215，P=0。399;P=0。052，P=0。261，P=0。240)。②大鼠右膝关节软骨组织病理变化。干预结束后，模型组大鼠右膝关节软骨萎缩、排列混乱;低剂量黄芩苷组、高剂量黄芩苷组、高剂量黄芩苷联合抑制剂组大鼠右膝关节软骨萎缩、细胞排序情况均较模型组改善，且高剂量黄芩苷组大鼠的改善情况优于低剂量黄芩苷组和高剂量黄芩苷联合抑制剂组。③大鼠右膝关节软骨Mankin评分。干预结束后，低剂量黄芩苷组、高剂量黄芩苷组、高剂量黄芩苷联合抑制剂组大鼠右膝关节软骨Mankin评分均低于模型组(P=0。000，P=0。000，P=0。000)，高剂量黄芩苷组右膝关节软骨Mankin评分低于低剂量黄芩苷组和高剂量黄芩苷联合抑制剂组(P=0。000，P=0。000)，低剂量黄芩苷组大鼠右膝关节软骨Mankin评分低于高剂量黄芩苷联合抑制剂组(P=0。000)。④大鼠右膝关节软骨组织中TNF-α、IL-1β、IL-10、MMP-13表达水平。低剂量黄芩苷组、高剂量黄芩苷组、高剂量黄芩苷联合抑制剂组大鼠右膝关节软骨组织中TNF-α、IL-1β、MMP-13表达水平均低于模型组(P=0。000，P=0。000，P=0。000;P=0。000，P=0。000，P=0。000;P=0。000，P=0。000，P=0。000)，IL-10 表达水平均高于模型组(P=0。000，P=0。000，P=0。000);高剂量黄芩苷组大鼠右膝关节软骨组织中TNF-α、IL-1β、MMP-13表达水平均低于低剂量黄芩苷组和高剂量黄芩苷联合抑制剂组(P=0。000，P=0。000，P=0。000;P=0。000，P=0。000，P=0。000)，IL-10表达水平高于低剂量黄芩苷组和高剂量黄芩苷联合抑制剂组(P=0。000，P=0。000)。⑤大鼠右膝关节软骨组织细胞凋亡及Hippo-YAP信号通路相关蛋白表达水平。低剂量黄芩苷组、高剂量黄芩苷组、高剂量黄芩苷联合抑制剂组大鼠右膝关节软骨组织中Cleaved-Caspase-3、Bax表达水平均低于模型组(P=0。000，P=0。000，P=0。000;P=0。000，P=0。000，P=0。000)，BCL-2、YAP、TAZ 表达水平均高于模型组(P=0。000，P=0。000，P=0。000;P=0。000，P=0。000，P=0。000;P=0。000，P=0。000，P=0。000);高剂量黄芩苷组大鼠右膝关节软骨组织中Cleaved-Caspase-3、Bax表达水平均低于低剂量黄芩苷组和高剂量黄芩苷联合抑制剂组(P=0。000，P=0。000;P=0。000，P=0。000)，BCL-2、YAP、TAZ表达水平均高于低剂量黄芩苷组和高剂量黄芩苷联合抑制剂组(P=0。000，P=0。000;P=0。000，P=0。000;P=0。000，P=0。000)。结论:黄芩苷干预KOA大鼠，能够缓解膝关节肿胀，抑制炎症反应和细胞凋亡，延缓关节软骨退变，其作用机制可能与激活Hippo-YAP信号通路有关。
Outcome and mechanism of baicalin against knee osteoarthritis in rats:a Hippo-YAP signaling pathway-based experimental study
Objective:To observe the effects of baicalin(BC)on knee osteoarthritis(KOA)in rats,and to explore its mechanism.Methods:Fifty rats were randomized into sham(SH)group,model group,low-dose BC(L-BC)group,high-dose BC(H-BC)group,and H-BC combined inhibitor group,10 rats in each group.All rats but the ones in SH group were subjected to anterior cruciate ligament transection(ACLT)and medial meniscectomy on the right knees for inducing KOA;while the ones in SH group underwent surgeries for merely making incision on the medial side of right knee,and the incision was sutured instantly after exposing the articular cavity.After successful model-ing,the rats in L-BC group and H-BC group were intragastric administrated with 50 and 100 mg/kg BC physiological saline solution(PSS),respectively;the ones in H-BC combined inhibitor group with 100 mg/kg BC PSS,followed by intraperitoneal injection of 1 mg/kg XMU-MP-1 solution;while the ones in SH group and model group with the same dose of normal saline.All rats in the 5 groups were intervened once a day for consecutive 30 days.Before the drug intervention,on day 15 and 30 after the drug intervention,the degree of swelling in the bilateral knees was measured,respectively.After the end of drug intervention,the rats were sacrificed,and their right knee cartilage tissues were harvested and stained with hematoxylin-eosin(HE)and safranin O-fast green(SO-FG)for observing the histopathological changes;meanwhile,the degree of knee articular cartilage degeneration was evaluated by using Mankin scoring system.Furthermore,the expression levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,IL-10 and matrix metalloproteinase(MMP)-13 in the right knee cartilage tissues were detected by using the enzyme-linked immunosorbent assay(ELISA)kit,and the expression levels of the Cleaved-cysteine aspar-tic acid specific protease(Cleaved-Caspase)-3,Bcl2-associated X protein(Bax),B-cell lymphoma-2(Bcl-2)protein,Yes-associated protein(YAP)and Tafazzin(TAZ)in the synovial tissues were detected by using Western blotting.Results:①0n day 15 and 30 after the drug in-tervention,the degree of swelling in the right knee was lower in H-BC group compared to model group,L-BC group and H-BC combined in-hibitor group(P=0.000,P=0.000,P=0.000;P=0.000,P=0.000,P=0.000);further pairwise comparison among model group,L-BC group and H-BC combined inhibitor group showed no statistical significance(P=0.063,P=0.215,P=0.399;P=0.052,P=0.261,P=0.240).②After the end of drug intervention,the atrophic and disorderly arranged cartilage was observed in the right knee of rats in model group.The cartilage atrophy and cellular arrangement were improved in rats of L-BC group,H-BC group,and H-BC combined inhibitor group compared with that of model group,and the improvement was better in H-BC group in contrast to L-BC group and H-BC combined in-hibitor group.③After the end of drug intervention,the Mankin score of the right knee cartilage was lower in L-BC group,H-BC group and H-BC combined inhibitor group compared to model group(P=0.000,P=0.000,P=0.000),and was lower in L-BC group compared to H-BC combined inhibitor group(P=0.000),and was lowest in H-BC group(P=0.000,P=0.000).④The expression levels of TNF-a,IL-1β,and MMP-13 in the right knee cartilage tissues were lower in L-BC group,H-BC group and H-BC combined inhibitor group compared to model group(P=0.000,P=0.000,P=0.000;P=0.000,P=0.000,P=0.000;P=0.000,P=0.000,P=0.000),and was lower in H-BC group compared to L-BC group and H-BC combined inhibitor group(P=0.000,P=0.000,P=0.000;P=0.000,P=0.000,P=0.000);while the expression level of IL-10 was higher in L-BC group,H-BC group and H-BC combined inhibitor group compared to model group(P=0.000,P=0.000,P=0.000),and was higher in H-BC group compared to L-BC group and H-BC combined inhibitor group(P=0.000,P=0.000).⑤The Cleaved-Caspase-3 and Bax were lowly expressed in the right knee cartilage tissues of rats in L-BC group,H-BC group and H-BC combined inhibitor group compared to model group(P=0.000,P=0.000,P=0.000;P=0.000,P=0.000,P=0.000),and were lowly expressed in H-BC group compared to L-BC group and H-BC combined inhibitor group(P=0.000,P=0.000;P=0.000,P=0.000);while the BCL-2,YAP and TAZ were highly expressed in L-BC group,H-BC group,and H-BC combined inhibitor group compared to model group(P=0.000,P=0.000,P=0.000;P=0.000,P=0.000,P=0.000;P=0.000,P=0.000,P=0.000),and were highly expressed in H-BC group compared to L-BC group and H-BC combined inhibitor group(P=0.000,P=0.000;P=0.000,P=0.000;P=0.000,P=0.000).Conclusion:BC can alleviate knee swelling,inhibit inflammatory response and apoptosis,and delay articular cartilage degeneration in KOA rats.It may work by activating Hippo-YAP signaling pathway.
万方数据
维普
