骨痹止痛丸对肾阳虚大鼠软骨调控作用机制研究

Study on theregulation mechanism of Gubi Zhitong Wan on cartilage of kidney-yang deficiency rats

目的探讨骨痹止痛丸对肾阳虚大鼠软骨的保护作用及调控机制。方法根据随机数字表法将30只SPF级6周龄雄性大鼠分为对照组、肾阳虚组、骨痹止痛丸组,每组10只。对照组大鼠后肢肌肉注射0.9%生理盐水0.3 mL,每日1次,连续22天。肾阳虚组大鼠后肢肌肉注射氢化可的松混悬液2.5 mg/100 g,每日1次,连续进行22天。骨痹止痛丸组大鼠后肢肌肉注射氢化可的松混悬液2.5 mg/100 g,每日1次,连续22天,从第8天开始,以1.05 g/(kg·d)的剂量给予骨痹止痛丸水溶液灌胃给药,每日1次,连续进行15天,直至第22天。给药期间记录大鼠体质量变化。连续给药22天后麻醉大鼠,腹主动脉取血;处死大鼠,取肾脏和膝关节软骨组织,以苏木精—伊红(hematoxylin-eosin,HE)染色法进行病理切片观察。实时荧光定量PCR(real-time quantitative PCR,qRT-PCR)法检测各组大鼠膝关节软骨中Col2a1、Col9a1、Col10a1、Col11a1、Acan、Comp、Hapln1、Ibsp、Wwp2、Sox9、Sox5、Sox4等基因的表达水平。酶联免疫吸附法(enzyme cinked immunosorbent assay,ELISA)法检测各组大鼠血清促肾上腺皮质激素(adreno cortico tropic hormone,ACTH)、皮质醇(cortisol,CORT)、促黄体生成素(luteinizing hormone,LH)和睾酮(testosterone,T)水平。结果(1)与对照组比较,肾阳虚组血清ACTH、T、LH、CORT水平显著降低(P<0.05),经骨痹止痛丸给药治疗后,以上指标显著上升(P<0.05)。(2)膝关节软骨病理切片结果:肾阳虚组大鼠膝关节软骨层表面塌陷,凹凸不平,软骨层明显变薄,软骨细胞固缩,软骨基质成分明显减少。骨痹止痛丸组软骨层增厚,表面光滑连续,相比肾阳虚组软骨层中软骨细胞数量、软骨基质和同源软骨细胞数目增加,软骨层下骨小梁成分增宽,分布数量增加。肾脏病理切片结果:对照组肾脏肾小管和肾小球均未见异常变化;肾阳虚组肾小球扩张,近曲小管变性,远曲小管扩张。骨痹止痛丸组肾小球扩张程度减轻,肾小管功能状态改善,近曲小管未见明显变性,远曲小管未见明显扩张。(3)qRT-PCR检测结果:肾阳虚组较对照组的Col2a1、Col9a1、Col10a1、Col11a1、Acan、Comp、Hapln1、Ibsp、Wwp2、Sox9、Sox5、Sox4等基因表达明显下调(P<0.05);与肾阳虚组相比,骨痹止痛丸组以上基因表达明显上调(P<0.05)。结论骨痹止痛丸能够有效改善大鼠肾阳虚的病理状态,并通过调控Col2a1、Col9a1、Col10a1、Col11a1、Acan、Comp、Hapln1、Ibsp、Wwp2、Sox9、Sox5、Sox4等基因的表达,从而达到保护软骨的效果。

Objective To explore the protective effect and regulation mechanism of Gubi Zhitong Wan on cartilage of rats with kidney yang deficiency.Methods According to the method of random number table,30 SPF 6-week-old male rats were divided into control group(n=10),kidney yang deficiency group(n=10)and Gubi Zhitong Wan group(n=10).Rats in control group were intramuscularly injected with 0.9%saline 0.3 mL once a day for 22 days.Rats in kidney yang deficiency group were injected with hydrocortisone suspension 2.5 mg/100 g once a day for 22 days.Rats in Gubi Zhitong Wan group were injected with hydrocortisone suspension 2.5 mg/100 g into the hindlimb muscle once a day for 22 days.From the 8th day,rats in Gubi Zhitong Wan group were given 1.05 g/(kg·d)aqueous solution by intragastric administration,once a day for 15 consecutive days until the 22nd day.The changes of body mass of rats were recorded during administration.After continuous administration for 22 days,rats were anesthetized and the blood was taken from the abdominal aorta,rats were killed,and the cartilage tissues of the kidney and knee joint were taken,and the pathological sections were observed by hematoxylin-eosin(HE)staining.The expression levels of Col2a1,Col9a1,Col10a1,Col11a1,Acan,Comp,Hapln1,Ibsp,Wwp2,Sox9,Sox5 and Sox4 in knee joint cartilage of rats in each group were detected by real-time quantitative PCR(qRT-PCR).The levels of adreno cortico tropic hormone(ACTH),cortisol(CORT),luteinizing hormone(LH)and testosterone(T)were measured by ELISA.Results(1)Compared with control group,the serum levels of ACTH,T,LH and CORT in kidney yang deficiency group decreased significantly(P<0.05),and the above indexes increased significantly after treatment with Gubi Zhitong Wan(P<0.05).(2)Pathological section results of knee cartilage:in kidney yang deficiency group,the surface of the cartilage layer of the knee joint collapsed,the cartilage layer became thinner,the chondrocytes pyknotic,and the cartilage matrix decreased significantly.In Gubi Zhitong Wan group,the cartilage layer was thickened and the surface was smooth and continuous.Compared with kidney yang deficiency group,the number of chondrocytes,cartilage matrix and homologous chondrocytes in the cartilage layer increased,and the trabecular components under the cartilage layer widened and the number of distribution increased.The results of renal pathological sections:there were no abnormal changes in renal tubules and glomeruli in control group,and glomeruli dilatation,proximal convoluted tubule degeneration and distal convoluted tubule dilatation in kidney yang deficiency group.In Gubi Zhitong Wan group,the degree of glomerular dilatation was reduced,the functional state of renal tubule was improved,there was no obvious degeneration of proximal convoluted tubule and no obvious dilation of distal convoluted tubule.(3)The expression of Col2a1,Col9a1,Col10a1,Col11a1,Acan,Comp,Hapln1,Ibsp,Wwp2,Sox9,Sox5,Sox4 and other genes in kidney yang deficiency group was significantly lower than that in control group(P<0.05),and the expression of the above genes in Gubi Zhitong Wan group was significantly higher than that in kidney yang deficiency group(P<0.05).Conclusion Gubi Zhitong Wan can effectively improve the pathological state of kidney yang deficiency in rats and protect cartilage by regulating the expression of Col2a1,Col9a1,Col10a1,Col11a1,Acan,Comp,Hapln1,Ibsp,Wwp2,Sox9,Sox5 and Sox4.