左归丸对去卵巢致骨质疏松症模型大鼠破骨细胞活性及miR-133b-3p/RhoA表达的影响

Effects of Zuogui Pill on osteoclast activity and expression of miR-133b-3p/RhoA in postmenopausal osteoporosis rats

目的研究左归丸对去卵巢致骨质疏松症大鼠破骨细胞中miR-133b-3p、RAS同源基因家族成员A(RhoA)表达的影响,探讨其作用机制。方法将SD雌性大鼠按随机数字表法分为正常组、模型组、假手术组、左归丸组,每组6只。模型组、左归丸组采用卵巢切除术构建大鼠骨质疏松症模型。左归丸组灌胃左归丸水煎液10 g/kg,连续灌胃12周。采用比色法测定大鼠血清钙、磷水平,ELISA法检测ALP水平;骨密度仪检测各组大鼠股骨骨密度。培养各组破骨细胞,采用Western blot法检测RANKL、RUNX2蛋白表达。对各组大鼠骨组织进行miRNA测序及差异表达分析。采用miR-133b-3p类似物及其对照处理破骨细胞,采用细胞计数试剂盒-8(CCK-8)方法检测破骨细胞增殖活力,采用抗酒石酸酸性磷酸酶染色检测破骨细胞活性,双荧光素酶报告基因实验检测miR-133b-3p与RhoA的靶向关系,采用miR-133b-3p类似物及RhoA共表达的"挽救"实验研究左归丸影响破骨细胞活性的分子调控机制。结果与模型组比较,左归丸组骨密度增加(P<0.05或P<0.01),血清钙、磷水平增加(P<0.05),ALP水平下降(P<0.05),RANKL蛋白表达下降(P<0.01),RUNX2蛋白表达增加(P<0.05)。测序结果显示,左归丸组骨质疏松大鼠破骨细胞中rno-miR-133b-3p表达下调幅度最大(P<0.01)。左归丸组破骨细胞miR-133b-3p表达低于模型组。miR-133b-3p过表达可促进破骨细胞增殖和分化,RhoA过表达可逆转由miR-133b-3p过表达所引起的破骨细胞过度增殖和分化。结论RhoA是miR-133b-3p调控的靶基因,左归丸通过调控miR-133b-3p/RhoA抑制破骨细胞活性,从而缓解骨质疏松症状。

Objective To study the effects of Zuogui Pills on the expressions of miR-133b-3p and RhoA in osteoclasts of postmenopausal osteoporosis rats;To discuss its potential mechanism.Methods SD female rats were randomly divided into normal group,model group,sham-operation group,and Zuogui Pills group using a random number table method,with 6 rats in each group.The model group and Zuogui Pills group were treated with oophorectomy to construct a rat model of osteoporosis.Zuogui Pills group was orally administered with Zuogui Pills decoction at a concentration of 10 g/kg for 12 consecutive weeks.Colorimetric method was used to measure the serum calcium and phosphorus levels of rats,and ELISA method was used to detect ALP levels.Bone density meter was used to measure the bone density of the femurs of rats in each group.The osteoclast of each group were cultured,and the expressions of RANKL and RUNX2 protein were detected by Western blot.MiRNA sequencing and differential expression analysis were performed on bone tissues of rats.Osteoclasts were treated with miR-133b-3p mimic and its negative control.The cell proliferation activity of osteoclasts was detected by cell counting kit-8(CCK-8).The osteoclast differentiation activity was detected by the tartrate-resistant acid phosphatase staining.The dual-luciferase reporter assay was used to detect the relationship between miR-133b-3p and RhoA.The"rescue"experiment of miR-133b-3p mimic and RhoA co-expression were used to study the molecular regulatory mechanism of Zuogui Pills on osteoclast activity.Results Compared with the model group,the bone mineral density of Zuogui Pills group significantly increased(P<0.05,P<0.01),the levels of calcium and phosphorus in serum increased,the level of alkaline phosphatase ALP decreased(P<0.05),the expression of RANKL protein decreased,and the expression of RUNX2 protein increased.Sequencing results showed that rno-miR-133b-3p was down-regulated in osteoclasts of postmenopausa osteoporosis rats treated with Zuogui Pills with the maximum difference(P<0.01).Q-PCR results showed that the expression of miR-133b-3p in osteoclasts of Zuogui Pills group was significantly lower than that of the model group.The upregulation of miR-133b-3p could significantly promote the cell proliferation and differentiation of osteoclasts.RhoA overexpression could reverse the excessive proliferation and differentiation of osteoclasts caused by miR-133b-3p overexpression.Conclusions RhoA is the target gene regulated by miR-133b-3p.Zuogui Pills can inhibit the activity of osteoclasts by regulating miR-133b-3p/RhoA axis,relieving the symptoms of osteoporosis.