Effects of Zuogui Pill on osteoclast activity and expression of miR-133b-3p/RhoA in postmenopausal osteoporosis rats / 国际中医中药杂志

ABSTRACT

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.