| Objective1.To observe the clinical efficacy of Zhuanggu Zhitong Capsule in the treatment of postmenopausal osteoporosis.According to the results of relevant data analysis of clinical medical records and the results of linear correlation analysis of autophagy-related protein mTOR,an integrated analysis was conducted to explore the correlation between postmenopausal osteoporosis and autophagy mediated by AMPK/mTOR signaling pathway.2.The rat model of postmenopausal osteoporosis was established by ovariectomy to further clarify the therapeutic effect of Zhuanggu Zhitong Capsule on ovariectomized rats,and to analyze whether the results of animal experiments were consistent with those of clinical studies.3.To clarify the molecular mechanism of Zhuanggu zhitong Capsule in the treatment of postmenopausal osteoporosis by regulating autophagy through AMPK/mTOR signaling pathway.Methods1.Clinical research(1)Study object,grouping and treatment:From January 2021 to June 2022,70 patients with postmenopausal osteoporosis(liver-kidney yin deficiency,blood stasis type)who met the inclusion criteria were selected from the outpatient and inpatient departments of the Orthopedic Traumatology Department of Liuzhou Traditional Chinese Medicine Hospital of Guangxi Zhuang Autonomous Region.They were divided into two groups:Zhuanggu Zhitong Capsule group and alendronate sodium group in a sequential and random manner.On the basis of oral calcium and vitamin D,all the included patients were given oral Zhuanggu Zhitong Capsules 4 capsules/time,3 times/day,and alendronate sodium group 70 mg/time,1 time/week.There are 6 courses of treatment every 4 weeks.(2)Clinical observation indexes:①BMD:BMD of lumbar spine was measured by dual energy X-ray absorptiometry before and after drug treatment.②TCM syndrome score:According to the TCM syndrome quantitative grading scale,the clinical symptoms were graded and quantified,and the changes of scores before and after treatment were recorded and compared between the two groups.③Serum Ca2+,E2,IL-6 and TNF-α:The levels of serum Ca2+and E2 before and after treatment were detected by fluorescence quantitative PCR.Serum IL-6 and TNF-αlevels were measured by enzyme-linked immunosorbent assay(EILSA).④Serum bone turnover markers:EILSA method was used to detect the levels of bone alkaline phosphatase,parathyroid hormone,osteocalcin,type I procollagen amino terminal propeptide,β-linked degradation products and 25-hydroxyvitamin D in the two groups before and after drug treatment.⑤mTORC1 content:The serum mTORC1 content of the two groups of patients before and after treatment was measured by EILSA method,and it was recorded and linearly correlated with the relevant clinical efficacy data to explore the correlation between autophagy marker mTORC1 and postmenopausal osteoporosis.2.Animal experiment(1)Experimental animals,modeling,grouping and drug intervention:60 SPF level healthy female 6-month old SD rats were used to establish a rat model of postmenopausal osteoporosis by bilateral oophorectomy.According to the body weight,Rand function of Excel table was used to randomly divide the rats into two groups:18 rats in the normal control group and 42 rats in the ovaries removed by surgery.Three months later,6 rats in each group were randomly selected to verify whether the model was successful.After the model was successful,the remaining rats in the surgery group were randomly divided into three groups.All rats were divided into 4 groups:Control group,OVX group,ZGZT group and Estrogen group,with 12 rats in each group.After successful modeling,rats in each group were given corresponding drug intervention every day.The rats in the normal control group and castrated group were given 10ml/kg normal saline by gavage every day,and the rats in the Zhuanggu Zhitong Capsule group were given 0.56g/kg Zhuanggu Zhitong Capsule by gavage once a day;Estrogen group was given 0.104mg/kg compound estradiol tablets by gavage once a day.The drug was administered continuously for 3 months.After 3 months of drug intervention,the samples were taken for relevant indicators testing.(2)Test indexes:①BMD:BMD of the whole body and lumbar vertebrae of rats before and after drug intervention was detected by dual energy X-ray bone mineral density detector.②Micro CT:Micro-CT was used to scan and analyze the bone tissue parameters of L3 vertebral body in vitro,and the cross-sectional view of lumbar spine was obtained to observe the microstructure of bone tissue.③Histomorphology:TRAP staining of lumbar 2 vertebrae and HE staining of lumbar 3 vertebrae were selected for histomorphological observation and analysis.④Serum bone turnover markers,serum inflammation-related factors,serum calcium concentration:The effects of serum inflammatory factors(TNF-α,IL-17,IL-1β),bone turnover markers(PINP,BALP,β-CTX)and serum calcium concentration were detected by enzyme-linked immunosorbent assay(EILSA).⑤The expression of autophagy-related genes and protein levels in the upstream and downstream of AMPK/mTOR signaling pathway:qPCR and Western Blotting were used to detect the levels of autophagy-related markers AMPK,mTOR,LC3-Ⅱ,Atg5,Beclin-1 and ULK1 in the upstream and downstream of AMPK/mTOR signaling pathway in lumbar vertebrae.Results1.Clinical research results(1)Bone mineral density:Zhuanggu Zhitong Capsule can significantly increase the bone density of the lumbar spine.The difference in bone density of the lumbar spine before and after treatment suggests that the alendronate group is more obvious.(2)TCM syndrome score:Zhuanggu zhitong Capsule can significantly reduce the TCM syndrome score,TCM syndrome score difference before and after treatment was significantly better than alendronate sodium group.(3)VAS score of low back pain:Zhuanggu Zhitong Capsule can significantly reduce the VAS score of low back pain,and there is no statistical difference compared with alendronate sodium group.(4)Serum Ca2+ E2,IL-6 and TNF-α levels:Zhuanggu Zhitong Capsule can significantly increase serum Ca2+,E2 levels,significantly reduce IL-6,TNF-α levels,Zhuanggu Zhitong Capsule to improve E2 levels significantly better than alendronate sodium group.(5)Bone metabolism related marker content:Zhuanggu Zhitong Capsule can significantly improve the level of bone formation related indicators(BALP,PTH,OC,PINP).There was no statistical difference between the increase difference of BALP,PTH and OC and the alendronate group,while the difference of PINP was better than that of the Zhuanggu Zhitong Capsule group.Zhuanggu Zhitong Capsule group can significantly improve the level of bone resorption index(β-CTX),β-CTX difference before and after treatment Zhuanggu Zhitong Capsule group than alendronate sodium group significantly.In addition,Zhuanggu Zhitong Capsule can significantly increase the level of 25-OH-VD.(6)Analysis of mTORC1 content and its correlation with clinical information:Zhuanggu Zhitong Capsule can significantly reduce mTORC1 content in serum.There was a significant negative correlation between mTORC1 content and BMD,but no significant correlation with 25-OH-VD content.2.Animal experimental results(1)Bone mineral density:Ovariectomy can significantly reduce bone mineral density in rats.Zhuanggu Zhitong Capsule can significantly increase the bone mineral density of lumbar vertebrae in OVX rats,but there is no significant change in bone mineral density of the whole body.(2)Micro-CT:Zhuanggu Zhitong Capsule could significantly increase the lumbar spine bone surface density(BS/TV)and trabecular thickness(Tb.Th)in OVX rats.Cross-sectional view shows:Zhuanggu Zhitong Capsule can increase the number of OVX rats trabecular bone,trabecular bone arranged more closely,and neat and orderly.(3)Histomorphological evaluation:TRAP staining:Ovariectomy can increase the number of osteoclasts per unit area of cancellous bone in rats.Zhuanggu Zhitong Capsule can make the osteoclast staining deeper and the number of osteoclasts less in the lumbar spine of OVX rats.HE staining:Zhuanggu Zhitong Capsule can significantly increase the number of bone trabeculae in the bone marrow cavity of OVX rats,significantly reduce the number of adipocytes,significantly increase the number of osteoblasts on the surface of bone trabeculae,significantly reduce the number of osteoclasts,significantly reduce the number of adipocytes in the bone marrow,relatively thick and solid bone trabeculae,narrow the gap between bone trabeculae,more connected and maintained continuous.(4)The content of bone turnover markers:Ovariectomy can cause the serum PINP and BALP levels of rats to decrease significantly,and the level of β-CTX increased significantly.Estrogen has a significant effect on reducing β-CTX.There was no significant difference between Zhuanggu Zhitong Capsule and estrogen group,indicating that Zhuanggu Zhitong Capsule has a certain tendency to reduce β-CTX.(5)Serum calcium concentration:Ovariectomy can cause a downward trend in serum calcium concentration in rats,and Zhuanggu Zhitong Capsule has a certain trend of increasing calcium concentration in OVX rats.(6)Serum TNF-α,IL-17,IL-1 β:Ovariectomy can cause IL-17 and IL-1β serum levels were significantly increased,Zhuanggu Zhitong Capsule can significantly reduce OVX rats IL-17 and IL-1β levels.(7)The expression levels of genes related to mTOR/AMPK signaling pathway:Ovariectomy could significantly decrease the expression of ULK1,Beclin-1,Atg5,AMPK and LC3-II mRNA,and significantly increase the expression of mTOR mRNA.Zhuanggu Zhitong Capsule could significantly up-regulate the expression of ULK1,Beclin-1,Atg5,AMPK and LC3-II mRNA in OVX rats,and down-regulate the expression of mTOR mRNA.(8)mTOR/AMPK signaling pathway related protein levels:Ovariectomy can cause rats ULK1,Beclin-1,Atg5,AMPK,LC3-Ⅱ protein levels were significantly decreased,mTOR protein levels were significantly increased,Zhuanggu Zhitong Capsule can significantly reduce OVX rats ULK1,Beclin-1,Atg5,AMPK,LC3-Ⅱ protein levels,significantly increased its mTOR protein levels.Conclusion1.Zhuanggu Zhitong Capsule combined with Calcium and Vitamin D can effectively regulate bone metabolism,increase bone density,and alleviates osteoporosis pain and systemic care.There is a significant negative correlation between mTOR centered signaling pathway mediated autophagy and postmenopausal osteoporosis.2.Ovariectomy can cause the imbalance between bone formation and bone absorption,resulting in bone loss.Zhuanggu Zhitong Capsule can restore the changes of osteocyte differentiation induced by ovariectomy,effectively reduce the bone loss and abnormal changes of bone trabeculae induced by ovariectomy.It has a bone protective effect in ovariectomy induced postmenopausal osteoporosis,and its anti osteoporosis effect is basically consistent with the clinical research results.3.Ovariectomy can induce the activation of autophagic factors in the upstream and downstream of AMPK/mTOR signaling pathway.Zhuanggu Zhitong Capsule can regulate autophagy by changing the changes of the above related factors,reduce the negative effects of ovariectomy on osteoblasts and osteoclasts,and restore bone density and bone microstructure characteristics.At the same time,Zhuanggu Zhitong Capsule can also inhibit the release of inflammatory factors,so as to achieve the effect of relieving osteoporosis pain. |
