| Objectives1.Clinical research partIn this part,the objective is to reveal the characteristics of TCM syndrome types in patients with diabetic osteoporosis and its relationship with clinical data through a retrospective analysis of TCM syndrome types and clinical data of patients with diabetic osteoporosis,aiming at providing a referrence for better diagnosis and treatment of the disease and improving the clinical outcomes.2.Experimental research partIn previous studies,Zishen Jiangtang pill has been proved to prevent and treat diabetic osteoporosis by regulating Wnt/b-catenin signaling pathway and miRNA can regulate Wnt/b-catenin signaling pathway.The objective of this part is to further explore the mechanism that Zishen Jiangtang pill may prevent and treat diabetic osteoporosis by miRNA targeting Wnt pathway,thus providing a new theoretical basis for the prevention and treatment of diabetic osteoporosis with Zishen Jiangtang Pill.In this study,the effects of Zishen Jiangtang Pill on the glucose metabolism,bone metabolism,bone mineral density and differentially expressed miRNA targeting Wnt/b-catenin signaling pathway in diabetic rats are to be observed.Methods1.Clinical research partA retrospective investigation method was used to select eligible patients with diabetic osteoporosis who were hospitalized in Shenzhen Hospital of Traditional Chinese Medicine from January 2013 to December 2017.Then according to the standard of syndrome differentiation,a re-differentiation of syndromes was made based on their main clinical symptoms at admission and their clinical data such as gender,age,height,weight,body mass index(BMI),duration of diabetes,history of fracture,fasting plasma glucose(FPG),HbA1c,serum calcium,serum phosphorus and bone mineral density(BMD)were collected.Finally,the results of syndrome differentiation and clinical data were statistically analyzed.2.Experimental research partThe model of diabetic rats was established by intraperitoneal injection of streptozotocin(STZ).The rats were randomly divided into model group,TCM group.And the corresponding drugs were given by gavage for rats in different treatment group.Meanwhile,a control group was set in normal animals.There were 8 rats in each group.After treatment for 12 weeks,FPG,HbA1c,serum calcium,serum phosphorus,osteocalcin(OC),type I collagen carboxyterminal peptide(b-CTX)and BMD were detected.At the same time,miRNAs differentially expressed in bone tissue were detected by high-throughput sequencing and bioinformatics software was used to predict miRNAs targeting Wnt/b-catenin signaling pathway.Finally,quantitative real-time PCR(qPCR)was used to validate the differentially expressed miRNAs that were screened for Wnt pathway.Results1.Clinical research part(1)A total of 127 eligible DOP patients were collected in this study,including 54 males(42.5%)and 73 females(57.5%),with a ratio of men to women of 0.74:1.The average age of the patients was 66.58 years old and the age was mainly between 50-79(93.7%).The average duration of diabetes of was 9.02years and the average BMI was 21.05kg/m~2.There were 34 patients(26.8%)with history of fracture and 93 patients(73.2%)without history of fracture.The average BMD of the patients was-3.07,average FPG 8.86mmol/L,average HbA1c7.64%,average serum calcium 2.26mmol/L and average phosphorus 1.13mmol/L.(2)The most common syndrome of 127 patients was yin and yang deficiency with40 cases(31.5%),followed by qi stagnation and blood stasis with 37 cases(29.1%),liver and kidney loss with 26 cases(20.5%),spleen and kidney yang deficiency and blood stasis with 14 cases(11.0%).The least syndrome was kidney essence deficiency with 10 cases(7.9%).When these syndromes were divided into two kinds of syndrome types,the results showed that there were more cases of kidney deficiency syndrome with a total of 90 cases(70.9%)than non-kidney deficiency syndrome with a total of 37 cases(29.1%).(3)There was no significant difference in gender,BMI,FPG,HbA1c,serum calcium,serum phosphorus and history of fracture between kidney deficiency patients and non-kidney deficiency patients(P<0.05),but there was significant difference in age,duration of diabetes and BMD(P>0.05)and the patients with kidney deficiency had older age,longer duration of diabetes and lower BMD on average than non-kidney deficiency patients.Regression analysis results showed that the age and duration of diabetes were independent factors of kidney deficiency syndrome.2.Experimental research part(1)Compared with the normal group,the FPG,HbA1c,serum calcium,serum phosphorus in the model group were significantly increased(P<0.01).Compared with the model group,the FPG,HbA1c,serum calcium,serum phosphorus in the TCM group were significantly decreased(P<0.01).(2)Compared with the normal group,theb-CTX in the model group were significantly increased(P<0.01).Compared with the model group,theb-CTX in the TCM group were significantly decreased(P<0.01).Compared with the normal group,the OC in the model group were significantly decreased(P<0.01).Compared with the model group,the OC in the TCM group were significantly increased(P<0.01).(3)Compared with the normal group,the BMD in the model group were significantly decreased(P<0.01).Compared with the model group,the BMD in the TCM group were significantly increased(P<0.01).(4)The results of the length distribution of the clean tag showed that the peak values of the three groups were all concentrated at 22nt.The screening results of differentially expressed miRNA showed that there were 2significantly up-regulated miRNAs and 426 significantly down-regulated miRNAs in MOD/NOR and that there were 35 significantly up-regulated miRNAs and 141significantly down-regulated miRNAs in ZSW/MOD.Then seven qualified miRNAs were screened under the condition of MOD/NOR down regulation and ZSW/MOD up regulation or MOD/NOR up regulation and ZSW/MOD down regulation.Finally,five miRNAs targeting the Wnt/b-catenin signaling pathway were screened out after the bioinformatics software was used to predict the target genes of the above qualified miRNAs.(5)The results of qPCR validation showed that the expression of miR-26a-5p in the model group was up-regulated compared with the normal group(P<0.05),and the expression of miR-26a-5p in the TCM group was down-regulated compared with the model group(P<0.05).The expression of miR-200c-3p in the model group was up-regulated compared with the normal group(P<0.05),and down-regulated in the TCM group compared with the model group(P<0.01).The expression of miR-124-3p in the model group was down-regulated compared with the normal group(P<0.01),and up-regulated in the TCM group compared with the model group(P<0.05).There was no significant difference in the expression of miR-151-5p and miR-138-5p among the three groups(P>0.05).Conclusions1.Clinical research partThe most common syndrome type of patients with diabetic osteoporosis is yin and yang deficiency,followed by Qi deficiency and blood stasis,liver and kidney loss,spleen and kidney yang deficiency and blood stasis,at least kidney essence deficiency.The overall analysis results of the syndromes showed that kidney deficiency syndrome was more common than non-kidney deficiency syndrome and the patients with kidney deficiency syndrome have older age,longer duration of diabetes and lower BMD.The age and duration of diabetes are the independent risk factors of kidney deficiency syndrome.2.Experimental research partZishen Jiangtang Pill can improve glucose metabolism,bone metabolism,and increase BMD in diabetic rats.The effect of Zishen Jiangtang Pill on preventing and treating diabetic osteoporosis may be achieved by down-regulating miR-26a-5p and miR-200c-3p targeting Wnt pathway and up-regulating miR-124-3p targeting Wnt pathway to affect the process of osteogenic differentiation. |
PDF Full Text Request