| Objective1 A cross-section clinical study was conducted to observe whether there were differences in bone turnover markers between elderly patients with type 2 diabetes and non-diabetic patients,and to analyze the correlation between glycosylated hemoglobin and different bone turnover markers,so as to provide objective evidence for the early diagnosis and efficacy evaluation of type 2 diabetes osteoporosis.2 The rat model of type 2 diabetes was established through animal experiments in vivo,and the correlation between bone transition markers and type 2 diabetes osteoporosis was clearly defined.The animal model was interfered with by Bushen Tongluo method(Bushen Tongluo formula)guided by collateral-disease theory,and the influence of Bushen Tongluo formula on bone tissue of type 2 diabetes osteoporosis rats was observed.3 Based on Wnt/β-catenin signaling pathway,the mechanism of Bushen Tongluo formula to improve bone metabolism in type 2 diabetic osteoporosis was investigated in vivo.4 In vitro cell experiments further verified the mechanism of promoting osteogenic differentiation through regulating Wnt/β-Catenin signaling pathway by the drug-containing serum of Bushen Tongluo formula,providing preliminary experimental basis for the basic research and development and clinical transformation of Bushen Tongluo formula.Method1 The clinical study selected the elderly patients who were hospitalized in the orthopedic center of our hospital from June 2022 to June 2023 as the research objects.According to whether there was type 2 diabetes,32 cases were set up in the non-diabetes group(control group)and 32 cases in the type 2 diabetes group(T2DM group).Serum osteocalcin(OCN),tartrate-resistant acid phosphatase(TRACP),25-hydroxyvitamin D3(25OHD3),calcitonin(CT),blood calcium,blood phosphorus and alkaline phosphatase(ALP)in 2 groups were detected by enzyme immunoassay(Elisa).Glycosylated hemoglobin(HbAlc)level was measured in T2DM group.The relevant information of patients in the two groups was extracted,and a database was established to compare and analyze the differences in biochemical indexes of bone metabolism between the two groups and the correlation analysis between biochemical markers of bone conversion and HbAlc in the T2DM group.2 Diabetic fatty(ZDF)(fa/fa)rats were divided into 5 groups with 8 rats in each group by random stratified sampling method according to blood sugar and body weight.They were model group,metformin hydrochloride group,Bushen Tongluo formula high-dose,medium-dose and low-dose groups.Eight ZDF(fa/+)rats of the same week were set as normal control group.Dosage:metformin hydrochloride dose was 0.135 g/kg;The doses of Bushen Tongluo formula in high,medium and low groups were 2.5,1.25 and 0.625 g/kg,respectively;Model group and normal group were given equal volume of deionized water for 12 consecutive weeks.After drug intervention,serum glucose metabolism index Insulin(Insulin resistance index),insulin resistance index(HOMA-IR),advanced glycation end products(AGEs),insulin-like growth factor 1(IGF-1)contents of rats were determined serologically.Interleukin 1β(IL-1β),tumor necrosis factor α(TNF-α),bone metabolism markers OCN,type Ⅰ procollagen amino terminal peptide(PINP),type Ⅰ collagen cross-linked C-terminal peptide(CTX),parathyroid hormone(PTH);HE staining and saffron and solid green staining were performed on the femoral head of the proximal femur.Dual-energy X-ray and Micro-CT imaging were used to detect bone density and bone microstructure.Biomechanical three-point bending of femur was performed to measure bone strength.3 The expression levels of Bushen Tongluo formula in bone osteogenic differentiation related target gene ALP,type I collagen(Collal),osteogenic transcription factor(Osterix),OCN and dwarf transcription factor 2(Runx2)were detected by RT-PCR in vivo.The expression levels of Wntl,low density lipoprotein receptor-associated protein 5(LRP5)andβ-catenin gene in Wnt/β-catenin pathway were determined.Protein expression levels of Col1a1,OCN and Runx2 related to osteogenic differentiation of bone tissue were detected by Western Blot.The expression levels of Wnt/β-catenin pathway Wntl,phosphorylated glycogen synthase-3β(p-GSK-3β),p-GSK-3β/GSK-3β,β-catenin and LRP5 protein in bone tissue were determined.4 Forty male SD rats were randomly divided into normal group,Bushen Tongluo formula high-dose group,Bushen Tongluo formula medium-dose group and Bushen Tongluo formula low-dose group according to body weight after 3 days of adaptive feeding,with 10 rats in each group.Dosage:the doses of Bushen Tongluo formula Granule in high,medium and low groups were 2.5,1.25 and 0.625 g/kg,respectively;Rats in normal group were given the same volume of deionized water intragastric administration,once a day,after 7 days of anesthesia to obtain drug-containing serum from abdominal aorta.Osteoblasts were selected,and after cell resuscitation,passage,cryopreserved and identification,the proliferative activity of drug-containing serum on osteoblasts was detected by CCK8 method.ALP staining and alizarin red(ARS)staining were used to detect the differentiation and mineralization of osteoblasts.The expression of bone related target proteins and Wnt/β-catenin signaling pathway were detected by Western Blot.The in vivo results were further verified.Results1 Clinical Research(1)Comparison of patients’ general conditions:there were no statistical differences in age,height,weight,BMI,diastolic blood pressure and other general baseline levels between the control group and the T2DM group,indicating comparability between groups.There was no significant difference in serum calcium,phosphorus and ALP between the two groups.(2)Bone metabolism index detection:Compared with the control group,serum OCN level in T2DM group was significantly decreased(P=0.022).Compared with the control group,the other three TRACPs,25OHD3 and CT showed no statistical significance in T2DM group.(3)Correlation analysis with HbAlc:HbAlc was negatively correlated with OCN(β=-2.518,P=0.011).2 In vivo pharmacodynamic study(1)Detection of serum glucose metabolism:compared with model group,fasting serum INS,HOMA-IR and AGEs levels in Bushen Tongluo formula group decreased,and the decrease was most significant in Bushen Tongluo formula high group(P<0.01).Compared with model group,IGF-1 level in Bushen Tongluo formula group was increased,and the IGF-1 level in Bushen Tongluo formula high dose group was most significantly increased(P<0.01).(2)Detection of serum bone metabolism:compared with model group,serum PTH,OCN,PINP and CTX levels in Bushen Tongluo formula group were increased,and the increases were most significant in Bushen Tongluo formula high dose group(P<0.01).(3)Detection of serum inflammatory markers:Compared with model group,serum levels of IL-1β and TNF-α in Bushen Tongluo formula group were significantly decreased(P<0.01),the decrease of Bushen Tongluo formula high dose group was the most obvious.(4)Bone histomorphologic detection:HE staining showed that compared with the normal group,the trabecular bone of the femoral head tissue of rats in the model dose group became thinner,irregular and discontinuous,and the trabecular space widened and the network structure was lost.The normal microstructure of bone trabeculae in Bushen Tongluo formula group was orderly distributed in the mesh network,and the morphology and structure were similar to those in the normal group.Compared with normal group,the cartilage layer of femoral head in model group was thinner and the level of proteoglycan was significantly decreased by saffranine O-solid green staining.Compared with model group,Bushen Tongluo formla group increased cartilage thickness and proteoglycan content in femoral head bone tissue.(5)Bone tissue dual-energy X-ray and Micro-CT examination:compared with model group,BMD of lumbar spine and femur in Bushen Tongluo formula group increased.Compared with the normal group,the trabecular arrangement in the metaphyseal region of bone tissue in the model group was sparse,the trabecular structure was missing,broken,the reticular structure was incomplete,and there was a large cavity in the bone marrow cavity.Compared with model group,the trabecular structure of metformin group and Bushen Tongluo formula group was improved to some extent,and the trabecular structure of Bushen Tongluo formula high dose group was the best,which was closest to the normal group.The data obtained by Micro-CT three-dimensional scanning of distal metaphysis of femur of rats in each group were analyzed.Compared with model group,bone volume fraction,bone trabecular thickness and bone trabecular number of rats in Bushen Tongluo formula high dose group were significantly increased(P<0.05,P<0.01),the separation degree of bone trabeculae and structural model index were significantly decreased(P<0.01).Bushen Tongluo formula high dose group had the greatest improvement on bone microstructure of ZDF rats.(6)Biomechanical test of bone tissue:Compared with model group,maximum load and elastic modulus of femur tissue in Bushen Tongluo formula high dose group were significantly increased(P<0.01),the bending strength increased significantly(P<0.05).Compared with model group,the maximum loading of femur tissue in Bushen Tongluo formula middle dose group was significantly increased(P<0.05),the elastic modulus was significantly increased(P<0.01),there was no statistical difference in bending strength.Compared with model group,the maximum load,complete strength and elastic modulus of femur tissue in Bushen Tongluo formula low group had no statistical difference.3.In vivo mechanism study(1)Bone tissue RT-PCR detection:Compared with model group,mRNA expression levels of ALP,Collal,Osterix,OCN and Runx2 in bone tissue of rats in Bushen Tongluo formula group were increased,and the increases in Bushen Tongluo formula high dose group were most significant(P<0.01).Compared with model group,mRNA expression levels of Wnt1,LRP5 and β-catenin in bone tissue of rats in Bushen Tongluo formula group were increased,and the increases in Bushen Tongluo formula high dose group were most significant(P<0.01).(2)Bone tissue Western Blot detection:Compared with model group,the expression lelvels of Collal,OCN and Runx2 in bone tissue of Bushen Tongluo Formula group were increased,and the increases in Bushen Tongluo formula high dose group were most significant(P<0.05,P<0.01).Compared with model group,the expression levels of Col1a1,OCN and Runx2 protein in bone tissue of Bushen Tongluo formula low dose group were not statistically different.Compared with model dose group,the protein expression levels of Wnt1,p-GSK-3β,p-Gsk-3β/GSK-3β,β-catenin and LRP5 in bone tissue of rats in Bushen Tongluo formula group were increased,and the increases in Bushen Tongluo formula high dose group were most significant(P<0.01).4 In vitro cell studies(1)CCK8 cell viability test:In the experiment,there was no cytotoxicity in the serum containing Bushen Tongluo formula at low,medium and high concentrations.Compared with blank group,the cell proliferation activity of Bushen Tongluo formula drug-containing serum group was significantly increased(P<0.01),the cell vitality of Bushen Tongluo formula high dose group was the strongest.(2)osteoblast ALP and APS staining:compared with blank group,Bushen Tongluo formula high dose group showed the highest degree of ALP staining and the highest activity of osteoblast ALP under gross and microscopic observation.The ARS staining degree of cells in Bushen Tongluo formula high dose group was deep red,and the calcium deposition and mineralization degree of osteoblasts were the highest.(3)Western Blot analysis of osteoblasts:compared with blank group,the expressions of OCN and Runx2 of osteoblast-related proteins were significantly increased in Bushen Tongluo formula containing serum high and medium(P<0.01);The β-catenin expression level of bone cells in high and medium serum containing Bushen Tongluo formula was significantly increased(P<0.01);p-GSK-3β/GSK-3β ratio increased in Bushen Tongluo formula high dose group(P<0.05).Compared with blank group,the expression ofβ-catenin protein in Bushen Tongluo high dose group was significantly increased(P<0.01),the expression of β-catenin protein in Bushen Tongluo high dose group+KYA1797K group was significantly increased(P<0.01);Compared with Bushen Tongluo high dose group,β-catenin protein expression in KYA1797K group was significantly decreased(P<0.01),the expression of β-catenin protein in Bushen Tongluo high dose group+KYA1797K group was significantly decreased(P<0.05).Compared with blank group,the P-GSK-3β/GSK-3β ratio in B Bushen Tongluo high dose group was significantly increased(P<0.01);Compared with Bushen Tongluo high dose group,p-GSK-3β/GSK-3β ratio in KYA1797K group was significantly decreased(P<0.01),the expression of β-catenin protein in Bushen Tongluo Fang Gao+KYA1797K group was significantly decreased(P<0.05).Conclusion:1 Both elderly T2DM patients and ZDF rats showed low bone turnover,and there was a significant negative correlation between OCN and HbAlc level.OCN may be a candidate specific bone transition marker for osteoporosis in patients with T2DM.2 Tonifying kidney Tongluo decoction can improve glucose metabolism,bone metabolism,reduce inflammation,increase bone density,improve bone microstructure bone quality and bone strength in T2DM rats.2 Busshen Tongluo formula can improve glucose metabolism,bone metabolism,reduce inflammation,increase bone density,improve bone microstructure and bone strength in T2DM rats.3 Bushen Tongluo formula can up-regulate the expression levels of genes and molecular proteins related to osteogenic differentiation in bone tissue of T2DM rats,and promote osteogenic differentiation.Bushen Tonglou decoction can up-regulate the expression level of key molecular genes and proteins of Wnt/β-catenin pathway in bone tissue of T2DM rats.It is suggested that Bushen Tongluo decoction can promote osteogenic differentiation of bone tissue in T2DM rats by up-regulating Wnt/β-catenin signaling pathway.4 The drug-containing serum of Bushen Tongluo Formula promoted osteogenic differentiation and mineralization of rat osteoblasts by activating Wnt/β-catenin signaling pathway.Cell experiments further verified the mechanism of Bushen Tongluo formula promoting osteogenic differentiation by regulating Wnt/β-catenin signaling pathway. |
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