| Purpose:To investigate the pathogenesis of diabetic osteoporosis and the potential mechanism of Sanhuang Tangshenkang in its prevention and treatment from the perspective of bone metabolism.To observe whether Sanhuang Tangshenkang can improve the osteoporosis status of diabetic rats by up-regulating the expression levels of Wnt 3a,LRP-5 and β-catenin protein through Wnt/β-catenin signaling pathway.Material and method:1 Grouping,modeling and evaluation of experimental animalsFifty 3-month-old male SD rats were randomly divided into 5 groups:blank group,model group,Sanhuang Tangshenkang low-dose group,Sanhuang Tangshenkang high-dose group and Gushukang group.Except blank group,the rats in other groups were treated with high sugar and high fat diet for 4 weeks plus fasting intrabitoneal injection of 30 mg/kg streptozotocin to establish diabetic osteoporosis rat model.Starting the day after the successful modeling of diabetes,each medication group was given the corresponding drugs for 12w continuous intervention.The status of rats was observed,evaluated and recorded throughout the experiment.2 Test indicators(1)The blood glucose(FBG),blood calcium(Ca)and blood phosphorus(P)levels of rats in each group were detected by automatic biochemical instrument.(2)Serum bone specific alkaline phosphatase(BALP),serum tartrate-resistant acid phosphatase(TRAP)and fasting insulin(FINS)levels of rats in each group were detected by enzymal-linked immunosorbent assay(ELISA).(3)Mi-cro CT was used to observe the tissue morphology of left femur in each group and to detect bone mineral density(BMD).(4)Hematoxylin-eosin staining(HE)was used to observe the pathological morphology and structure of bone tissue in each group(5)The expression distribution of Wnt 3a,LRP-5 and β-catenin in bone tissues of rats in each group was observed by immunohistochemical staining(IHC)and semi-quantitative analysis was performed.(6)The protein expressions of Wnt 3a,LRP-5 and β-catenin in bone tissue of rats in each group were detected by Western blotting(WB).3 Statistical methods:SPSS 25,Image j and GraphPad Prism8 were used for data analysis,image analysis and mapping.P<0.05 was considered statistically significant,and P<0.01 was considered statistically significant.Results:1 General situation:After successful modeling,it was observed that compared with blank group,rats in model group showed obvious manifestations of "thirst dissipation" and "bone impotence" of traditional Chinese medicine,such as polydipsia,hypereating,hyperuria,weight loss,waist collapse,mental lethargy,dim eyes,sparse and dull body hair and yellow hair.After 12w of drug intervention,compared with model group,the performance of TCM"thirst quenching" and "bone impotence" of rats in all drug administration groups were improved to varying degrees.2 Biochemical indices of glucose metabolism:After 12 weeks of administration,FBG and FINS in model group were significantly increased compared with blank group(P<0.01).Compared with model group,FBG in low-dose and high-dose groups was decreased(P<0.05),while FBG in Gushukang group had a downward trend,but the difference was not statistically significant(P>0.05).Compared with model group,FINS were decreased in low-dose group(P<0.05).3 Biochemical indexes of bone metabolism:After 12 weeks of administration,Ca and P levels in model group showed a trend of decrease compared with blank group,but the difference was not statistically significant(P>0.05).Compared with model group,Ca and P levels in low-dose and high-dose groups showed an increasing trend,while P levels in Gushukang group showed an increasing trend,but the difference was not statistically significant(P>0.05).Compared with blank group,BALP in model group was significantly decreased(P<0.01).Compared with model group,BALP in low-dose and high-dose groups was increased(P<0.05).Compared with blank group,TRAP in model group was significantly higher(P<0.01).Compared with model group,TRAP in low-dose,high-dose and Gushukang groups was significantly decreased(P<0.01);Compared with Gushukang group,TRAP in low-dose group was significantly increased(P<0.01),and TRAP in high-dose group was increased(P<0.05).4 Changes in bone density and microstructure of bone tissue of rats:12 weeks after administration,Micro-CT scanning results showed that the cortical bone of bone tissue of rats in the blank group was uniform,and the bone trabeculae were connected to each other into a network,which was relatively dense and arranged in a regular way.Compared with blank group,the bone marrow cavity of rats in model group was significantly enlarged,and trabecular bone was fractured and sparse.Compared with model group,femoral bone trabeculae of rats in each administration group increased,thickened,arranged closely and neatly,and bone microstructure morphology was improved to some extent.After 12 weeks of administration,bone mineral density in model group was significantly decreased compared with blank group(P<0.01).Compared with model group,the low-dose group,high-dose group and Gushukang group were increased in different degrees(P<0.01 or P<0.05).5 Histopathological observation of rat femur:In the blank group,the femoral bone trabeculae were arranged regularly,tightly and structurally intact,and no obvious pathological changes were observed.Compared with blank group,the trabecular structure of femur bone in model group was loose,irregular,slender and thin,with fracture and increased lipid droplets.Compared with model group,femoral bone trabeculae of rats in high-dose,low-dose and Gushukang groups increased,thickened,arranged closely and neatly,the number of lipid droplets decreased,and the bone microstructure was improved.6 Immunohistochemical staining(IHC):Compared with blank group,the average optical density values of Wnt 3a,LRP-5 and β-catenin proteins in model group were significantly decreased(P<0.01).Compared with model group,the average optical density values of Wnt 3a,LRP-5 and β-catenin proteins in each medication group were increased to varying degrees.7 Western Blot(WB):Compared with blank group,the protein expressions of Wnt 3a,LRP-5 and β-catenin in model group were decreased(P<0.01);Compared with model group,the protein expressions of Wnt 3a,LRP-5 and β-catenin in all treatment groups showed an increased trend(P<0.01 or P<0.05).Conclusion:1 Sanhuang Tangshenkang can significantly increase the serum BALP content and reduce TRAP content in diabetic rats,and play a preventive role by improving the activity of osteoblasts,promoting bone formation,inhibiting the activity of osteoclasts and inhibiting bone resorption.2 Sanhuang Tangshenkang can effectively improve the bone mineral density of diabetic rats,among which low and high doses of Sanhuang TangShenkang have a certain effect,but low dose intervention effect is the best.3 Low and high doses of Sanhuang Tangshenkang can increase and thicken the trabeculae of femur bone in diabetic rats,and reduce the number of lipid drops,thus improving the microstructure of bone.4 Sanhuang Tangshenkang can up-regulate the protein expression levels of Wnt 3a,LRP-5 and β-catenin in bone tissue,suggesting that Sanhuang Tangshenkang may interfere with diabetic osteoporosis rats induced by high sugar and high fat diet combined with streptozocin through Wnt/β-catenin signaling pathway. |
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