Study On The Effect And Mechanism Of Tianhuang Formula On Bone Metabolism Under The Expression Of Adipose Tissue Specific NSREBP1c

Objective:By means of Micro-CT,biomechanics and bone histopathology,it was clear that a P2-n SREBP1 c mice had glycolipid bone metabolic disorder.Through the intervention of Tianhuang formula on a P2-n SREBP1 c mice for16 weeks,it was clear that Tianhuang formula could effectively improve the disorder of bone metabolism in mice.Secondly,starting from the related pathways of bone formation,bone resorption and insulin,find out how the bone derived factors regulate bone function and improve the overall energy homeostasis after the intervention of Tianhuang formula in a P2-n SREBP1 c mice,so as to provide a basic research basis for the theory of glycolipid metabolic disease.Methods:1.Study on bone function under the expression of adipose tissue specific n SREBP1 c.The content is: after determining the genotype of a P2-n SREBP1 c mice,they were fed to 12 weeks for oral glucose tolerance test(OGTT);blood phosphorus,blood calcium,glucose and lipid metabolism were measured.Observe the pathological states of white fat(i WAT),brown fat(BAT)and femur wered by hematoxylin eosin(HE)staining;The status of femoral osteoclasts was observed by trap staining;The formation of skull bone was observed by alizarin red staining.The m RNA levels of genes related to bone formation and bone resorption of femur were detected by real-time fluorescence quantitative method(RT-PCR),including Runx2,BGLAP and COL1A1 related to bone formation;OPG,RANKL and RANKL/OPG related to bone resorption.2.Study on the pharmacodynamic effect and mechanism of Tianhuang formula on improving bone metabolism in a P2-n SREBP1 c mice.The content is:2.1 12 week old a P2-n SREBP1 c mice were divided into four groups:littermate wild-type group(WT),transgenic group(TG),120mg/kg Tianhuang formula(THF)and 2mg/kg alendronate(ALN),with 6-8 mice in each group.The mice in the same nest wild-type group and transgenic group were given equal volume of distilled water by gavage for 16 weeks.Through the comprehensive animal energy monitoring system,observe the effect of Tianhuang formula intervention on energy metabolism of a P2-n SREBP1 c mice at the end of 14 weeks;The central body temperature of a P2-n SREBP1 c mice was measured at the end of 15 weeks after intervention with Tianhuang formula.2.2 at the end of the 16 th week after the intervention of Tianhuang formula,a P2-n SREBP1 c mice were dissected to observe the pathological changes of skull,femur,liver and white fat;Two-dimensional and three-dimensional reconstruction of femoral microstructure by Micro-ct;The femur was tested by universal mechanical testing machine.The contents of serum incomplete carboxylated Osteocalcin(uc OC),insulin-like growth factor-1(IGF-1)and C-terminal peptide(CTX-1)of type I collagen were detected by Elisa.2.3 at the end of the 16 th week after the intervention of Tianhuang formula,the m RNA levels of insulin mediated lipolysis and sugar transport related genes such as PI3 K,AKT1,GLUT2,SIRT1,LDLR and PPARα in liver were detected by RT-PCR;TNF-α,IL6,PPARα,Twist2 and f OXO1 m RNA levels of White fat.m RNA levels of genes related to femoral bone formation and bone resorption,Runx2,BGLAP,OPN,COL1A1 and ALP related to bone formation;OPG,RANKL,TCIRG1,Cath and ctsk related to bone resorption;m RNA levels of insulin related genes such as PI3 K,AKT1,SIRT1 and PRKAA1 in bone tissue.Results:1.12 week old a P2-n SREBP1 c transgenic mice showed the phenotype of glycolipid bone metabolism disorder,decreased oral glucose tolerance,increased blood calcium and decreased blood phosphorus;The formation of skull bone nodules increased,femoral osteoclasts increased,trabecular connectivity decreased and the gap increased;The m RNA levels of osteogenesis related genes such as Runx2,BGLAP and COL1A1 were up-regulated,and osteoclast RANKL/OPG were up-regulated,indicating hypermetabolism of bone.The brown fat of a P2-SREBP1 c transgenic mice turned white and the white fat had inflammatory infiltration.2.12 week old a P2-n SREBP1 c transgenic mice after 16 weeks of intervention with Tianhuang formula,the HDL and LDL of a P2-SREBP1 c transgenic mice decreased,the blood calcium increased,and the length and weight of femur increased.Micro-CT and three-point bending test showed that the intervention of Tianhuang formula could increase bone mineral density,reduce bone trabecular clearance and reduce fracture load.After the intervention of Tianhuang formula,the bone metabolism disorder of a P2-n SREBP1 c transgenic mice was improved to a certain extent,and the m RNA levels of osteogenesis related genes Runx2,BGLAP and OPN were up-regulated;Down regulate the m RNA levels of osteoclast related genes RANKL/OPG,Tcirg1 and ctsk;Increase the expression of insulin related m RNA such as PI3 K,AKT1 and SIRT1 in bone tissue.The results of ELISA showed that the intervention of Tianhuang formula increased the content of uc OC and IGF-1 in blood and decreased the content of ctx-1.After the intervention of Tianhuang formula,it can reduce the liver lipid accumulation and improve the liver glucose transport capacity of a P2-SREBP1 c transgenic mice;Reduce the inflammatory infiltration of white fat and partially improve the energy metabolism of mice.Conclusion:1.12-week-old a P2-n SREBP1 c transgenic mice showed glucose and lipid metabolism disorder and hypermetabolism of bone.2.Tianhuang formula can improve the nocturnal respiratory exchange rate of 28 week old a P2-n SREBP1 c transgenic mice.3.Tianhuang formula reduces TNF-α、IL-6 slows down the dissolution of bone matrix,down regulates the level of RANKL,reduces the downstream signal cascade of OPG/RANK/RANKL,and reduces bone resorption;Enhancing the expression of insulin conduction related genes in bone tissue can be one of the important factors to promote the expression of bone formation signals such as Runx2 and BGLAP.4.Tianhuang formula can improve the expression of insulin conduction related genes in the liver of a P2-n SREBP1 c mice,increase the content of IGF-1,and mediate osteoblasts to promote bone formation.5.Tianhuang formula can enhance uc OC content in a P2-n SREBP1 c mice,paracrine into liver and other organs,increase insulin production,improve insulin function between liver and bone,and maintain energy homeostasis.