Based On The TGF-β1 And Smad2/3 Signaling Pathways, The Mechanism Of Guilu Erxian Ointment Intervening In Glucocorticoid-induced Osteoporosis Rats With Kidney Deficiency Was Investigated

PurPose: 1.To observe the effect of Turtle and Deer Erxian Ointment on glucocorticoid-induced renal deficiency osteoporosis in rats.2.To observe the potential mechanism of action of Turtle and Deer Erxian Ointment in interfering with glucocorticoid-induced renal deficiency osteoporosis in rats.3.To observe whether Turtle and Deer Erxian Cream interferes with the effect of glucocorticoid-induced osteoporosis in rats with renal deficiency through the upregulation of TGF-β1 and Smad2/3 m RNA expression levels via TGF-β1 and Smad2/3 signaling pathways.4.To provide an experimental basis for enriching the theory of "kidney dominates bone" in TCM,and then provide a theoretical basis for the clinical treatment of glucocorticoid-induced renal deficiency osteoporosis with TCM precision medicine.Material and method: 1.Experimental animal grouping,modeling and evaluationSixty 3-month-old SD rats were selected and randomly divided into six groups: blank group(CON group),model group(DXMS group),low-dose group(LGLEX group),medium-dose group(MGLEX group),high-dose group(HGLEX group),and alendronate group(AL group).The glucocorticoid-induced renal deficiency osteoporosis rat model was constructed by the method of dexamethasone sodium phosphate injection in the hind limbs of rats(8w),except for the CON group,and the corresponding drug was given to each medication group for 6 w.The biological characterization information of rats was observed,evaluated and recorded throughout the experiment.2.Detection indexes(1)Bone mineral density(BMD)of the left isolated femur in each group was measured by dual-energy X-ray absorptiometry(DEXA).(2)The biomechanical parameters of the right isolated femur were measured by universal tensile testing machine.(3)Serum osteocalcin(BGP)and serum anti-tartrate acid phosphatase(TRACP-5b)were measured by enzyme-linked immunosorbent assay(ELISA).(4)Hematoxylin-eosin staining(HE)and Masson’s trichrome staining were used to observe the pathological morphological structure of bone tissue.(5)Immunohistochemical staining(IHC)was used to observe the distribution of TGF-β1 and Smad2/3 protein expression in bone tissues.(6)Real-time fluorescence quantitative nucleic acid amplification reverse transcription polymerase chain reaction(RT-PCR)was used to detect the m RNA content of TGF-β1 and Smad2/3 in rat bone tissues.(7)TGF-β1 and Smad2/3 protein expression in rat bone tissues were detected using a fully automated quantitative protein blotting analysis system(Sim Ple Wes).3.Statistical methods.SPSS26,Image Pro Plus and Gra Ph Pad Prism8 software were used for data analysis,image analysis and graphing.P<0.05 was considered statistically significant for differences,and p <0.01 was considered statistically significant for differences.Results: 1.Biological characterization information: After successful modeling,it was observed that the rats in the DXMS,LGLEX,MGLEX,HGLEX and AL groups significantly showed Chinese medicine "kidney deficiency" compared with the CON group.After 6 w of drug intervention,it was found that compared with DXMS group,LGLEX group,MGLEX group,HGLEX group and AL group could significantly improve the manifestation of "kidney deficiency" in Chinese medicine in the modeled rats.2.BMD of the left femur of the rats after modeling and treatment: After 8w of modeling,the BMD of the left femur of the rats in the normal and modeling groups was measured by DEXA,and it was found that the BMD of the modeling group decreased significantly(P<0.01).After 8w of treatment,the BMD values of the left side of the isolated femur of rats in each group were measured by DEXA and were significantly lower in the DXMS group compared with the CON group(P<0.01).The increase was more obvious in the MGLEX group.3.Biomechanics of the right femur of rats: compared with the CON group,the cross-sectional moment of inertia,elastic load,elastic radius,elastic modulus,maximum load,maximum radius,maximum bending stress,maximum bending strain,bending energy and bending strength of the femur of the DXMS group were significantly decreased(P<0.01);compared with the DXMS group,the LGLEX group,MGLEX group,HGLEX group and AL group were significantly decreased.The biomechanical parameters of the femur were significantly higher in the LGLEX,MGLEX,HGLEX and AL groups compared with the DXMS group(P<0.05).4.Biochemical indexes of bone metabolism.(1)Serum osteocalcin(BGP): compared with the CON group,the serum BGP level of rats in the DXMS group decreased significantly(P<0.01);compared with the DXMS group,the serum BGP level of rats in all drug groups increased(P<0.01),with the MGLEX group showing a more significant increase.(2)Anti-tartaric acid phosphatase(TRACP-5b): compared with the CON group,the serum TRACP-5b level of rats in the DXMS group increased significantly(P<0.01);compared with the DXMS group,the serum TRACP-5b level of rats in the LGLEX,MGLEX,HGLEX and AL groups decreased significantly(P<0.01),and the decrease was most significant in the MGLEX group(P<0.01).The decrease was most significant in the MGLEX group(P<0.01).5.Morphological observation of the distal femur of rats.(1)HE is staining of rat distal femur: compared with the CON group,the number of bone trabeculae and the total area occupied by bone trabeculae were significantly lower in the DXMS group,and the bone trabeculae were sparse,broken and disorganized;compared with the DXMS group,the number of bone trabeculae and the total area occupied by bone trabeculae were significantly lower in the MGLEX and HGLEX groups.The number of trabeculae and the total area occupied by trabeculae in the MGLEX and HGLEX groups were significantly higher,and the trabeculae were dense and had good continuity.(2)Masson staining of the distal femur of rats: compared with the CON group,the DXMS group showed disturbed cell arrangement and a small number of collagen fibers in the adjacent trabeculae;compared with the DXMS group,the MGLEX and HGLEX groups showed a large number of continuous collagen fibers in the adjacent trabeculae,which were filamentous and regularly arranged.6.Immunohistochemical staining(IHC): the mean values of TGF-β1 and Smad2/3 protein expression optical density measured in the DXMS group were significantly lower compared with the CON group;the mean values of TGF-β1 and Smad2/3 protein expression optical density in each dosing group were increased to different degrees compared with the DXMS group.7.Reverse transcription polymerase chain reaction(RT-PCR): compared with the CON group,the TGF-β1 and Smad2/3 m RNA contents were significantly decreased in the DXMS group(P<0.01);compared with the DXMS group,the TGF-β1 and Smad2/3 m RNA contents were significantly increased in the AL,MGLEX and HGLEX groups(P < 0.01).8.Fully automated protein blotting quantitative analysis system(Simple Wes): compared with the CON group,TGF-β1 and Smad2/3 protein expression in the DXMS group showed a decreasing trend(P<0.01);compared with the DXMS group,TGF-β1 and Smad2/3 protein expression in the LGLEX,MGLEX,HGLEX,and AL groups showed an increasing(P<0.01).Conclusion: 1.The rat model of glucocorticoid-induced renal deficiency osteoporosis can be successfully replicated by applying the modeling method of "intramuscular dexamethasone sodium Phosphate injection".2.Turtle and deer Erxian cream can effectively increase the BMD value of glucocorticoid-induced renal deficiency osteoporosis rats,in which low,medium and high doses of turtle and deer Erxian cream have good efficacy,but the medium dose has the best efficacy.3.The glucocorticoid-induced osteoporosis in rats can improve the biomechanical Properties,bone strength and fracture resistance.4.The essence of Turtle and Deer Erxian Cream can significantly increase the serum osteocalcin(BGP)content and reduce the content of anti-tartrate acid Phosphatase(TRACP-5b)in rats,thus increasing the activity of osteoblasts and Promoting bone formation,inhibiting the activity of osteoclasts and inhibiting bone resorption,and finally exerting therapeutic effects.5.The mRNA and Protein expression levels of TGF-β1 and Smad2/3 in bone tissues were up-regulated by Turtle and Deer Erxian Ointment,indicating that Turtle and Deer Erxian Ointment could exert its therapeutic effects on the treatment of glucocorticoid-induced renal deficiency osteoporosis rats through TGF-β1 and Smad2/3 signaling Pathways.