| Purpose:Based on the FTO-ALKBH5-PPARy signaling pathway,we investigated the efficacy and mechanism of Liang Yi Tang in the treatment of postmenopausal osteoporosis(PMOP).By observing the changes of FTO,ALKBH5 and PPARγ mRNA and their protein expression in the femur of PMOP model rats,the expression of FTO,ALKBH5 and PPARγprotein in the femur and gastrocnemius muscle,and the changes of FTO-ALKBH5-PPARγsignaling pathway related factors expression after 10 weeks of treatment with Two Yi Tang,the efficacy and mechanism of Two Yi Tang in treating PMOP were clarified.This study was conducted to clarify the efficacy and mechanism of Two Yi Tang in the treatment of PMOP,and to provide different ideas and experimental basis the clinical treatment of PMOP in Chinese medicine.Material and method:Female SPF-grade Wistar rats were randomly divided into normal group,model group,Calcium D group and Liang Yi Tang group.Except for the normal group,the model group and each dosing group underwent de-ovarianization and moulding surgery to establish the PMOP rat model.Gavage was started 24 hours after surgery and the drug was administered once daily for 10 weeks.After final gavage and anesthesia,blood was drawn from the abdominal aorta to prepare serum and the femur and gastrocnemius muscle of the left and light hind limibs were taken after execution.Masson staining was used to observe the pathological pattern of rat gastrocnemius muscle,real-time fluorescence quantitative PCR was used to detect the expression of FTO,ALKBH5 and PPARγ mRNA in rat bone tissue,Western Blot was used to detect the expression of FTO,ALKBH5 and PPARy in rat bone tissue,Immunohistochemistry(IHC)was used to detect the expression of FTO,ALKBH5 and PPARy in rat bone tissue.Immunohistochemistry(IHC)was performed to detect the expression of FTO,ALKBH5 and PPARγ in rat famur and gastrocnemius muscle.Results:Experiment 1:Pathological morphology of the femur and mnscles in the PMOP model rat.1.The results of HE staining of bone tissues:The number of trab eculae in the femur of the rats in the normal group was large,evenly distributed,small in spacing,continuous and intact,and connected to each other in a reticular pattern;the number of trab eculae in the femur of the rats in the model group,the Calcarea D group,and the Liang Yi Tang group was reduced,and the trabeculae in some areas were interrupted,and the bone tissue of the rats’ femur showed fat accumulation and destruction of trabecular structure.Compared with the model group,the number of bone trabeculae in the Liang Yi Tang group was increased,the distribution was more uniform,the femoral bone tissue was more intact,the vacuole structure was reduced,and the fat accumulation was also reduced.There was no significant difference in the distribution of bone trabeculae in the Calcium D group compared to the model group,and the fat accumulation was reduced,but it was inferior to that of the two Instrument group.2.Masson staining of muscle tissue:Severe muscle fibrosis was observed in the gastrocnemius muscle tissue of the rats in the model group compared with the normal group.Compared with the model group,the area of fibrosis in the gastrocnemius muscle tissue of rats was significantly reduced after treatm ent with Calcium D and Liang Yi Tang.Experiment 2:Changes in bane biochemical indicators ALP and TRACP in the PMOP model rats.The results of bone biochemical indexes:Compared with the normal group,the ALP level in the model group was significantly lower(P<0.01)and the TRACP level was significantly higher(P<0.01);compared with the model group,the ALP level in both the Calcium D group and the Liang Yi Tang group was significantly higher(P<0.01),and the Calcium D group was better than the Liang Yi Tang group;the TRACP level in both the Calcium D group and the Liang Yi Tang group was significantly lower(P<0.01),with the most significant reduction in the Liang Yi Tang group and the second most significant reduction in the Calcium D group(P<0.01).Experiment 3:Effects of Liaog Yi Tang on the changes of FTO,ALKBH5,PPARγmRNA and their protein expression in bone tis sue of rats in PMOP model.1.The effect of Liang Yi Tang on the changes of FTO:ALKBH5 and PPARγ mRNA expression in bone tissue of PMOP model rats.Compared with the normal group,there was a trend of increasing FTO and PPARγmRNA expression in the model group,but no significant difference(P>0.05),and a trend of decreasing ALKBH5 mRNA expression in the ALKBH5 group,but no significant difference(P>0.05);compared with the model group,there was a decrease in FTO and PPARγ mRNA expression in each medication group(P<0.05),and the Calcium D group There was a decreasing trend of ALKBH5 mRNA expression in the Calcium D group,but no significant difference(P>0.05),and a significant increase in ALKBH5 mRNA expression in the Liang Yi Tang group(P<0.01);compared with the Calcium D group,there was a decreasing trend of FTO and PPARγ mRNA expression in the Liang Yi Tang group,but no significant difference(P>0.05),and a significant increase in ALKBH5 mRNA expression(P<0.01).2.The effect of Liang Yi Tang on the changes of FTO,ALKBH5 and PPARγ protein expression in bone tissue of PMOP model rats.Compared with the normal group,bone tissue FTO and PPARγ protein expression in the model group was significantly higher(P<0.01)and ALKBH5 protein expression was sigruficantly lower(P<0.01);compared with the model group,bone tissue FTO protein expression in the Calcium D group was not significantly different(P>0.05),PPARγ protein expression was downregulated(P<0.05),bone tissue FTO and PPARγ protein expression in the Liang Yi Tang group was significantly lower(P<0.01);compared with the model group,the expression of ALKBH5 protein was significantly up-regulated in all drug groups(P<0.01):but there was no significant difference between the Calcium D group and the Liang Yi Tang group(P>0.05).Compared with the Calcium D group,the expression of FTO protein decreased(P<0.05),the expression of ALKBH5 protein did not differ significantly(P>0.0 5),and the expression of PPARγ protein decreased significantly(P<0.01).Experiment 4:Effects of Liang Yi Tang on the expression of FTO,ALKBH5 and PPARγproteins in bone and muscle tissues of rats in PMOP model.1.The effect of Liang Yi Tang on the expression of FTO:ALKBH5 and PPARγ protein in rat bone tissue.IHC staining results:Compared with the normal group,FTO and PPARγ proteins in the model group and each drug group showed brown-yellow positive expression in the areas of bone trabeculae and bone marrow of rats,and the positive expression increased;ALKBH5 protein positive expression decreased.Compared with the model group,the positive expression of FTO and PPARγ protein in bone trabeculae and bone marrow decreased in each drug group,among which the positive expressi on of FTO protein was the least in the Liang Yi Tang group,and the positive expression of PPARγ protein was not significantly different between the two drug groups;the positive expression of ALKBH5 protein increased in each drug group,and the positive expression was the most in the Liang Yi Tang group.The results of data analysis:Compared with the normal group,the expression of FTO and PPARγ protein in the model group was significantly higher(P<0.01)and ALKH5 protein expression was significantly lower(P<0.01);compared with the model group,the expressi on of FTO and PPARγ protein in each drug group was significantly lower(P<0.01)and ALKBH5 protein expression was significantly higher(P<0.01).0.01).The expression of FTO and ALKBH5 protein in the Liang Yi Tang group was better than that in the Calcium D group,and the expression of PPARγ protein was not significantly different from that in the Calcium D group(P>0.05).2.The effect of Liang Yi Tang on the expression of FTO:ALKBH5 and PPARγ protein in rat muscle tissue.IHC staining results:Compared with the normal group,the FTO and PPARγ proteins in the model group showed brownish-yellow positive expression in the cytoplasm and cell membrane of rat myocytes,ad the positive expression increased;the positive expression of ALKBH5 protein decreased.Compared with the model group,the positive expression of FTO and PPARγ protein in the cytoplasm and cell membrane of myocytes was reduced in each drug group,among which the positive expressi on of FTO protein was theleast in the Liang Yi Tang group,and the positive expression of PPARγ protein was not significantly different between the two drug groups;the positive expression of ALKBH5 protein wa.s increased in each drug group,and the positive expressi on was the most in the Liang Yi Tang group.The results of data.analysis:Compared with the normal group,the expression of FTO and PPARγ protein in the model group was significantly increas ed(P<0.01),and the expressi on of ALKBH5 protein was significantly decreased(P<0.01);compared with the model group,the expression of FTO protein in each drug group was significantly decreased(P<0.01);the expression of ALBH5 protein in the Calcium D group was increased(P<0.05),and the expression of ALKBH5 protein in the Liang Yi Tang group was significantly increased(P<0.01);the expression of ALKBH5 protein in the Calcium D group was significantly increased(P<0.05),and the expression of ALKBH5 protein in the Liang Yi Tang group was significantly increased(P<0.01).Conclusion:1.In PMOP model rats,the number of bone trabeculae decreased,some areas of bone trabeculae were intempted,fat accumulation in bone tissues appeared,areas of fibrosis in muscle tissues increased,bone formation decreased and bone resorption increased,indicating successful moulding.2.Filling the essence and benefiting the qi of the Chinese herbal medicine compound Liang Yi Tang can increase the number of bone trabeculae,reduce the fat accumulation,reduce the fibrosis of muscle tissue,increase the level of ALP and decrease the level of TRACP in the PMOP model rats This indicates that the herbal compound Liang Yi Tang has a certain improvement effect on the pathological changes of bones and muscles in PMOP model rats.3.he expression of FTO and PPARy mRNA and protein in the bone tissue of PMOP model rats tended to increase,while the expression of ALKBH5 mRNA and protein decreased,suggesting that the pathogenesis of PMOP is related to the abnormal expression of FTO-ALKBH5-PPARy signal pathway.4.By regulating FTO-ALKBH5-PPARγ signal pathway in bone and muscle tissues,filling essence and benefiting qi,the compound Liang Yi Tang decreased FTO and PPARy protein expression and increased ALKBH5 protein expression,thus promoting osteogenic differentiation and inhibiting lipogenic differentiation,which may be one of the molecular biological mechanisms for the prevention and treatment of PMOP. |
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