To Explore The Effect Of Jiaweiyanghe Decoction On Osteoblast Aging Based On AMPK Signaling Pathway

Objective:To observe the therapeutic effect of the drug-containing serum of Jiawei Yanghe Decoction（JWYHD）on H₂O₂-induced MC3T3-E1 cells,and to investigate the possible mechanism of this formula for the prevention and treatment of age-related osteoporosis based on the AMPK signaling pathway.Methods.1.Thirty SD male rats were randomly divided into a blank group and a group treated with Jia Wei Yang He Decotion（10g/kg/d）.15 rats in each group were gavaged daily in the morning and afternoon for 7 days,blood was collected from the abdominal aorta,left to stand for 2hs and the serum containing the drug was collected by centrifugation;2.MC3T3-E1 was incubated with different concentrations of H₂O₂（0μM,100μM,200μM,400μM,600μM,800μM）for 2h or 3h,and the optimal H₂O₂concentration and time were screened by CCK8 method to construct an oxidative stress model.The optimal concentration of H₂O₂ was screened by the CCK8 method.The optimal concentration of H₂O₂ was screened by incubating each group of cells with drug-containing serum（5%,10%,15%,20%and 25%）;3.Observation of the effect of J JWYHD-containing serum on H₂O₂-induced aging of MC3T3-E1 cellsCCK8 method was used to detect the effect of different concentrations（5%,10%and 15%）of JWYHD containing serum on H₂O₂-induced proliferation of MC3T3-E1cells;SA-β-Gal staining was used to detect the senescence of MC3T3-E1 cells in each group;Western blot method was used to detect the expression of p16,p21 and IL6proteins in MC3T3-E1 cells in each group;kits were used to detect the effect of oxidative stress indicators SOD and MDA in the supernatant of MC3T3-E1 cells in each group.4.Observation of the effect of JWYHD-containing serum on the differentiation of MC3T3-E1 cells induced by H₂O₂Osteogenic differentiation of MC3T3-E1 cells was induced with complete medium containing osteogenic induction solution（50μg/m L ascorbic acid,10 m M sodiumβ-glycerophosphate and 10 n M dexamethasone）.On day 5 of osteogenic induction culture,the alkaline phosphatase activity of each group of MC3T3-E1 cells was detected by BCIP/NBT staining kit;on day 21 of osteogenic induction culture,calcium deposition during osteogenic differentiation of each group of MC3T3-E1cells was detected by alizarin red S staining.5.Study on the mechanism of anti-aging of MC3T3-E1 cells induced by H₂O₂ by JWYHD-containing serum.The expression of LKB1,p-AMPK and FOXO3a,RUNX2 proteins in MC3T3-E1 cells of each group were detected by Western blot method.Results1.The results showed that the induction of MC3T3-E1 cells with 400μM H₂O₂for 3h was effective in constructing a model of oxidative stress in bone;5%-15%of the drug-containing serum of JWYHD had a certain promotion effect on the proliferation of MC3T3-E1 cells,but 20%and 25%of the drug-containing serum of JWYHD had an inhibitory effect on the proliferation of MC3T3-E1 cells（p<0.05）.2.The results of CCK8 showed that the inhibitory effect of H₂O₂ on MC3T3-E1was significantly improved in the 5%,10%and 15%serum-containing groups,p<0.01.The results of SOD and MDA in cell culture supernatant showed that the SOD activity was significantly increased and the MDA level was significantly decreased in the 5%,10%and 15%groups compared with the H₂O₂ group（p<0.01）.<The results of SA-β-Gal staining showed that compared with the H₂O₂ group,the staining of senescence-relatedβ-galactosidase was significantly weaker in the 5%,10%and 15%groups（p<0.01）,and the percentage of SA-β-Gal positive cells was significantly lower in each group（p<0.01）.The results of Western blot showed that 5%,15%and15%of JWYHD-containing serum significantly inhibited the expression of p16 and p21 proteins in MC3T3-E1 cells,p<0.01,and decreased the expression of IL-6protein,p<0.05;and the expression of p16 and p21 proteins and IL-6 protein in MC3T3-E1 cells showed concentration-dependent inhibition with the concentration of JWYHD-containing serum.3.The results of alkaline phosphatase staining showed that the activity of ALP was significantly higher in the 5%,10%and 15%serum-containing groups compared with the H₂O₂ group,p<0.01.The results of alizarin red staining showed that the mineralized area was significantly increased in the 5%,10%and 15%serum-containing groups compared with the H₂O₂ group,p<0.01,and the 15%serum-containing group was the most effective in promoting ALP activity and formation of mineralized area in MC3T3-E1 cells.The best effect was observed in the MC3T3-E1cells in terms of ALP activity and formation of mineralized areas.4.Western blot showed that 5%,10%and 15%drug-containing serum groups significantly promoted the expression of LKB1,p-AMPK and FOXO3a,RUNX2proteins in MC3T3-E1 cells,p<0.01,and the drug-containing serum of JWYHD promoted the expression of LKB1,p-AMPK and FOXO3a,RUNX2 proteins in MC3T3-E1 cells in a concentration-dependent manner.and RUNX2 protein expression in MC3T3-E1 cells.ConclusionJWYHD can effectively delay H₂O₂-induced senescence in MC3T3-E1 cells and protect their functions.By attenuating H₂O₂-induced oxidative stress in MC3T3-E1 cells,JWYHD could enhance ALP activity and promote mineralized nodule formation.It is possible that JWYHD may delay osteoblast senescence and reduce oxidative stress by regulating the expression of related proteins in the AMPK signaling pathway,thereby promoting osteogenic differentiation and preventing osteoporosis.