Study On The Effect Of Cortex Mori Combined With Rhizoma Anemarrhenae On Hyperlipidemia With Osteoporosis

ObjectiveTo investigate the effect of single drug and combination of Cortex Mori and Rhizoma Anemarrhizae on hyperlipidemia and osteoporosis.Methods1.The effect of improvement of Cortex Mori combined with Rhizoma Anemarrhenae on hyperlipidemia combined with glucocorticoid induced osteoporosis in rats72 SD male rats were fed adaptability for one week,and were randomly divided into the normal group,model group,Cotex Mori group,Rhizoma Anemarrhenae group,epimedium group,lipitor group,calcitriol group,Cotex Mori-Rhizoma Anemarrhenae(1:2)group,Cotex Mori-Rhizoma Anemarrhenae(2:1)group.The normal group was fed normal diet,and the other groups were fed high-fat diet.Nine weeks after modeling,normal saline was given to the normal group and the model group and corresponding drugs were given to the other groups.After 18 weeks of administration,glucocorticoids were intramuscutaneously injected into all groups except the normal group.After 24 weeks of administration,the effects of Cortex Mori and Rhizoma Anemarrhenae on body weight,Lee' s index,glucose and fat metabolism and bone metabolism of model rats were observed.?The contents of glucose(GLU),total cholesterol(TC),triglyceride(TG),LDL cholesterol(LDL-C),alkaline phosphatase(ALP)and tartaric acid phosphatase(TRAP)in rat serum were determined.?HE staining was used to detect the pathological changes of tibia in rats.?The expressions of PPAR-y and Runx-2 in rats were detected by immunofluorescence.?Western blot was used to detect the expression levels of Runx-2?Wnt3a?PPAR-? C-FOS protein in rat bone tissue.2.The effects of morusin combined saponins B? on MC3T3-E1 cellsMC3T3-E1 cells were cultured in vitro,which were divided into the following groups:blank group(Con):complete medium.Dexamethasone group(DEX):culture medium containing dexamethasone.Morusin group(Mor):DEX+Mor drug containing medium.Sanggenon C group(SanC):DEX+SanC drug containing medium.Saponin B? group(TB?):DEX+TB? drug containing medium.Icariin group(Ica):DEX+Ica drug containing medium.Morusin-saponin B?(1:1)group(Mor-TB ? 1:1):DEX+Mor-TB?(1:1)drug-containing medium.Morusin-saponin B?(1:2)group(Mor-TB? 1:2):DEX+Mor-T ?(1:2)drug-containing medium.Morusin-saponin B?(2:1)group(Mor-TB? 2:1):DEX+Mor-TB?(2:1)drug containing medium.?Cell proliferation activity was detected by CCK-8.?ALP content in cells was detected by alkaline phosphatase assay kit(ALP).?Alizarin red staining was used to detect the formation of mineralized nodules in MC3T3-E1 cells.?Real-time PCR was used to detect the expression of PPAR-?,Runx-2,Wnt3a,IGF,Osterix mRNA in MC3T3-E1 cells.?Western blotting was used to detect the protein expression of PPAR-y,Runx2,Wnt3a in MC3T3-E1 cells.3.The effects of morusin combined saponins B? on RAW264.7 cellsRAW264.7 cells were cultured in vitro,which were divided into the following groups:blank group(Con):complete medium.Model group(sRANKL):sRANKL containing medium.Sanggenon C group(SanC):sRANKL+SanC drug containing medium.saponins B? group(TB?):sRANKL+TB?drug containing medium.Icari in group(Ica):sRANKL+Ica drug containing medium.Morusin-saponin B?(1:1)group(Mor-TB1:1):sRANKL+Mor-TB?(1:1)drug-containing medium.Morusin-saponin B?(1:2)group(Mor-TB?1:2):sRANKL+Mor-TB?(1:2)drug-containing medium.Morusin-saponin B?(2:1)group(Mor-TB? 2:1):sRANKL+Mor-TB?(2:1)drug-containing medium.CDMTT assay of cell proliferation activity;OTartaric acid phosphatase detection kit(TRAP)was used to detect TRAP content in RAW264.7 cells.?Real-time PCR was used to detect the expressions of C-FOS and NFATcl mRNA in RAW264.7 cells.?Western-blot detection of C-FOS protein expression in RAW264.7 cells.Results1.The effect of improvement of Cortex Mori combined with Rhizoma Anemarrhenae on hyperlipidemia combined with glucocorticoid induced osteoporosis in rats?Compared with the normal group,body weight,abdominal circumference,Lee' s index and serum GLU,TC,TG,LDL-C and TRAP contents were significantly increased in the model group,while serum BALP and BGP contents were decreased.Compared with the model group,after the intervention of drugs,the body weight,abdominal circumference and Lee's index of the rats were significantly reduced,the contents of GLU,TC,TG,LDL-C and TRAP in the serum were significantly reduced,and the contents of BALP and BGP in the serum of the rats were significantly increased.?HE staining of rat tibia showed that,compared with the model group,the volume of adipocyte decreased and the number of osteoblasts were increased in both Cotex Mori and Anemarrhena Rhizome alone and in combination.?Immunofluorescence results showed that the combination of Cotex Mori and Anemarrhena Rhizome can increase the expression of Runx2 and decrease the expression of PPAR-?,and it can promote the formation of osteoblasts.?Western-blot results showed that Wnt/?-catein signaling pathways were activated.Compared with the model group,the expressions of Runx2,Wnt3a protein were increased,the expressions of PPAR-y and C-FOS protein were decreased.The combination of Cotex Mori and Anemarrhena Rhizome can regulate the expression of Runx2/PPAR-y proteins.2.The effects of morusin combined saponins B? on MC3T3-E1 cells?According to the CCK-8 test results,each administration group had a certain proliferation effect on MC3T3-E1 cell.According to the results of CCK-8,we chose morusin 2 ?M,saponins B? and Sanggenon C 32 ? M,epimedium glycoside of 16 ?M,morusin-saponin B?(1:1,1:2,2:1)4 ?M as subsequent experiment concentration.?The ALP content in the DEX group was higher than that in the Con group,and the ALP content in the DEX groups was higher than that in the DEX group.?Alizarin red staining showed that,compared with DEX,the number of mineralized nodules in MC3T3-E1 cells increased after administration.??Real-time PCR results showed that,compared with the DEX group,Runx2,IGF,Wnt3a,Osterix mRNA expression increased and PPAR-? mRNA expression decreased in each group.?The results of western-blot showed that compared with the DEX group,the expression of Wnt3a and PPAR-y protein was increased and the expression of PPAR-? protein was decreased.3.The effects of morusin combined saponins B? on RAW264.7 cells?According to the MTT test results:we chose morusin,Sanggenon C,saponins B?,epimedium glycoside,morusin-saponin B?(1:1,1:2,2:1)are all 2 ?M as subsequent experiment concentration.?According to the results of TRAP test:compared with con group,TRAP content in sRANKL group was increased,and the content in each group was decreased after drug intervention.?The Real-Time PCR results showed that compared with sRANKL group,the combination of morusin and saponin B ? can decrease the expression of C-FOS and NFATcl mRNA.? Western blot results showed that compared with sRANKL group,the combination of morusin and saponin B ? can decrease the protein expression of C-FOS.Conclusions1.For rats with high fat and glucocorticoid induced osteoporosis,The administration of Cortex Mori and Rhizoma Anemarrhenae alone or in combination can not only reduce the weight of rats,regulate the metabolism of glucose and lipid,but also reduce the contents of GLU,TC,TG and LDL-C.It can also regulate bone metabolism,and regulate the contents of BALP and TRAP in serum of rats,and reduce the pathological damage of bone tissue in rats.Compared with epimedium,which is commonly used in clinical anti-osteoporosis,Cortex Mori can not only regulate the disorder of lipid metabolism,but also effectively improve the changes of bone diseases.2.Combination of the morusin and saponins B? can activate the Wnt/?-catein signal pathway,and regulate expression of Runx-2/PPAR-?,and promote MC3T3-E1 cell proliferation,differentiation and mineralization.In addition,the combination of the two drugs can also inhibit the expression of key proteins in osteoclast differentiation and inhibit thetransformation of osteoclast precursor cells into osteoclasts.