| ObjectiveFrom the view of PI3K/Akt signal pathway of osteoblasts(OB)and osteoclasts(OC),this study used the combination of experiments in vivo and in vitro to reveal the gender difference and mechanism of Yougui Pills in the treatment of osteoporosis in ovariectomized and orchiectomized rats,to further clarify the scientific connotation of"kidney governing bone" and provide experimental basis for the treatment of osteoporosis with traditional Chinese medicine.To clarify the above problems,this study was conducted following four experiments.Experiment 1.The difference of therapeutic effect of Yougui Pill on osteoporosis and its effect on PI3K/Akt signal pathway in ovariectomized and orchiectomized ratsMethodsThree-month-old SPF Sprague-Dawley(SD)rats(78 male and 78 female)were selected.After adaptive feeding,female and male rats were randomly divided into basic group(n=12),normal control group(n=12),sham operation group(n=12)and model group(n=42).At the end of the grouping,the rats in the female and male basic group were killed after anesthesia,and the bone histomorphological index data of lumbar vertebrae and tibia were taken as the basic value.Then,according to the previous method of the research group,the female and male model groups were castrated respectively to prepare the "kidney deficiency"OP rat model.Thirteen weeks after castration,the rats in the model group were randomly divided into ovariectomized(OVX)group(n=13),estrogen group(n=12),female Yougui Pill group(n=12),orchiectomized(ORX)group(n=13),androgen group(n=13)and male Yougui Pill group(n=12).The rats in each treatment group were given intragastric administration respectively.The above groups were given once a day and rested for 1 day every 7 days for 13 weeks.At the end of administration,the rats in each group were killed under anesthesia,and the 4th lumbar vertebrae and bilateral tibia were taken for the detection of Micro-CT bone histomorphology index,and the frozen sections of decalcified bone were made.The expressions of p-PI3K and p-Akt protein in osteoblasts and osteoclasts in tibia bone marrow of rats in each group were detected by immunofluorescence.The expressions of BGP,RANKL,OPG,p-GSK-3 β,β-catenin,RUNX2,TRAP,TRAF-6,c-Src and NFATc1 in rat tibial bone marrow were detected by immunohistochemical method.Results1 Effect of Yougui Pill on the change rate of lumbar BMD in ovariectomized and orchiectomized ratsThe change rate of BMD of lumbar vertebrae in sham operation group was positive,which indicated that the bone mass increased,while the change rate of BMD in ovariectomized model group(OVX group/ORX group)was negative,indicating bone loss.The bone loss rate of lumbar vertebrae in model group(OVX group/ORX group)was significantly higher than that in sham operation group of the same sex(P<0.01),and the bone loss rate in OVX group was significantly higher than that in ORX group(P<0.05).The change rate of BMD in the positive drug group and Yougui Pill group showed a negative increase,although it was still in the state of bone loss,but it was significantly lower than that in the same sex model group(OVX group/ORX group)(P<0.01,P<0.05),and the bone loss in the male Yougui Pill group was significantly higher than that in the female Yougui Pill group(P<0.05).2 Effect of Yougui Pill on microstructure of tibia in ovariectomized and orchiectomized rats2.1 Effect of Yougui Pill on the change rate of tibial BV/TV in ovariectomized and orchiectomized ratsThe change rate of tibial BV/TV in the model group(OVX group/ORX group)was negative,while that in the sham operation group was positive,and the former was significantly higher than the latter of the same sex(P<0.01).Compared with ovariectomized and orchiectomized rats,the change rate of BV/TV in OVX group was significantly higher than that in ORX group.The change rate of BV/TV in the positive drug group and Yougui Pill group showed a negative increase,but it was significantly lower than that in the same sex model group(OVX group/ORX group)(P<0.01,P<0.05).Compared with the Yougui Pill group of different sexes,the male Yougui Pill group was significantly higher than the female Yougui Pill group(P<0.05).2.2 Effect of Yougui Pill on the change rate of tibial Tb.N in ovariectomized and orchiectomized ratsThe change rate of Tb.N in tibia of sham operation group was significantly higher than that of sham operation group,while the change rate of Tb.N in ovariectomized rat group was negative,which was significantly higher than that of sham operation group of the same sex(P<0.01).Compared with ovariectomized and orchiectomized rats,the change rate of Tb.N in ORX group was significantly lower than that in OVX group.The change rate of Tb.N in the positive drug group and Yougui Pill group showed a negative increase,but it was significantly lower than that in the same sex model group(OVX group/ORX group)(P<0.01,P<0.05).The change rate of Tb.N in female Yougui Pill group was significantly lower than that in male Yougui Pill group(P<0.05).2.3 Effect of Yougui Pill on the change rate of tibial Tb.Sp in ovariectomized and orchiectomized ratsThe change rate of Tb.Sp of tibial cancellous bone in sham operation group showed negative growth,indicating that the trabecular separation became smaller,and the Tb.Sp change rate of ovariectomized rats increased significantly,indicating that the trabecular separation became larger.The change rate of Tb.Sp in OVX group and ORX group was significantly higher than that in sham operation group of the same sex(P<0.01),and the former was significantly higher than the latter(P<0.05).The change rate of Tb.Sp in the positive drug group and Yougui Pill group was significantly increased,but significantly lower than that in the same sex model group(OVX group/ORX group)(P<0.01,P<0.05).The change rate of Tb.Sp in female Yougui Pill group was significantly lower than that in male Yougui Pill group(P<0.05).2.4 Effect of Yougui Pill on tibial SMI in ovariectomized and orchiectomized rats The SMI in the model group(OVX group/ORX group)was significantly higher than that in the sham operation group of the same sex(P<0.01),and the SMI in the ORX group was significantly lower than that in the OVX group(P<0.05).Compared with OVX group,estrogen group and female Yougui Pill group decreased significantly(P<0.01,P<0.05),androgen group and male Yougui Pill group decreased significantly compared with ORX group(P<0.01,P<0.05),but all positive drugs and Yougui Pill group were still significantly higher than sham operation group of the same sex(P<0.01,P<0.05).Between male and female,the SMI of female Yougui Pill group was significantly lower than that of male Yougui Pill group(P<0.05).3 Effects of Yougui Pill on OPG,RANKL protein expression and RANKL/OPG ratio in tibial bone marrow of ovariectomized and orchiectomized rats3.1 Effect of Yougui Pill on the expression of OPG protein in tibial bone marrow of ovariectomized and orchiectomized ratsThe expression of OPG protein in tibial bone marrow of model group(OVX group/ORX group)was significantly lower than that of sham operation group of the same sex(P<0.01).Compared with ovariectomized and orchiectomized rats,the expression of OPG protein in OVX group was significantly lower than that in ORX group(P<0.01).The expression of OPG protein of osteoblasts in tibia bone marrow in positive drug group and female Yougui Pill group was significantly higher than that in the same sex model group(OVX group/ORX group)(P<0.01,P<0.05),but there was no significant difference between male Yougui Pill group and ORX group.The expression of OPG protein in female Yougui Pill group was significantly higher than that in male Yougui Pill group(P<0.01).3.2 Effect of Yougui Pill on the expression of RANKL protein in tibial bone marrow of ovariectomized and orchiectomized ratsThe expression of RANKL protein in tibial bone marrow of model group(OVX group/ORX group)was significantly higher than that of sham operation group of the same sex(P<0.01).Compared with ovariectomized and orchiectomized rats,the expression of RANKL protein in OVX group was significantly higher than that in ORX group.(P<0.01)The expression of RANKL protein in tibial bone marrow in positive drug group and Yougui Pill group was significantly lower than that in the same sex model group(OVX group/ORX group)(P<0.01,P<0.05),but the expression in male Yougui Pill group was still higher than that in male sham operation group(P<0.05).The expression of RANKL protein in male Yougui Pill group was significantly higher than that in female Yougui Pill group(P<0.05).3.3 Effect of Yougui Pill on RANKL/OPG ratio in tibial bone marrow of ovariectomized and orchiectomized ratsThe ratio of RANKL/OPG in tibial bone marrow of model group(OVX group/ORX group)was significantly higher than that of sham operation group of the same sex(P<0.01).Compared with ovariectomized and orchiectomized rats,the ratio of RANKL/OPG in OVX group was significantly higher than that in ORX group(P<0.01).The RANKL/OPG ratio of osteoblasts in tibia bone marrow in positive drug group and Yougui Pill group was significantly lower than that in the same sex model group(OVX group/ORX group)(P<0.01,P<0.05),but the ratio in male Yougui Pill group was still higher than that in male sham operation group(P<0.05).The ratio of RANKL/OPG in male Yougui Pill group was significantly higher than that in female Yougui Pill group(P<0.01).4 Effect of Yougui Pill on BGP protein expression of osteoblasts in tibial bone marrow of ovariectomized ratsThe expression of BGP protein in tibial bone marrow of model group(OVX group/ORX group)was significantly higher than that of sham operation group of the same sex(P<0.01).Compared with ovariectomized and orchiectomized rats,the expression of BGP protein in OVX group was significantly higher than that in ORX group.(P<0.05).The expression of BGP protein in tibial bone marrow of rats in positive drug group and Yougui Pill group was significantly lower than that in OVX group/ORX group(P<0.01,P<0.05),but it was still higher in Yougui Pill group and androgen group than that in sham operation group of the same sex(P<0.01).The expression of BGP protein in female Yougui Pill group was significantly lower than that in male Yougui Pill group(P<0.01).5 Effect of Yougui Pill on PI3K/Akt signal pathway of osteoblasts in tibial bone marrow of ovariectomized and orchiectomized rats5.1 Effect of Yougui Pill on p-PI3K and p-Akt protein expression of osteoblasts in tibial bone marrow of ovariectomized and orchiectomized ratsThe fluorescent area of p-PI3K and p-Akt expression in tibial bone marrow osteoblasts in model group(OVX group/ORX group)was significantly higher than that in sham operation group of the same sex(P<0.01).Compared with ovariectomized and orchiectomized rats,the expression of p-PI3K and p-Akt in OVX group was significantly higher than that in ORX group(P<0.05).The fluorescence area of p-PI3K and p-Akt expression in tibial bone marrow osteoblasts in the positive drug group and Yougui Pill group was significantly lower than that in the same sex model group(OVX group/ORX group)(P<0.01,P<0.05).The expression of p-PI3K and p-Akt in female Yougui Pill group was significantly lower than that in male Yougui Pill group(P<0.05).5.2 Effect of Yougui Pill on p-GSK-3 β,β-catenin and RUNX2 protein expression of osteoblasts in tibial bone marrow of ovariectomized and orchiectomized rats The expressions of p-GSK-3 β,β-catenin and RUNX2 in tibial bone marrow of model group(OVX group/ORX group)were significantly higher than those of sham operation group of the same sex(P<0.01).Compared with ovariectomized and orchiectomized rats,the expression of these three factors in OVX group was significantly higher than that in ORX group(P<0.01).The expressions of p-GSK-3 β,β-catenin and RUNX2 in tibial bone marrow of rats in positive drug group and Yougui Pill group were significantly lower than those in OVX group/ORX group(P<0.01,P<0.05).The expression of p-PI3K and p-Akt in female Yougui Pill group was significantly lower than that in male Yougui Pill group(P<0.01).6 Effect of Yougui Pill on TRAP protein expression of osteoclasts in tibial bone marrow of ovariectomized ratsThe expression of TRAP protein in tibial bone marrow of model group(OVX group/ORX group)was significantly higher than that of sham operation group of the same sex(P<0.01).Compared with ovariectomized and orchiectomized rats,the number of rats in ORX group was significantly lower than that in OVX group(P<0.05).The expression of TRAP protein in tibial bone marrow in positive drug group and Yougui Pill group was significantly lower than that in OVX group/ORX group(P<0.01,P<0.05).The expression of TRAP in female Yougui Pill group was significantly lower than that in male Yougui Pill group(P<0.05).7 Effect of Yougui Pill on PI3K/Akt signal pathway of osteoclasts in tibial bone Marrow of ovariectomized and orchiectomized rats7.1 Effect of Yougui Pill on p-PI3K and p-Akt protein expression of osteoclasts in tibial bone marrow of ovariectomized and orchiectomized ratsThe fluorescence area of p-PI3K and p-Akt expression in tibial bone marrow osteoclasts in model group(OVX group/ORX group)was significantly higher than that in sham operation group of the same sex(P<0.01).Compared with ovariectomized and orchiectomized rats,the expression of p-PI3K and p-Akt in OVX group was significantly higher than that in ORX group(P<0.01,P<0.05).The fluorescence area of p-PI3K and p-Akt expression in tibial bone marrow osteoclasts in the positive drug group and Yougui Pill group was significantly lower than that in the same sex model group(OVX group/ORX group)(P<0.01,P<0.05).The expression of p-PI3K and p-Akt in female Yougui Pill group was significantly lower than that in male Yougui Pill group(P<0.01,P<0.05).7.2 Effect of Yougui Pill on TRAF-6,c-Src and NFATc1 protein expression of osteoclasts in tibial bone marrow of ovariectomized and orchiectomized ratsThe protein expression of TRAF-6,c-Src and NFATc1 in tibial bone marrow of model group(OVX group/ORX group)was significantly higher than that of sham operation group of the same sex(P<0.01).Compared with ovariectomized and orchiectomized rats,the expression of these three factors in OVX group was significantly higher than that in ORX group(P<0.01).The expressions of TRAF-6,c-Src and NFATc1 in tibial bone marrow of rats in positive drug group and Yougui Pill group were significantly lower than those in OVX group/ORX group(P<0.01,P<0.05).The expression of p-PI3K and p-Akt in female Yougui Pill group was significantly lower than that in male Yougui Pill group(P<0.01).Experiment 2.Effect of drug-containing serum of Yougui Pill in ovariectomized and orchiectomized rats on PI3K/Akt signal pathway of osteoblastsMethods(1)Preparation of drug-containing serum:A total of eighty 3-month-old SD rats,SPF grade,40 male and 40 female were selected.After adaptive feeding,female and male rats were randomly divided into sham operation group and model group The methods of modeling and grouping of model group were the same as those in experiment 1.At the end of the grouping,the rats in each group were given intragastric administration twice a day for 7 days,and 1 hour after the last administration.After anesthesia,blood was taken from the abdominal aorta under aseptic condition,and the serum was isolated,inactivated,filtered and packaged,and then frozen in the refrigerator at-80℃.(2)The mouse preparietal osteoblast cell line MC3T3-E1 was inoculated into a 6-well plate at 5×104 cells/well.After 24 hours of cell attachment,10%rat serum of each group was added for culture.The specific experimental groups are as follows:female sham operation group rat serum group,male sham operation rat serum group,OVX rat serum group,ORX rat serum group,estrogen serum+OB group,androgen serum group,female Yougui Pill drug-containing serum group,male Yougui Pill drug-containing serum group.After 48 hours of treatment,the cells were collected and the follow-up indexes were detected.Immunohistochemical method was used to detect the protein expression of BGP,RANKL and OPG in OB,Real-time PCR method was used to detect the expression of PI3K,Akt,GSK-3 β,β-catenin and Runx2 mRNA in OB.Western-Blot method was used to detect the expression of p-PI3K,PI3K,p-Akt,Akt,p-GSK-3 β,GSK-3β,β-catenin and RUNX2 in OB.Results1 Effect of drug-containing serum of Yougui Pill in ovariectomized and orchiectomized rats on the expression of BGP protein in osteoblastsCompared with female sham operation group,the expression level of BGP protein in O VX serum group,estrogen drug-containing serum group and female Yougui Pill drug-containing serum group was significantly higher than that in female sham operation group(P<0.01).Compared with male sham operation serum group,the expression level of BGP protein in ORX serum group,androgen drug-containing serum group and drug-containing serum group of male Yougui Pill was significantly higher than that in male sham operation serum group(P<0.01).Compared with male and female,the expression of BGP protein in OVX serum group was significantly higher than that in ORX serum group(P<0.05),and the expression of BGP protein in female Yougui Pill containing serum group was also significantly higher than that in male Yougui Pill group(P<0.05).2 Effects of drug-containing serum of Yougui Pill in ovariectomized and orchiectomized rats on OPG,RANKL protein expression and RANKL/OPG of osteoblastsCompared with the female sham operation group,the RANKL protein level and RANKL/OPG ratio of OB in the OVX serum group were significantly up-regulated(P<0.01).The OPG protein level was significantly down-regulated(P<0.01).Compared with the OVX serum group,the drug-containing serum and estrogen serum of female Yougui Pill could significantly reduce the RANKL protein and RANKL/OPG ratio of OB(P<0.01),and significantly promote the secretion of OPG of OB(P<0.01).The expression of RANKL protein and RANKL/OPG ratio of OB in ORX serum group were significantly higher than those in male sham operation serum group(P<0.01).The level of OPG protein was significantly decreased(P<0.05).The RANKL protein expression of OB in androgen serum and drug-containing serum of male Yougui Pill was significantly down-regulated as compared with that in ORX serum group(P<0.05,P<0.01),The expression of OPG protein in androgen group was significantly up-regulated(P<0.05),but there was no significant change in OPG in male Yougui Pill drug-containing serum group.The expression of RANKL protein and RANKL/OPG in OVX serum group was significantly higher than that in OVX group(P<0.05,P<0.01).The expression of OPG protein in ORX serum group was significantly higher than that in OVX serum group(P<0.05).RANKL protein level and RANKL/OPG in female Yougui Pill containing serum group were significantly lower than those in male Yougui Pill group(P<0.05,P<0.01),and OPG protein expression in female Yougui Pill containing serum group was significantly higher than that in male group(P<0.01).3 Effects of drug containing serum of Yougui pill in ovariectomized and orchiectomized rats on mRNA expression of PI3K,Akt,GSK3β,β-catenin and Runx2 in osteoblastsCompared with the female sham operation group,the expression levels of PI3K and Akt mRNA in OVX serum group,estrogen group and female Yougui Pill drug-containing serum group were significantly higher than those in the sham operation group(P<0.01).Compared with the male sham operation serum group,the expression of PI3KmRNA in the ORX serum group and the drug-containing serum group of male Yougui Pill increased significantly,and the expression of Akt mRNA in the ORX serum group,androgen group and the drug-containing serum group of Yougui Pill increased significantly(P<0.05,P<0.01).Compared with the OVX group,there was no significant difference in the expression of PI3K and Akt mRNA between the estrogen group and the drug-containing serum of female Yougui Pill.Between male and female model group,the expression of PI3K and Akt mRNA in OVX serum group was significantly higher than that in ORX group(P<0.05).Compared with the female sham operation group,the expression levels of GSK-3 β,β-catenin and Runx2 mRNA in the OVX serum group were significantly increased(P<0.01,P<0.05),and the expression levels of Runx2 mRNA in the estrogen group and female Yougui Pill group were also significantly higher than those in the female sham operation group(P<0.01).Compared with the male sham operation serum group,the expression levels of GSK3β,β-catenin and Runx2 mRNA were significantly increased in the ORX serum group(P<0.05),and the expression level of GSK-3βmRNA was also significantly increased in the male Yougui Pill group(P<0.05).Compared with the ORX group,the effects of androgen and drug-containing serum of male Yougui Pill on the expression of the above three kinds of mRNA were not statistically significant.There was no significant difference between male and female model groups.4 Effects of drug-containing serum of Yougui Pill in ovariectomized and orchiectomized rats on the expression of p-PI3K,PI3K,p-Akt and Akt proteins in osteoblastsCompared with the female sham operation group,the phosphorylated expression levels of p-PI3K and p-Akt proteins in OVX serum group,estrogen group and female Yougui Pill drug-containing serum group were significantly higher than those in sham operation group(P<0.01,P<0.05).Compared with male sham operation serum group,the phosphorylated expression level of p-PI3K protein in ORX group and androgen group was significantly higher than that in ORX group(P<0.01,P<0.05),androgen group and drug-containing serum group of male Yougui Pill(P<0.01,P<0.05),and the difference was statistically significant.The level of p-Akt protein in OVX serum group was significantly higher than that in ORX serum group between female and male model serum groups(P<0.01),but there was no significant difference between female and male Yougui Pill groups.There was no significant difference between PI3K and Akt groups.Compared with the female sham operation serum group,the expression levels of p-GSK3β,β-catenin and RUNX2 protein in the OVX serum group increased significantly(P<0.01,P<0.05),and the expression level of β-catenin protein in the estrogen group increased significantly(P<0.05),the difference is statistically significant.Compared with the OVX group,there was no statistically significant difference between the estrogen group and the female Yougui Pill-containing serum group.Compared with the male sham operation serum group,the expression levels of β-catenin and RUNX2 protein in the ORX group and the androgen group were significantly increased,and the difference was statistically significant(P<0.05).The protein expression levels of β-catenin in the male Yougui Pill group increased significantly,the difference was statistically significant(P<0.05).Compared with the ORX group,there was no statistically significant difference between the androgen group and the male Yougui Pill-containing serum group.There was no statistically significant difference between GSK3β groups.There was no statistically significant difference in indicators between male and female groups.Experiment 3.Effect of drug-containing serum of Yougui Pill in ovariectomized and orchiectomized rats on PI3K/Akt signal pathway of osteoclasts MethodsThe mouse mononuclear macrophage cell line RAW264.7 was planted into a 96-well plate at 2×103 cells/well or 6×104 cells/well in a 6-well plate.The 96-well plate was pre-placed with bone slices.After 24 hours of adherence,add differentiation medium(DMEM+10%FBS+50ng/mL RANKL),induce 48 hours,add 10%rat serum of each group(grouping method is the same as experiment 2),cell culture to the 5th day,to OC tartrate-resistant acid phosphatase(TRAP)staining was performed to observe and count the number of TRAP-positive cells(nucleus ≥3);the cells were cultured to the 9th day,and the bone slices were stained with toluidine blue for bone lacuna to calculate the bone Absorb the area of the lacuna.Immunohistochemical method was used to detect the expression of TRAP protein in OC;Real-time PCR method was used to detect the mRNA levels of PI3K,Akt,TRAF-6,c-Src,and NFATcl in OC,and Western-Blot method was used to detect p-PI3K,PI3K,and PI3K in OC.p-Akt,Akt,TRAF-6,c-Src,NFATc1 and other protein expression levels.Results1 Effect of drug-containing serum of Yougui Pill in ovariectomized and orchiectomized rats on osteoclast differentiationThe cells of OC stained by TRAP were purplish red and multinucleated.The TRAP+cells with nucleus≥ 3 were regarded as osteoclasts and counted.The number of TRAP+osteoclasts in OVX serum group was significantly higher than that in female sham operation group(P<0.01).The number of TRAP+ osteoclasts in ORX serum group was significantly higher than that in male sham operation group(P<0 01).The number of TRAP+osteoclasts in estrogen and female Yougui Pill drug-containing serum group was significantly lower than that in OVX group(P<0.01).The number of TRAP+osteoclasts in androgen group and male Yougui Pill drug-containing serum group was significantly lower than that in ORX group(P<0.05,P<0.01),but it was still significantly higher than that in sham operation group(P<0.01).Compared with ORX group,the number of TRAP+osteoclasts in OVX group was significantly higher than that in ORX group(P<0.05),while the number of osteoclasts in female Yougui Pill drug-containing serum group was significantly lower than that in male Yougui Pill drug-containing serum group(P<0.05).2 Effect of drug-containing serum of Yougui Pill on in ovariectomized and orchiectomized rats on bone resorption function of osteoclastsAfter the bone slices were stained with toluidine blue,the bone resorption lacunae formed by OC were blue and the shape was round,oval or sausage-shaped,indicating that the induced OC had the function of bone phagocytosis.The area of bone resorption lacunae in OVX/ORX group was significantly higher than that in sham operation group of the same sex(P<0.01).Compared with the model group of the same sex(OVX group/ORX group),the area of bone resorption lacunae in each drug containing serum group was significantly lower than that in the same sex model group(P<0.01).The drug-containing serum group of male Yougui Pill was higher than that of male sham operation group,and the difference was statistically significant(P<0.01).Compared with ORX group and OVX group,the area of bone resorption lacunae in OVX group was significantly higher than that in ORX group(P<0.05),while that in drug-containing serum group of ovariectomized rats was significantly lower than that in orchiectomized rats(P<0.05).3 Effect of drug-containing serum of Yougui Pill in ovariectomized and orchiectomized rats on the expression of TRAP protein in osteoclastsThe expression level of TRAP protein in OVX/ORX serum group was significantly higher than that in sham operation serum group of the same sex(P<0.01).The expression level of TRAP protein in each drug-containing serum group was significantly lower than that in the same sex model group(OVX group/ORX group)(P<0.05,P<0.01).There was significant difference between male and female model groups,and the expression level of TRAP protein in OVX group was significantly higher than that in ORX group(P<0.05).The drug-containing serum group of female castrated rats was significantly lower than that of male castrated rats(P<0.05).4 Effects of drug-containing serum of Yougui Pill in ovariectomized and orchiectomized rats on the expression of PI3K,Akt,TRAF6,c-Src and NFATc1 mRNA in osteoclastsCompared with the female sham operation group,the levels of PI3K and Akt mRNA in the OVX serum group were significantly higher than those in the female sham operation group(P<0.01).Compared with OVX serum group,the expression levels of PI3K and Akt mRNA in estrogen and female Yougui Pill drug-containing serum group were significantly lower than those in female Yougui Pill group(P<0.01).Compared with the male sham operation serum group,the expression levels of PI3K and Akt mRNA in the ORX group and the drug-containing serum group of male Yougui Pill were significantly increased(P<0.05,P<0.01).Compared with ORX serum group,the expression levels of PI3K and Akt mRNA in androgen group and male Yougui Pill drug-containing serum group were significantly lower(P<0.05,P<0.01).There were significant differences between male and female model groups,and the expression levels of PI3K and Akt mRNA in OVX serum group were significantly higher than those in ORX serum group(P<0.05).The level in the female Yougui Pill drug-containing serum group was significantly lower than that in the male Yougui Pill drug-containing serum group(P<0.05).Compared with the female sham operation group,the levels of TRAF6,c-Src and NFATc1 mRNA in the OVX serum group were significantly higher than those in the female sham operation group(P<0.01).Compared with OVX serum group,the expression levels of TRAF6,c-Src and NFATc1 mRNA in estrogen and female Yougui Pill group were significantly lower(P<0.01,P<0.05).Compared with male sham operation serum group,the expression levels of PI3K,Akt,TRAF6,c-Src and NFATc1 mRNA in ORX group were significantly increased(P<0.05).Compared with ORX serum group,the expression levels of TRAF6 and NFATc1 mRNA in androgen group and male Yougui Pill containing serum group were significantly lower than those in ORX group(P<0.05,P<0.01).The expression level of c-Src mRNA in drug-containing serum of male Yougui Pill was lower than that in ORX group.There was significant difference between male and female model group,and the expression levels of TRAF6,c-Src and NFATc1 mRNA in OVX serum group were significantly higher than those in ORX serum group(P<0.05).5 Effects of drug-containing serum of Yougui Pill in ovariectomized and orchiectomized rats on the expression of p-PI3K,PI3K,p-Akt,Akt,TRAF6,c-Src and NFATc1 proteins in osteoclastsCompared with the female sham operation group,the protein phosphorylation levels of p-PI3K and p-Akt in the OVX serum group,estrogen group and female Yougui Pill group were significantly higher than those in the sham operation group(P<0.01,P<0.05).Compared with the male sham operation serum group,the phosphorylated expression of PI3K and Akt protein in the ORX serum group and the drug-containing serum group of male Yougui Pill increased significantly(P<0.01).The protein phosphorylation levels of PI3K and Akt in each drug-containing serum group were significantly lower than those in the same sex model group(OVX group/ORX group)(P<0.05,P<0.01).There were significant differences between female and male model group and Yougui Pill group.The phosphorylated expression levels of PI3K and Akt proteins in OVX group were significantly higher than those in ORX group(P<0.05,P<0.01).The level in the female Yougui Pill drug-containing serum group was significantly lower than that in the male Yougui Pill drug-containing serum group(P<0.01).Compared with the female sham operation group,the protein expression levels of TRAF6,c-Src and NFATc1 in the OVX serum group were significantly higher than those in the female sham operation group(P<0.01).Compared with OVX serum group,estrogen and drug-containing serum of female Yougui Pill could significantly reduce the protein expression levels of TRAF6,c-Src and NFATc1(P<0.01).Compared with the male sham operation serum group,the protein expression levels of TRAF6,c-Src and NFATc1 in the ORX group were significantly higher than those in the sham operation serum group(P<0.01).Compared with ORX group,the protein expression levels of TRAF6,c-Src and NFATc1 in androgen group and male Yougui Pill group were significantly decreased(P<0.05,P<0.01).Compared between male and female,the level of NFATc1 protein in OVX group was significantly higher than that in ORX group(P<0.05).The TRAF6 of drug-containing serum group of ovariectomized rats was significantly lower than that of orchiectomized rats(P<0.05).Experiment 4.Effects of drug-containing serum of Yougui Pill in ovariectomized and orchiectomized rats on the osteoclast differentiation,function and PI3K/Akt signal pathway under the action of PI3K agonistsMethodThe mouse mononuclear macrophage cell line RAW264.7 was planted into a 96-well plate at 2×103 cells/well or 6×104 cells/well in a 6-well plate.The 96-well plate was pre-placed with bone slices.After the cells adhered to the wall for 24 hours,the differentiation medium(DMEM+10%FB S+50ng/mL RANKL)was added,induced for 48h,added 10μM PI3K agonist 740Y-P to the culture wells of the group contain... |
PDF Full Text Request