Study On The Mechanism Of Action Of Zuogui Pills And Yougui Pills On Differentiation Of BMSCs And Osteoclasts In PMOP Rats Based On AMPK/mTOR Pathwa

Purpose:Clarify the theoretical significance of the theory of five internal organs essence,qi,yin and yang in the pathogenesis and prevention of postmenopausal osteoporosis.Based on the theory of kidney essence,qi,yin and yang,the effects of Zuogui pills and Yougui pills on the proliferation activity of BMSCs were studied;the effects of Zuogui pills and Yougui pills on the differentiation of BMSCs and osteoclasts in PMOP rats mediated by AMPK/mTOR signaling pathway were studied.Explain the scientific connotation of"invigorating the kidney and filling the essence"and"mutual benefit of yin and yang"by studying the effects of Zuogui Pills and Yougui Pills on the proliferation activity of BMSCs and the differentiation trend of bone marrow stem cells in PMOP rats.Material and method:1 Traditional chinese medicine theory researchFrom the ancient literature of traditional Chinese medicine to the syndrome differentiation and treatment of postmenopausal osteoporosis,sort out the connotation and logical relationship of the five organs essence,qi,yin and yang,clarify the theoretical significance of the theory of kidney essence,qi,yin and yang in the occurrence and prevention of postmenopausal osteoporosis,and clarify the kidney tonifying theoretical basis for the prevention and treatment of postmenopausal osteoporosis by Zuogui Pills and Yougui Pills,which can invigorate the kidney essence,yin and yang.2 Effects of Zuogui Pills and Yougui Pills on AMPK/mTOR signaling pathway-mediated osteogenic differentiation of BMSCs in PMOP ratsSeventy SPF 2-month-old female nulliparous SD rats were randomly divided into blank group（KB group）,10 rats,sham operation group（SHAM group）,10 rats,and modeling group,50 rats,according to the random number method.In the modeling group,bilateral ovaries were removed to establish the PMOP rat model.In the SHAM group,only the adipose tissue near the ovaries and the same volume of ovaries were removed.After operation,the living rats in the modeling group were randomly divided into model group（OVX group）,Bujiale group（BJL group）,Yougui Pill group（YGW group）and Zuogui Pill group（ZGW group）.After 7 days,each administration group was administered by intragastric administration at a dose of 6.3 times the adult dose（based on unit body weight）,once a day,for 12W consecutively.10 rats that did not meet the inclusion criteria due to factors such as modeling surgery,improper gavage,and malnutrition were excluded,and a total of 60 rats were included in the experiment,with 10 rats in each group.After the last administration,the rats were fasted for 12 hours.Their lower limbs were dissociated,soaked in 75%ethanol for 15 minutes,the fur and muscles were stripped,and the femur and tibia were separated.The bone marrow tissue in the bone marrow cavity was washed into a petri dish with penicillin and streptomycin-containingα-MEM medium,centrifuged at 1000 rpm for 3 min,the supernatant was discarded,and the cells were resuspended inα-MEM medium containing 10%FBS,and then inoculated to the culture flask was moved to a CO₂constant temperature incubator for culture,and the medium was changed for the first time on the fifth day.After that,the medium was changed every 3 days.When the cells were confluent to 85%to 90%,the cells were passaged,and then passed to the third generation to be performed cell identification.The growth curve of BMSCs was drawn by CCK-8 method,and the effects of Zuogui Pills and Yougui Pills on the proliferation of BMSCs in PMOP rats were determined.The P3 generation BMSCs were taken for osteogenic differentiation induction,and after9 days of induction,the related indexes of BMSCs were detected.Alizarin red staining was used to observe the formation of mineralized nodules after osteogenic induction of BMSCs,alkaline phosphatase（ALP）activity detection kit was used to detect the ALP activity in each group,and q RT-PCR method was used to detect Runx2,ALP,COLⅠ,BSP,OPG gene expression,Western blotting method was used to detect the expression of Runx2,ALP,COLⅠ,BSP,OPG,AMPK/mTOR signaling pathway related proteins.3 Effects of Zuogui Pills and Yougui Pills on AMPK/mTOR signaling pathway-mediated adipogenic differentiation of BMSCs in PMOP ratsThe P3 generation BMSCs were used for adipogenic differentiation induction,and 9 days after induction,the related indexes of BMSCs were detected.Oil red O staining was used to detect the formation of lipid droplets in BMSCs after adipogenic induction.q RT-PCR was used to detect the expression of PPARγ,FABP4,LPL,C/EBPα,C/EBPβgenes,and Western blotting was used to detect PPARγ,FABP4,LPL,C/EBPα,C/EBPβ,AMPK/mTOR signaling pathway related proteins.4 Effects of Zuogui Pills and Yougui Pills on AMPK/mTOR signaling pathway-mediated osteoclast differentiation of bone marrow monocytes/macrophages in PMOP ratsAfter the last administration,the rats were fasted for 12 hours.Their lower limbs were removed and soaked in 75%alcohol for 10 to 15 minutes.the fur and muscles were stripped,and the femur and tibia were separated.The bone marrow tissue in the bone marrow cavity was washed into a petri dish with penicillin and streptomycin-containingα-MEM medium,filter with a 100-mesh sieve,centrifuge at 1000 rpm for 5 min,discard the supernatant;add 10times the cell volume（about 5 ml）of 1×red blood cells The lysate was lysed on ice for 5 min after blowing evenly,centrifuged at 1000 rpm for 5 min,and the supernatant was discarded;washed twice with serum-freeα-MEM;resuspended in 5 ml ofα-MEM medium containing1%penicillin and streptomycin、10%FBS,and inoculated into culture flasks,incubated in CO₂incubator for 24 hours;collected the supernatant,and rinsed the culture flasks twice with serum-freeα-MEM medium;25μg/L M-CSFα-MEM medium was resuspended and inoculated in a petri dish;after 3 days of culture,the supernatant was discarded,the petri dish was washed twice with serum-freeα-MEM medium,and 1%penicillin and streptomycin、10%FBS、25μg/L M-CSF inα-MEM medium,until the cells proliferate to an appropriate density.The isolated monocytes/macrophages were inoculated into cell culture flasks/plates,and after induction of osteoclast differentiation,the TRAP activity was detected by tartrate-resistant acid phosphatase detection kit,the expression of TRAP gene was detected by q RT-PCR,and the expression of TRAP,RANKL,AMPK/mTOR signaling pathway related proteins were detected by Western blotting.Results:1.The theory of essence,qi,yin and yang of five organs has important guiding significance in the interpretation of pathological phenomena.Deficiency of kidney essence and qi is the fundamental basis for the occurrence of postmenopausal osteoporosis,and the imbalance of yin and yang is the basic pathogenesis change.The prevention and treatment of PMOP from tonifying the kidney and filling the essence and adjusting yin and yang has a profound theoretical basis,and the curative effect is reliable.2.Effects of Zuogui Pills and Yougui Pills on AMPK/mTOR signaling pathway-mediated osteogenic differentiation of BMSCs in PMOP rats2.1 Effects of Zuogui Pills and Yougui Pills on the proliferation of BMSCs in PMOP ratsThe experimental results showed that at 24h,72h,and 120h after cell inoculation,compared with the KB group,there was no obvious difference in the SHAM group（P>0.05）,and the cell proliferation ability of the OVX group was decreased（P<0.05）.The cell proliferation ability of drug groups was enhanced（P<0.05）;at 24h,there was no obvious difference among the drug groups（P>0.05）;at 72h and 120h,the cell proliferation ability of the BJL group and the ZGW group was higher than that of the YGW group.2.2 Effects of Zuogui Pills and Yougui Pills on the formation of mineralized nodules after osteogenic induction of BMSCs in PMOP ratsThe results showed that there was no obvious difference in the number of mineralized nodules between the KB group and the SHAM group;compared with the KB group,the number of mineralized nodules in the OVX group was obviously reduced;compared with the OVX group,the number of mineralized nodules in each drug group was obviously increased,and ZGW group was better than YGW group,and YGW group was better than BJL group.2.3 Effects of Zuogui Pills and Yougui Pills on ALP activity after osteogenic induction of BMSCs in PMOP ratsThe results showed that there was no obvious difference in the ALP activity between the KB group and the SHAM group（P>0.05）.Compared with the KB group,the ALP activity in the OVX group was obviously decreased（P<0.01）;All three drug groups could increase ALP activity（P<0.05 or P<0.01）;compared with each drug group,ZGW group had the most obvious effect of increasing ALP activity,and there was no obvious difference between BJL group and YGW group（P>0.05）.2.4 Effects of Zuogui Pills and Yougui Pills on the expression of osteogenesis-related genes and proteins after osteogenic induction of BMSCs in ovariectomized ratsThe results showed that there was no obvious difference in the gene and protein expression levels of Runx2,ALP,COLⅠ,BSP and OPG between the KB group and the SHAM group（P>0.05）.Compared with the KB group,the gene and protein expression levels of Runx2,ALP,COLⅠ,BSP and OPG in the OVX group were decreased（P<0.05 or P<0.01）.Compared with the OVX group,the gene and protein expression levels of Runx2,ALP,COLI,BSP and OPG in the three drug groups were increased to varying degrees（P<0.01）.Compared with each drug group,the ZGW group had the most obvious effect on the expression of osteogenesis-related genes and proteins after osteogenic induction of BMSCs in ovariectomized rats.2.5 Effects of Zuogui Pills and Yougui Pills on the expression of AMPK/mTOR pathway-related proteins after osteogenic induction of BMSCs in ovariectomized ratsThe experimental results showed that there was no obvious difference in the phosphorylation level of AMPKαprotein,the expression level of TSC2 protein,the phosphorylation level of mTORC1 protein and the phosphorylation level of p70S6K1 protein between the KB group and the SHAM group（P>0.05）;compared with the KB group,the AMPKαprotein phosphorylation level and the TSC2 protein expression level in the OVX group increased（P<0.01）,and the mTORC1 and p70S6K1 protein phosphorylation levels decreased（P<0.01）;compared with the OVX group,the phosphorylation levels of AMPKαprotein in the three drug groups were reduced to varying degrees（P<0.01）,and the expression level of TSC2 protein was obviously reduced（P<0.05 or P<0.01）,mTORC1 and p70S6K1protein phosphorylation levels increased（P<0.01）.3.Effects of Zuogui Pills and Yougui Pills on AMPK/mTOR signaling pathway-mediated adipogenic differentiation of BMSCs in PMOP rats3.1 Effects of Zuogui Pills and Yougui Pills on the formation of lipid droplets after induction of adipogenic BMSCs in PMOP ratsThe experimental results showed that the cells of BMSCs gradually changed from long spindle to oval or round after adipogenic induction.After staining with Oil Red O staining solution,the formation of red lipid droplets with dark color and uneven size was observed under the microscope.Compared with the SHAM group,the number and color of lipid droplets in the KB group were not obviously different;compared with the KB group,the number of lipid droplets in the OVX group increased obviously,and the color of the overlap was deepened;compared with the OVX group,the number of lipid droplets in the three drug groups decreased obviously,and Less than KB group and SHAM group;ZGW group and BJL group had no obvious difference,the number of lipid droplets in YGW group was more than that in BJL group and ZGW group.3.2 Effects of Zuogui Pills and Yougui Pills on the gene and protein expressions associated with adipogenic differentiation after adipogenic induction of BMSCs in PMOP ratsThe results showed that there was no obvious difference in the gene and protein expression levels of PPARγ,FABP4,LPL,C/EBPα,C/EBPβbetween the KB group and the SHAM group（P>0.05）.Compared with the KB group,the gene and protein expression levels of PPARγ,FABP4,LPL,C/EBPαand C/EBPβin the OVX group were increased（P<0.01）;compared with the OVX group,the gene and protein expression levels of PPARγ,FABP4,C/EBPα,C/EBPβin the three drug groups were decreased to varying degrees（P<0.05 or P<0.01）;the expression levels of LPL gene and protein in BJL group and ZGW group decreased（P<0.01）,The expression levels of LPL gene and protein in YGW group were increased（P<0.05）.Compared with each drug group,the expression levels of lipid-related genes and proteins in YGW group were higher than those in BJL group and ZGW group except C/EBPβ（P<0.05 or P<0.01）.3.3 Effects of Zuogui Pills and Yougui Pills on the expression of AMPK/mTOR pathway-related proteins after adipogenic induction of BMSCs in PMOP ratsThe experimental results showed that there was no obvious difference in the phosphorylation level of AMPKαprotein,the expression level of TSC2 protein,the phosphorylation level of mTORC1 protein and the phosphorylation level of p70S6K1 protein between the KB group and the SHAM group（P>0.05）.Compared with the KB group,the AMPKαprotein phosphorylation level and the TSC2 protein expression level in the OVX group were obviously decreased（P<0.01）,and the mTORC1 and p70S6K1 protein phosphorylation levels were increased（P<0.01）.Compared with the OVX group,the phosphorylation level of AMPKαprotein and the expression level of TSC2 protein in the BJL group and the ZGW group were increased（P<0.05）,the phosphorylation level of AMPKαprotein and the expression level of TSC2 protein in the YGW group were increased（P<0.01）;the phosphorylation level of mTORC1 protein in the three treatment groups was decreased（P<0.01）,and the phosphorylation level of p70S6K1 protein was decreased（P<0.05）.4.Effects of Zuogui Pills and Yougui Pills on AMPK/mTOR signaling pathway-mediated osteoclast differentiation of bone marrow monocytes/macrophages in PMOP rats4.1 Effects of Zuogui Pills and Yougui Pills on TRAP activity after induction of osteoclast differentiation of bone marrow monocytes/macrophages in PMOP ratsThe experimental results showed that there was no obvious difference in TRAP activity between the KB group and the SHAM group（P>0.05）;compared with the KB group,the TRAP activity level of the OVX group increased（P<0.01）;compared with the OVX group,the TRAP activity levels of the three drug groups decreased to varying degrees（P<0.01）,and the TRAP activity of the BJL group was lower than that of the ZGW group,with a significant difference（P<0.05）,and the ZGW group was lower than the YGW group,with a significant difference（P<0.01）.4.2 Effects of Zuogui Pills and Yougui Pills on the expression of TRAP gene and protein after induction of osteoclast differentiation of bone marrow monocytes/macrophages in PMOP ratsThe experimental results showed that there was no obvious difference in the expression levels of TRAP gene and protein between the KB group and the SHAM group（P>0.05）;compared with the KB group,the expression levels of TRAP gene and protein in the OVX group were increased（P<0.01）.Compared with the OVX group,the expression levels of TRAP gene and protein in the three drug groups decreased to varying degrees（P<0.05 or P<0.01）,and the expression of TRAP gene and protein in BJL group and ZGW group decreased most significantly（P<0.01）.4.3 Effects of Zuogui Pills and Yougui Pills on the expression of RANKL protein after induction of osteoclast differentiation of bone marrow monocytes/macrophages in PMOP ratsThe experimental results showed that there was no obvious difference in the expression level of RANKL protein between the KB group and the SHAM group（P>0.05）;compared with the KB group,the expression level of RANKL protein in the OVX group was increased（P<0.01）.Compared with the OVX group,the expression levels of RANKL protein in each drug group decreased（P<0.05 or P<0.01）.The expression level of RANKL protein in BJL group and ZGW group was lower than that in YGW group,and there was a significant difference（P<0.01）.4.4 Effects of Zuogui Pills and Yougui Pills on the expression of AMPK/mTOR pathway-related proteins after induction of osteoclast differentiation of bone marrow monocytes/macrophages in PMOP ratsThe experimental results showed that there was no obvious difference in the phosphorylation level of AMPKαprotein,the expression level of TSC2 protein,the phosphorylation level of mTORC1 and p70S6K1 protein between the KB group and the SHAM group（P>0.05）.Compared with the KB group,the AMPKαprotein phosphorylation level and the TSC2 protein expression level in the OVX group were obviously decreased（P<0.01）,and the mTORC1 and p70S6K1 protein phosphorylation levels were increased（P<0.01）.Compared with the OVX group,the phosphorylation level of AMPKαprotein and the expression level of TSC2 protein in the three drug groups were increased（P<0.01）;the phosphorylation levels of mTORC1 and p70S6K1 proteins in the three drug groups were decreased（P<0.05或P<0.01）.Conclusion:1.Based on the theory of essence,qi,yin and yang of the kidney in traditional Chinese medicine,it is explained that the deficiency of essence and qi in the kidney is the fundamental basis for the occurrence of PMOP,and the imbalance of yin and yang on the basis of deficiency of essence and qi is the basic pathogenesis change.Invigorating the kidney and filling the essence and adjusting yin and yang are the basic methods to prevent and treat PMOP.2.The osteogenic differentiation ability of BMSCs in PMOP rats was attenuated,and it was related to the regulation of AMPK/mTOR signaling pathway;Zuogui Pills could interfere with AMPK/mTOR signaling pathway to enhance the osteogenic differentiation of BMSCs in PMOP rats,and the effect of Zuogui Pills was better than that of Yougui Pills.3.The ability of adipogenic differentiation of BMSCs in PMOP rats was enhanced,and it was related to the regulation of AMPK/mTOR signaling pathway;Zuogui Pills could interfere with AMPK/mTOR signaling pathway to inhibit the adipogenic differentiation of BMSCs in PMOP rats.Zuogui Pills were better than Yougui Pills in inhibiting adipogenic differentiation,but Yougui Pills were better than Zuogui Pills in regulating AMPK/mTOR signaling pathway.4.The osteoclast differentiation ability of bone marrow monocytes/macrophages in PMOP rats was enhanced,and it was related to the regulation of AMPK/mTOR signaling pathway;Zuogui Pills could interfere with AMPK/mTOR signaling pathway to inhibit osteoclast differentiation in PMOP rats.Zuogui Pills were better than Yougui Pills in inhibiting osteoclast differentiation,but Yougui Pills were better than Zuogui Pills in regulating AMPK/mTOR signaling pathway.5.Zuogui Pills and Yougui Pills enhanced the proliferation activity of BMSCs in PMOP rats;and changed the differentiation trend of bone marrow stem cells in PMOP rats through the AMPK/mTOR signaling pathway,enhanced the differentiation and maturation of osteoblasts,at the same time attenuated the differentiation of adipocytes and osteoclasts.Then this may be related to age-related senescence against bone marrow stem cells,to a certain extent,it reveals the scientific connotation of"invigorating the kidney and filling the essence"and"cooperating with yin and yang".Zuogui Pills mainly focus on nourishing kidney yin,and tend to promote bone,inhibit bone breakage,and promote"Yin formation";Yougui Pills mainly warm and invigorate kidney yang,and tend to promote lipid metabolism and promote"yang transform qi".