Discovery Mechanisms Of Chlorogenic Acid On Bone Metabolism In Ovariectomized Mice

IntroductionOsteoporosis（OP）is the most common type of bone metabolic disease characterized by decreased bone strength,degraded bone microstructure,and increased bone fragility,which greatly increases the risk of fracture.However,the pathogenesis of osteoporosis is still unclear,and mechanisms for the onset of various drugs are also to be studied.Therefore,elucidating the pathogenesis of osteoporosis and finding new drug targets are the key scientific issues in the prevention and treatment of osteoporosis.Recent studies have shown that the p38 MAPK pathway is an important signaling pathway involved in the regulation of inflammatory response,and its signaling pathway plays an important role in the proliferation and differentiation of mesenchymal stem cells into osteoblasts.After activation of the p38 MAPK pathway,osteoblast-specific factor Cbfa1 expression is increased.The P38 MAPK pathway controls the expression of gene activities of various transcription factors,such as NF-κB,MAX,SAP-1,and HSF-1.Among them,some transcription factors p38 direct substrates,and some are indirect substrates.The mechanism is still unclear.Previous studies by the research group have shown that chlorogenic acid can promote the osteogenesis of bone marrow mesenchymal stem cells and inhibit their differentiation into fat.We also found that chlorogenic acid treatment of osteoporosis model rats can increase the osteogenic differentiation of BMSCs,increase the number of osteoblasts,and increase the bone mineral density and trabecular bone number in osteoporosis model rats.However,the mechanism of chlorogenic acid to promote osteogenic differentiation of BMSCs,increase the number of osteoblasts and inhibit osteoporosis is still unclear.In order to explore the anti-osteoporosis mechanism of chlorogenic acid,the study is divided into three parts.The first part: gene chip spectrum analysis to find possible pathways of target genes and gene production;the second part: the role of target genes in in vitro experiments;the third part: the role of target genes in in vivo experiments.Text 1 Gene Chip Profiling for the Anti-osteoporosis Mechanism of Chlorogenic Acid in Ovarian Ovariectomized Mice Model Background:Osteoporosis is a clinically common metabolic disease,mainly characterized by loss of bone mass and destruction of trabecular bone microstructure.After menopause,due to the lack of estrogen in the body,the immune system is activated,the inflammatory factor continues to increase chronically,and the osteogenesis and osteoclast balance breaks.Osteoclast activity is higher than osteogenesis,which ultimately leads to further aggravation of osteoporosis.At present,the clinical treatment of osteoporosis drugs is mainly hormone-based,they all have certain side effects,so they are looking for alternative drugs.The research team has been working on.It was found that Eucommia ulmoides has anti-osteoporosis effect,and chlorogenic acid is one of the main components of Eucommia.The previous study of the research group showed that chlorogenic acid can promote the osteogenesis of bone marrow mesenchymal stem cells and inhibit the formation of fat.We also found that chlorogenic acid treatment of osteoporosis model mice can increase the osteogenic differentiation of BMSCs,increase the number of osteoblasts,and increase the bone mineral density and trabecular bone number in osteoporosis model rats.However,the mechanism of chlorogenic acid to promote osteogenic differentiation of BMSCs,increase the number of osteoblasts and inhibit osteoporosis is still unclear.In this experiment,the differential gene in ovarian castrated mouse model after chlorogenic acid intervention was analyzed by gene chip analysis and its possible mechanism of anti-bone metabolism was analyzed,which provided an effective clinical basis for the development of traditional Chinese medicine for postmenopausal osteoporosis.Purpose:Gene chip analysis of gene changes in ovarian castrated mouse models after cga intervention and signal pathways that genes may affect.Method:（1）Thirty BALB/c mice were randomly divided into three groups: sham operation group（Sham group）,model group（OVX group）,and chlorogenic acid intervention group（OVXT group）,with 10 mice in each group.Sham group and OVX group were given normal saline,and OVXT group was given chlorogenic acid 45mg/kg.d.Once a day,10 W continuous.（2）The mice were sacrificed by cervical dislocation for 10 weeks,and the bilateral femur and tibia were completely removed after alcohol soaking and disinfection.（3）Bone marrow mesenchymal stem cells（BMSCs）were isolated and identified from the tibia and identified.（4）The femoral fixative was fixed and sent to the Micro-CT to detect the changes of the trabecular microstructure.（5）Microarray maps were used to identify gene expression of bone marrow mesenchymal stem cells and verified by q PCR.（6）KEGG analysis and expression of various kinases in each group of cells was detected by the Wb method.（7）The results were statistically analyzed using spss20.0 software.Result:（1）Flow cytometry lines showed: CD₂₉ and CD₄₄ is were positive;CD₃₄ and CD₄₅ is was negative.The extracted cells were BMSCs cells.（2）Micro-CT showed the largest trabecular bone in the Sham group,followed by the OVXT and the OVX group.Bone micro-structures such as BV/TV,Tb.N,Tb.Th,BMD and other indicators were different between the two groups（P<0.05）.（3）The gene expression of bone marrow mesenchymal stem cells was identified by microarray mapping.There were 121 differentially expressed genes between OVX group and OVXT group,of which 36 genes were significantly expressed（change multiples > 2 and P < 0.05）.RGD1564664,Pdlim3,Myh11,Ear11 and Nnat were the five genes with the most significant difference in expression.The expression differences of Acss2,Cd24,Nnat and Pkp2 were the most significant among 36 genes verified by q PCR.（4）KEGG and MAPK signaling pathway analysis,Hspa1 a,Fgfr2,Gadd45 a,Tgfb3,Hspa1 b and other five genes are located in the MAPK pathway.The classical protein validation bioinformatics results of MARK pathway showed an increase in ERK and p38 phosphorylation in the OVXT group compared to the OVX group.Conclusion:（1）.CGA can effectively resist osteoporosis in ovarian castrated mice and improve bone mineral density.（2）Nnat may play a role in CGA against osteoporosis.（3）CGA anti-osteoporosis may be achieved by activating the MAPK pathway.Text2 Effects of Nnat on Osteogenic Differentiation of BMSCs in Ovariectomized Mice and Its Mechanism Background:Studies have shown that Nnat can activate the expression of inflammatory factors such as NF-κB and also induce phosphorylation of p38,Jun,and AKT kinases,while PI3 K and p38 inhibitors prevent Nnat from activating NF-κB.In addition,Nnat is considered to be Activation of NF-κB,MAPK,and FAK signaling pathways.Signal transduction mediated by mitogen-activated protein kinases（MAPKs）,including extracellular signal-regulated kinase（ERK）,c-Jun N-terminal kinase（c-Jun）N-terminal kinase,JNK)and p38 have been found to be critical for the differentiation and activation of normal osteoclasts and osteoblasts.Various exogenous stimuli（such as toll-like receptor agonists）and endogenous stimuli（such as growth factors and inflammatory cytokines）help determine the adhesion,migration,fusion,and survival of MAPKs to osteoclasts and osteoblasts,And the effect of bone resorption by osteoclasts25-28.In the first part of our study,we found that CGA has an anti-osteoporosis effect.Compared with the OVX group,the Nnat gene in the OVXT group was significantly increased,and ERK and p38 phosphorylation were increased.We speculate that Nnat plays a role in osteogenic differentiation of ovarian castrated mice.And it may be that it promotes osteogenic differentiation by activating MAPK associated with ERK and P38.Objective: To investigate the effect and mechanism of Nnat on osteogenic differentiation induced by BMSCs in ovarian ovariectomized mice.Method:（1）The fourth generation BMSCs were taken and osteogenic differentiation culture was carried out with osteoblast culture medium.The transcription level of Nnat in cells was detected by q PCR on the 0th,3rd,5th,7th,10 th and 14 th day after osteogenic differentiation.（2）The fourth generation BMSCs were divided into normal culture group（NC group）,Nnat overexpression（Nnat group）,empty plasmid group（KZ group）and common osteogenic culture（NC group）,Nnat knockdown（si RAN group）.Transfer to the empty plasmid group（KZ group）.（In this experiment,Nnat over-expressed and knocked down asynchronously）.（3）The transcription level of Runx2 m RNA was detected by q PCR.（4）The activity of ALP in each group of cells was analyzed by PNPP method.（5）Alizarin red staining was used to measure the mineralized nodules and evaluate the osteogenic ability of cells.（6）The expression of various kinases in each group of cells was detected by the Wb method.（7）Statistical analysis was performed using SPSS20.0 software and image J software.Result:（1）With the induction of osteogenic differentiation of BMSCs,the expression of Nnat in cells increased gradually,which was statistically significant.（2）The expression of Runx-2 in cells of each group was detected by q PCR.Compared with the normal induced osteogenesis group（NC group）,the expression of Runx-2 after overexpression of Nnat（Nnat group）was significantly increased.After knocking down Nnat with si RNA（si RNA group）,the expression of Runx-2 was significantly decreased,and the difference was statistically significant（p < 0.05）.There was no significant difference in Runx-2 expression between KZ group and NC group,and there was significant difference in Runx-2 expression between KZ group and NC group（p < 0.05）.（3）The PNPP method was used to detect the activity of alkaline phosphatase（ALP）in BMSCs after induction of osteoblasts.Compared with the control group（NC group）,after Nnat overexpression（Nnat group）,ALP activity was significantly increased,using si RNA.After knocking down Nnat（si RNA group）,ALP activity decreased significantly,and the difference was statistically significant（p<0.05）.（4）.Alizarin red staining showed that the number of intracellular mineralized nodules in BMSCs after induction of osteogenesis was significantly increased after overexpression of Nnat（Nnat group）compared with control group（NC group）.After knocking down Nnat with si RNA（si RNA group）,the number of mineralized nodules was significantly reduced,and the difference was statistically significant（p < 0.05）.（5）The expression of various kinases in each group was detected by WB method.Compared with the control group（NC group）,the expression of ERK1/2,JNK,p38,c-Fos and c-Jun in Nnat group was significantly increased,si RNA The expressions of ERK1/2,ERK5,p38 and c-Jun were significantly decreased,and the difference was statistically significant（p<0.05）.Conclusion:1.Nnat can regulate the osteogenic differentiation of BMSCs in ovarian ovariectomized mice.Increased expression of Nnat promotes osteogenic differentiation,decreased expression of Nnat,and inhibits osteogenic differentiation.2.Nnat regulates BMSCs to induce osteogenic differentiation through the MAKP signaling pathway.Text 3 Effects of Nnat on bone metabolism in ovarian castrated mouse models Background:Animal experiments are an important medium for humans to directly understand life phenomena.It is indisputable and irreplaceable for people to understand the various laws of the organic world.Medical animal experiments are an important link to achieve scientific research from molecular cell levels to clinical research.In the previous part of the experiment,it was found that Nnat can regulate the osteogenic differentiation of BMSCs in OVX mice.In order to clarify the role of Nnat in the body,we conducted animal experiments.Purpose:To investigate the effect of Nnat on bone metabolism in ovariectomized mice.Method:（1）Seventy BALB/c mice were randomly divided into 7 groups according to their body weight.Group A: sham operation group（Sham group）;Group B: model group（OVX group）;Group C: OVX+Nnat overexpression group: Group D: OVX+ empty plasmid group;Group E: OVX+CGA group;Group F : OVX+CGA+Nnat overexpression group;Group G: OVX+CGA+ KZ group,10 in each group.There was no significant difference in the initial mean body weight between the groups（P>0.05）.（2）Injecting the corresponding intervention fluids from the tail vein of the rats on the day of surgery and the 5th week after surgery: Group C and F were overexpressed with Nnat virus（10 ul/time/time）;Groups D and G were injected with empty plasmid（10 ul）/ Times / only);Group A,Group B and Group E were injected with normal saline(（10 ul / time / only）.（3）On the second day after surgery,the E,F,and G groups were intragastrically administrated with chlorogenic acid solution（45 mg/kg/day）,and A,B,C,and D were converted to the same amount of 0.9% sodium chloride injection.Stomach,free drinking,feeding.The mice were observed daily for various conditions and the body weight was measured weekly.（4）The mice were sacrificed by cervical dislocation for 10 weeks,and the bilateral femur and tibia were completely removed after alcohol soaking and disinfection.（5）Bone marrow mesenchymal stem cells（BMSCs）were isolated and cultured from the femur.After being qualified,the expression of Nnat was examined by q PCR.（6）The micro-structural changes of the trabecular bone were detected by tibia micro-CT.（7）The results were statistically analyzed using SPSS 20.0 software.Result:（1）Micro-CT scan showed that compared with the Sham group,the number of trabecular bone in the metatarsal metatarsal of all groups of ovarian castrated mice was significantly reduced,and the OVX group and the OVC+KZ group were the most reduced;compared with the OVX group: OVX+Nnat group,The number of trabecular bone in OVX+CGA group,OVX+CGA+Nnat group and OVX+CGA+KZ group increased,and the increase in OVX+CGA+Nnat group was more obvious,but it was still different from Sham group.There was no significant difference in trabecular bone number between OVX group and OVX+KZ group.There was no significant difference in trabecular bone number between OVX+CGA group and OVX+CGA+KZ group.（2）The microstructural parameters（BMD,BV/TV,Tb.N,Tb.Th）of the micro-CT scan showed that the Sham group was the highest,and the other groups were significantly different,and the difference was statistically significant（p<0.05）.The OVX group and the OVC+KZ group were the lowest;compared with the OVX group,the OVX+Nnat group,the OVX+CGA group,the OVX+CGA+Nnat group,and the OVX+CGA+KZ group were significantly increased,of which OVX+CGA+Nnat group The increase was more pronounced and the difference was statistically significant（p < 0.05）.The difference between OVX group and OVX+KZ group was not obvious,and the difference between OVX+CGA group and OVX+CGA+KZ group was not obvious.（3）The expression of Nnat in q PCR showed that there was no significant difference between the OVX+KZ group and the OVX+Nnat group,OVX+Nnat group,OVX+CGA group,OVX+CGA+Nnat group and OVX+CGA+KZ group value.There was a significant increase in the OVX+CGA+Nnat group,and the difference was statistically significant（p<0.05）.Compared with the OVX+CGA group,there was no significant difference in the OVX+CGA+KZ group,and the difference was not statistically significant.The expression of Nnat in OVX+CGA+Nnat group was significantly increased,and the difference was statistically significant（p<0.05）.Conclusion:（1）Enhanced Nnat expression can improve the bone structure of ovarian ovariectomized mice and increase the BMD,BV/TV,Tb.N,Tb.Th values of the tibia of ovarian castrated mice.（2）Chlorogenic acid can up-regulate the expression of Nnat in the ovary of ovariectomized mice,and then increase the BMD,BV/TV,Tb.N,Tb.Th values of the tibia of ovariectomized mice.