| Objective: 1.This study explores the genes related to ketogenic diet(KD)and osteoporosis(OP)in the public databases by bioinformatics analysis;2.This study researches the role and potential mechanism of CDK1 gene in osteoclasts(OCs)differentiation and bone resorption by animal experimental studies.Methods:1.Bioinformatics analysis: ⑴ the data set GSE35959 was selected and downloaded in GEO database(GEO database),and the differential expression gene(DEG)was identified by the GEO2 R tool to compare the gene expression profile between the OP group and the control group.Then,we carried out the weighted gene co-expression network analysis(WGCNA),and the module with the strongest correlation with OP was selected,and the gene contained in the selected module was used as the hub gene(HG)for further analysis;⑵The selected HGs were constructed by gene functional enrichment analysis(GFEA)and protein-protein interaction network(PPI network);⑶ From the Gene Cards database,we used the keywords "ketogenic diet","ketone body" and“ β-hydroxybutyric acid" to retrieve the gene related to KD(KDRG);⑷We Intersected HGs and KDRGs to obtain common genes(CG)that are associated with OP and KD at the same time,and incorporate CGs into machine learning for further analysis to obtain the final key genes(KG).2.Animal experiment verification: ⑴ Establishment of ketogenic diet animal model(KDAM): 30 mice were divided into three groups according to complete randomization:standard diet group(SD group),ketogenic diet group(KD group)and ketogenic diet + NF-κB signaling pathway inhibitor pyrrolidine dithiocarbamate(PDTC)intervention group(KD+PDTC group).SD group was fed with standard feed,KD group and KD+PDTC group were fed with ketogenic feed,and KD+PDTC group were given intraperitoneal injection of PDTC solution every week for a total of 8 weeks.During the feeding period,the changes of body weight,food and water consumption,blood glucose and blood ketone levels of mice among each group were observed.After feeding,take the femur of mice for the next experiment.⑵We used vernier caliper to measure the length of mouse femur and observe the difference of the length of mouse femur among each group.⑶Micro-CT was used to detect the changes of bone metabolism indexes of mice among each group.⑷Western Blot was used to detect the protein expression of osteoclast-related genes RANK and Ctsk,the protein expression of CDK1 in KGs and the protein expression of p65 and Bcl-3 in NF-κB signaling pathway.Results: 1.Bioinformatics analysis: ⑴ A total of 2875 DEGs related OP were found from the GEO database,including 2219 up-regulated genes and 656 down-regulated genes.After introducing OP group and control group into WGCNA analysis,135 HGs with the strongest correlation with OP were screened;⑵HGs was analyzed by gene ontology(GO)and Kyoto Encyclopedia of genes and genomes(KEGG).The results showed that "cell cycle process" appeared many times in the two enrichment analysis processes,and may play a key role in the OP progress.⑶After the intersection of HGs and KDRGs,16 CGs were obtained: AURKA,BIRC5,BRCA1,CCNA2,CCNB1,CDC20,CDC7,CDK1,CDKN3,HEK1,HELLS,HMGB2,NEK2,PLK4,RAD51,TTK,and the expression of these CGs in OP tissue all decreased significantly(P<0.05).The obtained CGs are further analyzed in machine learning,and the final 7 KGs are obtained: BIRC5,BRCA1,CDC7,CDK1,CDKN3,HELLS,and HMGB2.2.Animal experiment verification: ⑴During the construction of KDAM,the weight of mice in three groups gradually increased.From the start of the second week,the weight of mice in SD group started to be significantly higher than that in the other two groups(P<0.0001).In the two groups fed with ketogenic diet,the body weight of KD+PDTC group mice was slightly higher than that of KD group mice(P>0.05).⑵The blood ketone level of KD group and KD+PDTC group was significantly higher than that of SD group(P<0.01),which meant that the establishment of KDAM was successful.Meanwhile,the blood glucose level of the three groups of mice had no difference(P>0.05).⑶ The measurement of the mouse femur length showed that compared with SD group,the femur length of KD group and KD+PDTC group decreased(P<0.001).⑷ Micro-CT results showed that compared with SD group,the bone mineral density of KD group and KD+PDTC group mice decreased.With the regard to cancellous bone parameters,compared with SD group,the bone volume fraction,the number of bone trabeculae and the thickness of bone trabeculae in KD group and KD+PDTC group decreased significantly,while the bone trabecular separation increased,which further explained the negative effect of KD on the bone structure of mice(P<0.05).However,between KD group and KD+PDTC group,the bone structure damage of KD group mice was more serious(P>0.05).As for the changes of cortical bone parameters,the cortical bone area,total cortical bone area and cortical bone thickness of KD and KD+PDTC group mice were also significantly lower than those of SD group,but the damage of KD+PDTC group was more serious than that of KD group.⑸Western Blot showed that the protein expression of osteoclast-related genes Cts K in KD group was significantly higher than that of SD group(P<0.05),while the expression of Cts K in KD+PDTC group did not change much(P>0.05),It was lower than KD group(P<0.01).The protein expression of osteoclast-related genes RANK in KD group(P<0.01),and KD+PDTC group(P<0.05)was significantly higher than that of SD group,while the expression of RANK in KD+PDTC group was lower than KD group(P<0.05).Compared with SD group,the protein expression of p65 in NF-κB signal pathway of KD group increased(P<0.001),while the expression of KD+PDTC group was lower than that of SD group(P<0.01)and KD group(P<0.01).As for the result of Bcl-3 protein expression,compared with SD group,the expression of Bcl-3 in KD group increased(P<0.01).The expression of that in KD+PDTC group decreased(P<0.05).When it comes to the expression of CDK1 in HGs,it decreased in KD group(P<0.05)while increased in KD+PDTC group(P>0.05)regard to SD group.The expression of CDK1 in KD+PDTC group was lower than that in KD group(P<0.05).Conclusion: 1.Bioinformatics analysis found 7 KGs: BIRC5,BRCA1,CDC7,CDK1,CDKN3,HELLS,and HMGB2.The decreased expression level of the KGs plays an important role in the development of KD-induced OP.2.KD can damage bone mass and bone structure of mice;3.KD can down-regulate the expression of CDK1 and promote osteoclast differentiation and enhance bone absorption process in mice by activating Bcl-3/NF-κB/p65 signaling.4.PDTC can prevent the occurrence of OP caused by KD through inhibiting NF-κB signaling pathway,and this effect may play a role in cancellous bone. |
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