Study On The Mechanism Of ATP6V1H Against Bone Loss In Mice Under Microgravity Environment

Objective:Osteoporosis is a common bone disease with high incidence and is difficult to cure completely.However,previous studies have focused on studies under the action of estrogen,drugs,exercise,and microgravity,and most of them are single-sex studies,and there are few studies on the mechanism of bone loss in both genders.With the further development of aerospace medicine in my country,bone loss caused by the microgravity environment in outer space has gradually become a new focus in osteoporosis research in China.Many scholars use microgravity models to study the mechanism of bone loss,among which the rat tail suspension model is more commonly used.However,the tail suspension model is mainly used in experimental rats.Because of its small size and poor adaptability,it is difficult to establish a mouse microgravity model.ATP6V1H is a bone-related gene reported in recent years.Population surveys and experiments have verified that ATP6V1H is involved in bone loss,and its molecular mechanism needs to be further studied.Transcriptome sequencing is one of the most widely used sequencing technologies.It has been used in many fields,such as medicine and genetics.It can help people study the expression of genes in cells or tissues and discover new signaling pathways.Therefore,this study will establish a mouse microgravity model to explore the bone loss changes and molecular mechanism of Atp6v1h against the microgravity effect.Methods:1.Establishment and characteristic analysis of mouse tail suspension simulated microgravity model:3-month-old wild-type male and female mice（WT）were used as experimental objects,and they were usually reared and tail suspension.During the experiment,the behavioral and appearance characteristics of the normal-raised and suspension-raised mice were observed and compared,and their body weight changes were recorded.After the experiment,the femurs of mice were taken for micro-CT scanning and sectioned H＆E staining.The applicability of the tail suspension microgravity model was judged from the above results of bone morphology,etc.2.Effects of Atp6v1h knockout on bone loss in mice under microgravity:3-month-old wild-type male and female mice（WT）and Atp6v1h knockout(Atp6v1h^+/-)male and female mice were used as experimental subjects,and normal rearing and tail suspension rearing.The genotypes of the experimental mice were identified before the experiment,and the bodyweight of the mice in each group was recorded during the experiment.After the experiment,the femurs of mice were taken for micro-CT scanning and sectioned H＆E staining.The effect of the ATP6V1H gene on osteoporosis in microgravity was judged from the above results of bone morphology.3.Transcriptome sequencing analysis:Extract the bone marrow RNA of male WT and Atp6v1h knockout heterozygous mice,and entrust the company to conduct transcriptome sequencing of the bone marrow RNA.Transcriptome data were analyzed from genotype comparison,normal-suspension comparison,key signaling pathways,and critical genes were screened,and finally,crucial genes were verified by q-PCR.Results:1.Establishment and characteristic analysis of mouse tail suspension simulated microgravity model.（1）The behavior and appearance of mice reared with tail suspension were normal,with no accidental death.（2）The body weight of suspension-reared mice decreased in the second or third week and showed a fluctuating upward trend.（3）Compared with the normal feeding,BMD,BV/TV,Tb.Th and Tb.N of suspended feeding mice bone parameters decreased to varying degrees in the micro-CT test(33%-57%in male mice;33%-57%in female mice;BS/BV and Tb.Sp increased in varying degrees（11%-51%in male mice;4%-32%in female mice）.（4）Compared with the control group,the bone cortex and cartilage tissue of suspension-feeding mice became thinner in H＆E staining of bone tissue,the number of trabecular bones decreased significantly,and the thickness became light dispersion increased.2.The effect of a low dose of Atp6v1h on bone loss in the microgravity environment.（1）The body weight of Atp6v1h^+/-mice was not significantly different from that of WT mice,and the bodyweight of Atp6v1h^+/-mice decreased in the second week after suspension-feeding and then showed a fluctuating upward trend.（2）Compared with WT mice,the bone parameters BMD,BV/TV,Tb.Th and Tb.N of Atp6v1h^+/-mice were decreased to varying degrees in micro-CT detection（male mice 19%-57%;female mice 19%-57%;3%-33%of mice）,BS/BV,Tb.Sp increased to varying degrees（male mice 11%-51%;female mice 4%-32%）.Compared with the normal group,the bone parameters BMD,BV/TV,Tb.Th and Tb.N of the WT mice in the suspension group decreased to varying degrees（19%-59%in male mice;31%-71%in female mice）,BS/BV,Tb.Sp increased to varying degrees（male mice 45%-63%;female mice 50%-58%）;while the bone parameters BMD,BV/TV of Atp6v1h^+/-mice suspension group,Tb.Th and Tb.N decreased to varying degrees（male mice 62%-8%;female mice 31%-71%）,and BS/BV and Tb.Sp increased to varying degrees（male mice 13%～60%;female mice7%～20%）.The 95%confidence intervals of bone parameters BMD,BV/TV,BS/BV,Tb.Th,Tb.N,Tb.Sp in WT mice and Atp6v1h^+/-mice were greater than 0.05.（3）Compared with WT mice,the bone morphology of Atp6v1h^+/-mice showed irregular changes in bone tissue H＆E staining,and the cortical and cartilage tissues were slightly thinner trabecular bone was significantly reduced,and the dispersion was increased.Compared with the normal group,the cortical bone and cartilage tissue of WT mice became thinner after suspension,and the number and thickness of trabecular bone were significantly reduced,while the cortical bone and cartilage tissue of Atp6v1h^+/-mice were slightly thinner after suspension,and the bone was smaller.The number of beams decreases,the thickness decreases,and the dispersion increases.Female mice had smaller overall bone morphology,thinner cortical bone and cartilage tissue,fewer trabecular bone numbers,and thinner thickness compared with male mice.3.Transcriptome analysis of effects of Atp6v1h and microgravity on bone mass.（1）The quality of transcriptome sequencing samples met the standard,and there were different numbers of differential genes in different comparison groups.Among them,a total of 393 genes（142 up-regulated genes and 251 down-regulated genes）were compared between male WT mice and Atp6v1h^+/-mice,and a total of 276 genes（75up-regulated genes and 251 down-regulated genes）were compared between normal-suspension male WT mice（75 up-regulated genes and 251 down-regulated genes）.201),a total of 196 genes（146 up-regulated and 50 down-regulated genes）in the normal-suspension comparison of male Atp6v1h^+/-mice.In addition,there are only 20 common genes in the comparison of the differential genes between male WT mice and Atp6v1h^+/-mice and the normal-suspension differential genes of male Atp6v1h^+/-mice.（3）Differential gene functional annotation enrichment analysis.（1）Screening and analysis of critical pathways:The top 10 pathways with the highest significance in the three comparison groups were all significantly expressed in bone,and immune system,such as hematopoietic cell lineage and platelet activation,IL-17signaling pathway,TNF signaling pathway,NF-（?）B signaling pathway,rheumatoid arthritis,PI3K-Akt signaling pathway,MAPK signaling pathway,etc.（2）Screening and analysis of critical genes:Significantly different genes in critical pathways are essential genes,all of which are related to immunity and bone,such as Fos,Jun,Il1b,Atp6v0d2,Ibsp,Col6a1,etc.;（4）Experiments verified the changes of downstream bone-related genes regulated by Fos,such as down-regulation of Ctsk,up-regulation of Tgfb1,Mmp8,Mmp9,etc.Conclusion:1.Mice can be adapted to tail suspension condition and have obvious bone loss,which can be used as a research model for bone diseases such as osteoporosis,so the mouse microgravity model was successfully established;2.Atp6v1h dose is insufficient in a microgravity environment.There was no significant change in bone mass,so the knockout of Atp6v1h has the effect of resisting bone loss in microgravity;3.The single effect or interaction mechanism of a microgravity environment and low dose of Atp6v1h is related to the immune system and bone-related pathways.