The Effect Of Acupuncture On DEGs In Skeletal Muscle Injury Mice And Its Regulation Of UPR Related Pathways

BackgroundSkeletal muscle is the key to health,and it is very important for the body to play its normal function if its structure and function are intact.Clinically,skeletal muscle injury is directly related to a variety of acute and chronic diseases.Genes are the internal factors that determine the health of life.It supports the basic structure and performance of life and deduces the important physiological processes of life such as reproduction,cell division and protein synthesis.Although acupuncture is widely used in the treatment of diseases related to skeletal muscle injury,the effect of acupuncture on gene expression after skeletal muscle injury is still unclear,and it is urgent to adopt advanced research methods to explore.RNA sequencing is a cutting-edge method for detecting gene expression changes,which can objectively analyze the changes of differentially expressed genes under specific interventions(such as acupuncture intervention),and explore the related biological functions and signal pathways of differentially expressed genes that play an important role in the intervention process.Therefore,exploring the mechanism of acupuncture treatment of skeletal muscle injury diseases by transcriptome sequencing can provide a solid experimental basis for the clinical promotion of acupuncture treatment of skeletal muscle injury-related diseases.Unfolded protein response(UPR)is a main mechanism for maintaining the normal folding of proteins when proteins with abnormal endoplasmic reticulum folding in cells accumulate in large quantities.Skeletal muscle injury often causes endoplasmic reticulum stress of skeletal muscle and activates UPR pathway,and three branch pathways of UPR play a key role in regeneration and repair of damaged skeletal muscle.Combined with transcriptome sequencing results,it is worth studying whether acupuncture can promote the repair of damaged skeletal muscle by regulating UPR-related pathways.ObjectiveTo observe the effects of acupuncture on morphology and differentially expressed genes of injured muscle,so as to explore the effect of acupuncture on the gene levels after skeletal muscle injury.To study the UPR-related biological signal pathway suggested by differentially expressed genes,so as to reveal some mechanisms of acupuncture in the treatment after skeletal muscle injury.Methods1.The effect of acupuncture at "Ashi" on gene expressions in mice with gastrocnemius muscle injury.108 C57BL/6J mice were divided into four groups according to random number table,namely,WT group,Sham group,Inju group and Acup group.The mice in the Inju group and Acup group received the injection of bupivacaine into the bilateral gastrocnemius muscle.The mice in the Acup group received acupuncture treatment 24h after modelling and the treatment was conducted for only one time.After the treatment,morphological changes of gastrocnemius muscles in each group were assayed by HE-staining and gene expressions of gastrocnemius muscles were assayed by RNA-Sequencing.2.The effect of acupuncture at BL40 on gene expressions in mice with multifidus muscle injury.72 C57BL/6J mice were divided into four groups according to random number table,namely,WT group,BPVC group,Acup group and EA group.The mice in the EA group and Acup group received the injection of bupivacaine into the bilateral multifidus muscle.The mice in the EA and Acup group received acupuncture treatment 24h after modelling and the treatment was conducted for three times,20min per time.The exercise balance ability of mice in each group was tested by rotating bar test,the morphological changes of lumbar multifidus muscle in each group were observed by HE staining,and the gene expressions of multifidus muscle in each group was detected by RNA-Sequencing technique.3.The regulatory effect of acupuncture on UPR-related pathway of skeletal muscle injured mice.42 C57BL/6J mice were divided into seven groups according to random number table,namely,Sham-G group,Inju-G group,Acup-Ashi group,Sham-M group,Inju-M group,Acup-BL40 group and EA-BL40 group.The mice in the Sham-M and Sham-G group received the injection of physiological saline into the local muscle,while the mice in the remaining five groups received the injection of bupivacaine into the local muscle.The mice in the Acup-Ashi group received acupuncture treatment 24h after modelling and the treatment was conducted for only one time.The mice in the Acup-BL40 and EA-BL40 group received acupuncture treatment 24h after modelling and the treatment was conducted three times,20min per time.After treatment,the levels of proteins and mRNA in the signal pathways of UPR were detected by Western-blot and Real-time PCR assay.Results1.The effect of acupuncture at "Ashi" on gene expressions after gastrocnemius muscle injury.HE staining results showed that 24 hours after modeling,compared with the normal group,it showed severe muscle fiber swelling and breakage in the model group.Compared with the model group,it showed a better regeneration effect on 48h,72h and 7d,with reduced muscle fibers swelling,fewer broken muscle fibers,and more new muscle fibers with nuclei located in the middle of the muscle fibers after the intervention in the acupuncture groupTranscriptomics analysis showed that compared with Sham group,the Inju group had up-regulated 987 DEGs and down-regulated 73 DEGs;Compared with the Inju group,the Acup group had up-regulated 623 DEGs and down-regulated 65 DEGs.These DEGs after gastrocnemius injury were related to actin cytoskeleton,and signal pathways including chemokine signal pathways through further assay;These DEGs after acupuncture were related some main biological functions including organelle inner membrane,and signal pathways including thermogenesis and oxidative phosphorylation-related pathways.2.Effect of acupuncture/electroacupuncture at BL40 on gene expressions after lumbar multifidus muscle injuryThe Rota-rod test showed that compared with the WT group,after multifidus muscle injury,mice’s Rota-rod performance decreased slightly in the BPVC group,but with no statistically differences(P>0.05).Compared with BPVC group,acupuncture/electroacupuncture intervention,the Rota-rod performance in the Acup group and EA group increased slightly,but there was no statistical differences(P>0.05).HE staining showed that compared with WT group,it showed severe inflammatory infiltration and the rupture of muscle fiber after 48 hours of modeling in the BPVC group;compared with BPVC group,after acupuncture/electroacupuncture intervention,inflammatory infiltration and broken muscle fibers were significantly reduced in the Acup group and EA group.Transcriptomics results showed that compared with the normal group,the model group had up-regulated DEGs of 2063 and down-regulated DEGs of 1181;compared with the model group,after acupuncture or electroacupuncture intervention,the up-regulated DEGs of the acupuncture group were 80 and 93 DEGs were down-regulated;DEGs up-regulated in the electroacupuncture group were 59,and DEGs down-regulated were 126.Further analysis revealed that among the DEGs that were upregulated due to lumbar multifidus injury,48 DEGs reversed by acupuncture at BL40,and 21 DEGs reversed by electroacupuncture at BL40;among the DEGs that were downregulated by injury,there were 23 DEGs reversing by acupuncture at BL40 and 7 DEGs were reversing by electroacupuncture at BL40.The main biological functions of DEGs induced by lumbar multifidus muscle injury include actin cytoskeleton.The signaling pathways involved in the regulation of these DEGs include chemokine signaling pathways.The main biological functions of DEGs induced by acupuncture at BL40 include innate immune response and chemokine activity.The signal pathways that these DEGs participate in the regulation also include chemokine signaling pathway.The main biological functions of DEGs induced by electroacupuncture at BL40 include NADH dehydrogenase activity,and the significant regulation pathways are thermogenesis and oxidative phosphorylation.3.Effect of acupuncture on UPR-related pathway proteins and genes after skeletal muscle injuryWestern blot and Real-time PCR results of in gastrocnemius injury model mice showed that p-IRE1/IRE1 in Inju-G group was significantly higher than that in Sham-G group(P<0.01).Compared with Inju-G group,the expression of XBP1 mRNA and protein in Acup-Ashi group was increased(P<0.05,P<0.05).Western blot and Real-time PCR results of in multifidus muscle injury model mice showed that the expression of IRE 1 protein and p-eIF2α/eIF2α1 decreased significantly(P<0.01,P<0.01)compared with Sham-M group.Compared with the Inju-M group,the p-IRE1/IRE1 of Acup-BL40 group increased significantly(P<0.01),the expression of IRE1 mRNA increased(P<0.05),and the expression of ATF6 protein increased(P<0.05),the expression of CHOP mRNA and CHOP increased(P<0.05,P<0.05);compared with the the Inju-M group,the p-IRE1/IRE1 of the EA-BL40 group increased significantly(P<0.01),the expression of IRE1 mRNA and XBP1 mRNA increased significantly(P<0.01,P<0.01),the expression of ATF6 mRNA and ATF6 protein increased(P<0.01,P<0.05),and p-eIF2α/eIF2α increased significantly(P<0.01),the expression of eIF2α mRNA increased(P<0.05),and the expression of CHOP mRNA and CHOP increased(P<0.05,P<0.01).Conclusions(1)Intramuscular injection of bupivacaine can cause inflammatory reaction and induce a large number of differentially expressed genes(DEGs).GO/KEGG enrichment analysis suggested that intramuscular injection of bupivacaine might induce skeletal muscle injury by affecting actin cytoskeleton and chemokine signaling pathway.(2)Acupuncture at "Ashi" can further up-regulate the expression of DEGs induced by gastrocnemius injury.GO/KEGG enrichment analysis suggests that acupuncture at "Ashi"may promote the repair of damaged skeletal muscle by regulating organelle inner membrane,thermogenesis and oxidative phosphorylation.(3)Acupuncture/electro-acupuncture at BL40 can reverse the expression of some DEGs induced by lumbar multifidus muscle injury.GO/KEGG enrichment analysis suggests that acupuncture at BL40 may promote the repair of damaged skeletal muscle by regulating innate immune response,chemokine activity and chemokine signal pathway;electroacupuncture at BL40 may regulate NADH dehydrogenase activity,Oxidative phosphorylation and thermogenesis promote the repair of damaged skeletal muscle.(4)The study found that acupuncture and electroacupuncture can regulate genes and proteins in the branches of IRE1,ATF6,and PERK in the UPR pathway to varying degrees.It is suggested that the regulation of UPR related pathways by acupuncture may be one of the important mechanisms for acupuncture to promote the repair of skeletal muscle injury.