To Study The Mechanism Of RyR1 In Sepsis-induced Diaphragm Dysfunction Based On Transcriptome Sequencing In An Experimental Rat Model Of Sepsis

Objective:To study the effect of sepsis on the transcriptome sequencing of the diaphragm in rats,and to investigate the role of Ryanodine Receptor 1（Ry R1）expression and phosphorylation in Sepsis-induced diaphragma dysfunction（SIDD）based on transcriptome sequencing results.MethodsPart 1Twenty SPF adult male Sprague-Dawley（SD）rats were randomly divided into Sham operation group（Sham group,n=10）and sepsis 24 h group（CLP-24 h group,n=10）.The rats in Sham group only underwent abdominal exploration without cecal ligation and puncture.The sepsis model was established by Cecal ligation and puncture（CLP）in the CLP-24 h group.After modeling,the survival rate and weight of the rats were recorded.Then the rats were anesthetized,and the apical blood of the rats was collected to measure the levels of IL-6 and TNF-αby Enzyme linked immunosorbent assay（Elisa）.The diaphragm of the rats was dissected for transcriptome sequencing analysis.RNA was extracted using TRIzol method,the purity of RNA was detected using Nano Drop^TMOne/One C,and the RNA was accurately quantified using Life Invitrogen Qubit（?）3.0 fluorescence quantitative instrument.RNA integrity was measured with an Agilent 4200 Tape Station system.Illumina PE150 sequencing was performed after library construction.Part 2Thirty SPF adult male SD rats were randomly divided into Sham group（n=6）and sepsis group（CLP group）.Then CLP group was divided into four subgroups:CLP-6 h group（n=6）,CLP-12 h group（n=6）,CLP-24 h group（n=6）,and CLP-24 h+KN-93 group（n=6）.In the Sham group,only abdominal exploration was performed without cecal ligation and puncture.The sepsis model was established by cecal ligation and puncture（CLP）in CLP-6 h group,CLP-12 h group,CLP-24 h group and CLP-24 h+KN-93 group.In CLP-24 h+KN-93group,a single intraperitoneal injection of KN-93 was given immediately after operation.The indexes were detected and diaphragm samples were collected at the corresponding time points after modeling in each group.RM6240 biological information acquisition system was used to measure the Compound muscle action potential（CMAP）of the diaphragm,calculate the fatigue index of the isolated diaphragm,and fit the frequency contraction curve.Western blotting（WB）was used to detect the expression Ca MKⅡand Ry R1 and phosphorylation of Ry R1in the diaphragm.The function of Ry R1 was evaluated by the release rate of Ry R1 calcium ion using a multifunctional microplate reader.ResultsPart 1（1）Compared with Sham group,the survival rate and body weight of CLP group rats were significantly decreased,while the IL-6 and TNF-αof CLP-24h group rats were significantly higher than those of Sham group（P<0.05）.（2）A total of 4368 differentially expressed genes were detected in the Sham group and the CLP-24 h group,of which 1788 genes were significantly up-regulated and 2580 genes were significantly down-regulated（P<0.05）.Through functional enrichment,it was found that diaphragm dysfunction caused by sepsis was related to diaphragm fibers,calcium signaling pathway and muscle contraction,et al.The expression of NCX,PMCA,ORA1,Ca V2,ROC,RYR,SERCA,CALM and MLCK genes changed significantly,which may be related to SIDD.Part 2（1）With the extension of sepsis modeling time,the diaphragm CMAP amplitude gradually decreased,the duration gradually prolonged,the fatigue index gradually increased,and the frequency contraction curve gradually decreased（P<0.05）.（2）With the development of sepsis,the expression level of Ry R1gradually decreased,and the CLP-24 h group was significantly lower than the Sham group（P<0.05）.（3）With the extension of sepsis time,the level of P-Ry R1 gradually increased,and the differences between Sham group and CLP group were statistically significant（P<0.05）.（4）With the development of sepsis,the expression level of Ca MKⅡin CLP-24 h group was significantly higher than that in Sham group（P<0.05）.After addition of KN-93,the CLP-24 h+KN-93 group had a significantly higher amplitude,a significantly shorter duration,and a significantly higher frequency contraction curve than the CLP-24 h group（P<0.05）.There was no significant difference in fatigue index between CLP-24 h+KN-93 group and CLP-24 h group（P>0.05）.There was no significant difference in Ry R1 expression between CLP-24 h group and CLP-24 h+KN-93 group（P>0.05）.The expression levels of Ca MKⅡand P-Ry R1 in CLP-24 h+KN-93 group were significantly decreased（P<0.05）.（5）With the extension of sepsis time,the Ca²⁺release rate of Ry R1 in Sham CLP-6 h group,CLP-12 h group and CLP-24 h group decreased gradually（P<0.05）.The Ca²⁺release rate of CLP-24 h+KN-93 group was significantly higher than that of CLP-24 h group（P<0.05）.Conclusion（1）In this study,sepsis led to diaphragm dysfunction,and the gene level of the diaphragm was significantly changed.The expression of RYR gene in the diaphragm of sepsis rats was significantly decreased,and the change of Ry R gene expression was closely related to muscle contraction and calcium ion balance.Combined with the existing literature,it was speculated that RYR was a key candidate gene for the occurrence and development of SIDD,so Ry R was selected as the follow-up study object.（2）Sepsis can damage the function of Ry R1 through the decrease of Ry R1expression and the phosphorylation of Ry R1 mediated by Ca MKⅡ,leading to the release of Ca²⁺in diaphragm cells at rest.When Ry R1 is activated,the release of Ca²⁺is insufficient,resulting in contractile dysfunction of diaphragm.