Effects Of Sepsis And Mechanical Ventilation Interaction On Functional Impairment Of Rat Diaphragm

Objective:（1）To study the effects of different durations of mechanical ventilation on diaphragm functional impairment in rats in the same sepsis background;and to compare with non-septic ventilated rats to observe the relationship between the duration of mechanical ventilation and diaphragm functional impairment.（2）To explore the effect of different levels of sepsis on the functional impairment of rat diaphragm at the same duration of mechanical ventilation.Methods:Part I48 adult male Sprague-Dawley（SD）rats were selected and randomly divided into two major groups.The sham-operated group（SHAM+MV group）and the sepsis group（CLP+MV group）.Rats in the SHAM+MV group underwent open abdominal exploration but not ligation,and were mechanically ventilated for 3,6,9 and 12 h after sepsis level scoring at 12 h postoperatively,divided into 4 groups（n=6）:SHAM+MV3h group,SHAM+MV6h group,SHAM+MV9h group and SHAM+MV12h group;rats in the CLP+MV group underwent cecal ligation and puncture（CLP）with 50%cecum ligation and mechanical ventilation for 3,6,9 and 12 h after sepsis degree scoring at 12 h postoperatively,divided into 4 groups（n=6）:CLP+MV3h group,CLP+MV6h group,CLP+MV9h group and CLP+MV12h group.Rats were executed immediately at the end of mechanical ventilation and diaphragms and serum specimens were taken.Blood was collected from the internal carotid artery for arterial blood gas analysis,and p H,Pa O₂,Pa CO₂ and lactate（Lac）values were measured.Compound muscle action potential（CMAP）was measured by fitting the stimulation frequency-contraction curve with a biosignal acquisition system;the diaphragm specimens were stained with HE to observe the cross-sectional area（CSA）of muscle fibers;apoptosis was detected by TUNEL method.Apoptosis was detected by TUNEL;serum inflammatory factors（TNF-ɑ,IL-1βand IL-6）levels were measured by ELISA.The expression levels of MAFbx and Mu RF-1 were measured by Western blot.Part II30 adult male SD rats were randomly divided into 5 groups:（1）sham-operated group（SHAM group,n=6）rats underwent open cecum exploration only;（2）moderate sepsis group（CLP 50%group,n=6）rats underwent CLP and 50%cecum ligation;（3）moderate sepsis+mechanical ventilation for 6h group（CLP 50%+MV6h group,n=6）rats underwent 50%cecum ligation and perforation to construct（3）moderate sepsis+mechanical ventilation for 6 h group（CLP50%+MV6h group,n=6）rats underwent 50%cecum ligation and perforation to construct a moderate sepsis model,and mechanical ventilation for 6 h 12 h after surgery;（4）severe sepsis group（CLP75%group,n=6）rats underwent 75%cecum ligation and perforation,and mechanical ventilation for 6 h 12 h after surgery to construct a severe sepsis model;（5）severe sepsis+mechanical ventilation for 6 h group（CLP75%+MV6h group,n=6）rats underwent 75%cecum ligation and perforation,and mechanical ventilation for 6 h 12 h after surgery.The severity of sepsis was scored.Each group of rats was executed after 18 h and diaphragm and serum specimens were taken.Blood was collected from the internal carotid artery for arterial blood gas analysis,and p H,Pa O₂,Pa CO₂ and Lac values were measured.The contractile force of the diaphragm was measured by a biosignal acquisition system,the stimulation frequency-contraction curve was fitted,and the compound muscle action potential（CMAP）was detected;the diaphragm specimens were stained by HE to observe the cross-sectional area of muscle fibers（CSA）;apoptosis was detected by TUNEL;serum inflammatory factors（TNF-ɑ,IL-1βand IL-6）levels were measured by ELISA.The expression levels of muscle atrophy-related proteins MAFbx and Mu RF-1 were detected by Western blot.ResultsPart I（1）Arterial blood p H,Pa O₂,Pa CO₂ and Lac of rats were compared between sham-operated groups,and the differences were not statistically significant（P>0.05）.The differences were not statistically significant（P>0.05）when comparing arterial blood p H,Pa O₂,Pa CO₂ and Lac in rats between the sepsis groups.The degree of sepsis score was higher in the CLP+MV group compared with the SHAM+MV group,with a statistically significant difference（P<0.05）.（2）Compared with each ventilation group in the SHAM+MV group,each ventilation group in the CLP+MV group had elevated levels of TNF-α,IL-1βand IL-6,with statistically significant differences（P<0.05）.（3）Compared with SHAM+MV3h and SHAM+MV6h groups,CMAP amplitude was decreased and time course was prolonged in SHAM+MV9h and SHAM+MV12h groups,and the difference was statistically significant（P<0.05）.The CMAP time course was prolonged in the SHAM+MV12h group compared with the SHAM+MV9h group,and the difference was statistically significant（P<0.05）.Compared with the CLP+MV3h group,the CMAP amplitude was reduced and the time course was prolonged in the CLP+MV6h group,and the difference was statistically significant（P<0.05）.Compared with the CLP+MV3h and CLP+MV6h groups,the CLP+MV9h and CLP+MV12h groups had reduced CMAP amplitude and prolonged time course,and the difference was statistically significant（P<0.05）.Compared with the CLP+MV9h group,the CMAP time course was prolonged in the CLP+MV12h group,and the difference was statistically significant（P<0.05）.（4）Compared with the SHAM+MV3h group and SHAM+MV6h group,the mean contraction force of rat diaphragm under each frequency stimulation was reduced in the SHAM+MV9h group and SHAM+MV12h group,and the difference was statistically significant（P<0.05）.Compared with the CLP+MV3h group,the mean contraction force of rat diaphragm under each frequency stimulation was reduced in the CLP+MV6h,CLP+MV9h and CLP+MV12h groups,and the difference was statistically significant（P<0.05）.Compared with the CLP+MV6h group,the mean contraction force of the rat diaphragm was reduced in the CLP+MV12h group at each frequency stimulation,and the difference was statistically significant（P<0.05）.（5）Compared with the SHAM+MV3h,SHAM+MV6h and SHAM+MV9h groups,the CSA of rat diaphragm fibers was reduced in the SHAM+MV12h group,and the difference was statistically significant（P<0.05）.Compared with the CLP+MV3h and CLP+MV6h groups,the CSA of rat diaphragm fibers was reduced in the CLP+MV9h and CLP+MV12h groups,and the difference was statistically significant（P<0.05）.Compared with the CLP+MV9h group,the CSA of rat diaphragm fibers was reduced in the CLP+MV12h group,and the difference was statistically significant（P<0.05）.（6）The expression of MAFbx and Mu RF-1 was increased in the SHAM+MV12h group compared with the SHAM+MV3h,SHAM+MV6h and SHAM+MV9h groups,and the difference was statistically significant（P<0.05）.There was a statistically significant difference in the expression of MAFbx and Mu RF-1 in the CLP+MV9h and CLP+MV12h groups compared with the CLP+MV3h and CLP+MV6h groups（P<0.05）.The differences in the expression of MAFbx and Mu RF-1 in the CLP+MV12h group were statistically significant compared with the CLP+MV9h group（P<0.05）.（7）There was no statistically significant difference in apoptosis of rat diaphragm cells between SHAM+MV groups（P>0.05）.There was no statistically significant difference in apoptosis of rat diaphragm cells between CLP+MV groups（P>0.05）.Part II（1）There was no statistically significant difference in p H,Pa O₂ and Pa CO₂ between the groups（P>0.05）.Lac was higher in septic rats than in the SHAM group,and the level of Lac was significantly higher in the CLP75%group,and the difference was statistically significant.The difference in sepsis scores between the SHAM group,the cecum ligation 50%group and the cecum ligation 75%group was statistically（P<0.05）.（2）TNF-αlevels increased most significantly in the CLP75%+MV6h group,which were statistically significant when compared with the remaining four groups（P<0.05）.IL-1βlevels in the SHAM group were significantly lower than those in the sepsis modeling group rats（P<0.05）,and IL-1βlevels increased significantly in the CLP75%+MV6h group,which were statistically significant when compared with the remaining four groups（P<0.05）.The difference in IL-6 levels in rats in the sepsis modeling group was statistically significant compared with the SHAM group（P<0.05）.IL-6 was increased in the CLP50%+MV6h group and CLP75%group compared with the SHAM group and CLP50%group,and the difference was statistically significant（P<0.05）.IL-6 was significantly increased in the CLP75%+MV6h group compared with the rest of the groups,and the difference was statistically significant（P<0.05）.（3）Compared with the SHAM group,the CMAP amplitude in the rat diaphragm was reduced and the duration was prolonged in the sepsis modeling group,and the difference was statistically significant（P<0.05）.Compared with the CLP50%group,the CMAP amplitude was reduced in the CLP75%group,and the difference was statistically significant（P<0.05）.Compared with the CLP75%group,the CLP75%+MV6h group had reduced CMAP amplitude and prolonged time course,and the difference was statistically significant（P<0.05）.Compared with the CLP50%+MV6h group,the CLP75%+MV6h group had reduced CMAP amplitude and prolonged time course,and the difference was statistically significant（P<0.05）.（4）Compared with the SHAM group,the frequency-contraction force effect curves were reduced in all groups.Compared with the CLP50%group,the diaphragm contractility was reduced in the CLP75%group and the CLP50%+MV6 h group,and the difference was statistically significant（P<0.05）.Compared with the CLP75%group and the CLP50%+MV6h group,the contractile force of rat diaphragm was reduced in the CLP75%+MV6h group under 80,100 and 120 Hz frequency stimulation,and the difference was statistically significant（P<0.05）.（5）Compared with the SHAM group,the rat diaphragm fiber CSA was reduced in the sepsis modeling group,and the difference was statistically significant（P<0.05）.Compared with the CLP75%group,the CSA was reduced in the CLP75%+MV6h group,and the difference was statistically significant（P<0.05）.Compared with the CLP50%+MV6h group,the CSA was reduced in the CLP75%+MV6h group,and the difference was statistically significant（P<0.05）.（6）The expression of MAFbx and Mu RF-1 was increased in the CLP50%+MV6h and CLP75%groups compared with the SHAM and CLP50%groups,and the difference was statistically significant（P<0.05）.The expression of MAFbx and Mu RF-1 was increased in the CLP75%+MV6h group compared with the rest of the groups,and the difference was statistically significant（P<0.05）.（7）The apoptosis of diaphragm cells was increased in the CLP75%group compared with the SHAM group,CLP50%group and CLP50%+MV6h group in rats with a statistically significant difference（P<0.05）.Conclusion:（1）In the case of systemic inflammation in sepsis,the longer the duration of mechanical ventilation,the more severe the diaphragm dysfunction,with a decrease in contractility occurring at 6 h of mechanical ventilation and an increased expression of atrophy protein at 9 h of mechanical ventilation.（2）The severity of sepsis affects the diaphragm dysfunction caused by mechanical ventilation.The more severe the degree of sepsis,the earlier the onset of diaphragmatic dysfunction and the more severe the symptoms.