Study On The Effect Of The Vagus Nerve In Rats Excited By Berberine And Acupuncture To Cerebral Hemorrhage-Induced Multiple Organ Dysfunction Syndrome

Objective:Establish a model of intracerebral hemorrhage-induced multiple organ dysfunction syndrome(CHMODS)companied by vagotomy or not in rats,intervention with acupuncture and Berberine,investigate changes in tissue pathology of lung?heart?intestines?liver and kidney,to discuss the mechanism of vagus nerve in the development of Cerebral hemorrhage-induced multiple organ dysfunction syndrome(CHMODS),clear the role of vagus-never cholinergic anti-inflammatory pathway(VCAP)of the vagus nerve in rats excited by berberine and acupuncture to CHMODS.Methods:1.The SD rats were randomly divided into 10 groups:normal control group(n=6)?sham-operative group(n=6)?MODS group?MODS+acupuncture group?MODS+berberine group?MODS+ acupuncture+ berberine group?MODS+ SDV group?MODS+SDV+ acupuncture group?MODS+ SDV+ berberine group?MODS+ SDV+ acupuncture+berberine group.The final eight groups include 24h?72h?7d?14d four time points and every time point have 6 rats.The CHMODS rat model was made by injecting autologous blood into caudate nucleus,vagus nerve vagotomy was used to block the vagus nerve in CHMODS rats.Berberine and the electroacupuncture of Zusanli and concha area were given daily after the operation.2.Observe the symptoms and signs and detect the commom blood?function of liver and kidney?cardiac enzyme and endotoxin at each time point after the operation.Pathological changes of lung?heart?liver?intestines and kidney were recorded and pictures were taken through optical microscope.3.Determine the levels of the serum endotoxin by the application of modified matrix coloration method;Detect the protein and mRNA expression of CD14?TLR4?NF-?B P65 by using immunohistochemical and RT-PCR method;Detect the expression of TNF-? and Ach by the application of ELISA method;Detect the protein expression of a7nAchR by using immunohistochemical method;Observe the pathological changes of surrounding organs by using HE staining method;Evaluate the neural function of rats by the use of longa method and do statistical analysis about mortality rate in every group,analyze the incidence and mortality of SIRS in each group statistically.4.The mean and standard deviation with the experimental data(x±s),apply SPSS16.0 statistical software for statistical analysis,the data showed significant differences between the test using t test,analysis of variance,P<0.05 had significant difference.Results1.There were various degrees of inflammatory damage in each organ of rats in each points after cerebral hemorrhage.Compared with normal control group,there was a slight nonspecific inflammation in the lungs and liver occasionally in sham operation group.In MODS group,Lung,small intestine,liver,kidney and heart had obvious nonspecific inflammatory lesion in 24h,remained at a high level in 72h,damage mitigation and close to normal in 7d-14d.Inflammatory damage by tissue was lighter than MODS group in MODS +electroacupuncture group and MODS+ berberine group and higher than MODS +electroacupuncture + berberine group in 24h-72h,close to normal in 7d-14d.Inflammatory damage was at peak in MODS + electroacupuncture+ berberine group in 24h,more serious than normal control group and sham operation group in 24h,significantly reduced in 72h,and close to normal in 7d-14d.Inflammatory damage was more serious than MODS group in MODS+SDV group in 24h-72h,still obvious inflammatory reaction in 7d-14d.Inflammatory damage of small intestine,liver and kidney was the same as MODS+SDV group in MODS+ SDV+ electroacupuncture group?MODS+ SDV+ berberine group?MODS+SDV+ electroacupuncture + berberine group.Inflammatory damage of heart and lung was the same as MODS+SDV group in MODS+ electroacupuncture group?MODS+berberine group?MODS+ electroacupuncture + berberine group.2.Compared with normal control group,WBC?ALT?AST?Cr?BUN?CK in sham operation group was no significant difference(P>0.05);each index above in other groups was significantly higher than those in sham operation group and normal control group(P<0.05).Those index in MODS+ acupuncture group and MODS+berberine group was significantly lower than those in normal control group and sham-operative group.Those index in MODS+ acupuncture+ berberine group was significantly lower than those in MODS+ acupuncture group and MODS+berberine group in 14d?WBC?ALT?AST?Cr and BUN in MODS+SDV group,MODS+SDV+acupuncture group,MODS+ SDV+ berberine group and MODS+ SDV+ acupuncture+berberine group was significantly higher than those in MODS group,MODS+acupuncture group,MODS+ berberine group and MODS+ acupuncture+ berberine group at each phase point(P<0.05);there was no significant difference in CK(P>0.05)?there was no significant difference in WBC?ALT?AST?Cr and BUN in MODS+ SDV+ acupuncture group,MODS+ SDV+ berberine group and MODS+ SDV+acupuncture+ berberine group compared with MODS+ SDV group(P>0.05),CK was lower significantly at 14d.3.Compared with normal control group,endotoxin content in Sham operation group was no significant difference(P>0.05).Endotoxin Level was at peak in MODS group in 24h,and still high level in 72h and 7d,normal in 14d.The lever in MODS + electroacupuncture group and MODS+ berberine group was lower than that in MODS group(P<0.05),higher than MODS + electroacupuncture+ berberine group(P<0.05),and different with normal control groupand Sham operation group(P<0.05),normal in 14d.MODS + electroacupuncture+ berberine group was higher than normal control group and sham operation group(P<0.05)and lower than MODS + electroacupuncture group and MODS+ berberine group(P<0.05),normal in 7d.MODS+SDV group was higher than MODS group,normal control group and sham operation group in each point(P<0.05).MODS+ SDV+ electroacupuncture group,MODS+ SDV+ berberine group and MODS+ SDV+ electroacupuncture+berberine group was the same as the MODS+SDV group in each point(P>0.05).4.Western blot results:compared with normal control group,there was no significant difference in the expression of CD14,TLR4 and nfkbp65 protein in lung,small intestine,liver,kidney and heart in sham operation group(P>0.05).The expression of CD14,TLR4 and nfkbp65 in each organ of MODS group was at peak and higher than that of normal control group and sham operation group in 24h(P<0.05),decreased at 72h,reach to normal at 14d.The expression of each protein in MODS+ electroacupuncture group?MODS+ berberine group was less than MODS group at 7d.The expression of each protein in MODS+electroacupuncture group + berberine group was lower than that in MODS group at 72h(P<0.05),reach to normal at 7d.The expression of CD14,TLR4 and nfkbp65 in small intestine,liver and kidney tissues in MODS+SDV group were higher than those in MODS group in 24h-72h(P<0.05),still higher than normal group in 7d and 14d;there was no difference from MODS group in the expression level of each protein in heart and lung tissue at different time points(P>0.05).The changes of CD14,TLR4 and nfkbp65 protein expression in small intestine,liver and kidney were the same as MODS+SDV group in MODS+ SDV+ electroacupuncture group?MODS+ SDV+ berberine group?MODS+ SDV+ electroacupuncture + berberine group(P>0.05);the changes in heart and kidney was the same as MODS+electroacupuncture group?MODS+berberine group in MODS+ SDV+electroacupuncture group?MODS+ SDV+ berberine group(P>0.05);the changes in heart and kidney was the same as MODS+ electroacupuncture +berberine group in MODS+ SDV+ electroacupuncture + berberine group(P>0.05).5.The change of CD14,TLR4 and nfkbp65 mRNA in each organ was the same as the results of protein about these.6.TNF-? results:compared with normal control group,there was no significant difference in the expression of TNF-? protein in lung,small intestine,liver,kidney and heart in sham operation group(P>0.05).The expression of TNF-? in each organ of MODS group was at peak and higher than that of normal control group and sham operation group in 24h(P<0.05),decreased at 72h,kidney and heart were reach to normal,there was no significant difference with normal and sham groups in lung,small intestine and liver in 14d.The expression of TNF-a in MODS+ electroacupuncture group?MODS+ berberine group was lower than that in MODS group at 72h(P<0.05),reach to normal at 7d.The expression of TNF-a in small intestine,liver and kidney tissues in MODS+SDV group were higher than those in MODS group in 24h-72h(P<0.05),still higher than normal group in 7d and 14d;there was no difference from MODS group in the expression level of each protein in heart and lung tissue at different time points(P>0.05).The changes of TNF-a expression in small intestine,liver and kidney were the same as MODS+SDV group in MODS+ SDV+electroacupuncture group?MODS+ SDV+ berberine group?MODS+ SDV+electroacupuncture + berberine group(P>0.05);the changes in heart and kidney was the same as MODS+ electroacupuncture group?MODS+berberine group in MODS+SDV+ electroacupuncture group?MODS+ SDV+ berberine group(P>0.05);the changes in heart and kidney was the same as MODS+ electroacupuncture+berberine group in MODS+ SDV+ electroacupuncture + berberine group(P>0.05).7.Ach results:compared with normal control group,there was no significant difference in the expression of Ach in lung,small intestine,liver,kidney and heart in sham operation group(P>0.05).The expression of Ach in each organ of MODS group was at peak and higher than that of normal control group and sham operation group in 72h(P<0.05),reach to normal at 7d and 14d.The expression of Ach in MODS+ electroacupuncture group?MODS+berberine group was higher than that in MODS group at 72h(P<0.05)and lower than that in MODS+ berberine +electroacupuncture group,higher than normal at 7d and 14d.The expression of Ach in MODS+ berberine +electroacupuncture group was higher than MODS+ electroacupuncture group and MODS+ berberine group at 24h(P<0.05),higher than normal at 7d and 14d(P<0.05).The expression of Ach in small intestine,liver and kidney tissues in MODS+SDV group were lower than those in MODS group in 24h-14d(P<0.05);the expression of Ach in lung and heart was the same as MODS group at each point(P>0.05)?The changes of Ach expression in small intestine,liver and kidney were the same as MODS+SDV group in MODS+ SDV+ electroacupuncture group?MODS+ SDV+ berberine group?MODS+ SDV+ electroacupuncture + berberine group(P>0.05);the changes in heart and kidney was the same as MODS+ electroacupuncture group,MODS+berberine group and MODS+ electroacupuncture + berberine group in MODS+ SDV + electroacupuncture group,MODS+ SDV+ berberine group and MODS+SDV+ electroacupuncture + berberine group(P>0.05).8.The change of ? 7nAChR in each organ in every group was the same as the results of Ach.9.Comparison of longa nerve function scores in each group:The Longa scores of the normal control group and the sham operation group were 0 points.Rats in MODS group,severe neurological deficits occurred after 24h,and gradually recovered to normal at 7d.24 hours after the operation,all the rats suffered from varying degrees of limb and limb dysfunction,and recovered normal after treatment in MODS+ electroacupuncture group,MODS+ berberine group and MODS+ electroacupuncture group+ berberine group.The functional changes of the acupuncture group at each time point were approximately the same,and they were all more serious than those in the MODS group,but recovered poorly after treatment in MODS+ SDV+ electroacupuncture group?MODS+ SDV+berberine group?MODS+ SDV+ electroacupuncture + berberine group.10.SIRS incidence and group mortality statistics:The incidence of SIRS in each group was 100%.The mortality rates of each group are as follows:MODS group 11.11%,MODS+ electroacupuncture group 7.69%,MODS+ berberine group 4.00%,MODS+ electroacupuncture group+ berberine group 4.00%;MODS+ SDV group 22.58%,MODS+ SDV+ electroacupuncture group 20.00%,MODS+ SDV+ berberine group 14.29%,MODS+ SDV+ electroacupuncture + berberine group 14.29%.11.Endotoxin,TNF-a,a7nAchR,CD14,TLR4,NF-kappa B P65mRNA and protein were positively correlated with plasma WBC,ALT,AST,Cr,BUN,CR and Longa grade;ach and a7nAChR was negatively correlated with WBC,ALT,AST,Cr,BUN,CR and Longa grade.Conclusion:1.VCAP has a protective effect on the surrounding organs of rat cerebral hemorrhage in CHMODS.2.It' s better using using electro-acupuncture and berberine together.It can better to activate vagal cholinergic anti-inflammatory pathway,reduce inflammation,increase the expression of Ach and a7nAChR,reduce inflammation and damage to the surrounding organs and improving CHMODS prognosis.