Role Of The Cholinergic Anti-inflammatory Pathway In The Protective Effect Of Electroacupuncture At Zusanlipoint(ST36)on Sepsis-induced Acute Lung Injury In Rats

Acute Lung Injury(ALI)is a common syndrome,which can induce Acute Respiratory Distress Syndrome(ARDS)in critical ill patients.Sepsis is one of the leading causes of ALI.ALI is a big challenge in critical care medicine for the high incidence rates and mortality rates.So far,only Lung Protetive Ventilation Strategy(LPVS)and Retrict Liquid Management are evidence-based practice that might be associated with prognosis improvement of ALI.More effective therapy for ALI is still urgently needed.Over the long history of human society,infectious diseases are the leading cause of fight with illness.Since thousands of years ago,Traditional Chinese medicine has been used for sepsis in China.Mutiple internal and external treatments have been developed for treating sepsis-induced ALI.According to review of studies on treating sepsis-induced ALI,electroacupuncture at Zusanli(ST36)might have protection effect.By systematic review of acupuncture at ST36 in treating sepsis-indeced ALI in animal models,we evaluated the efficacy and mechanisms of it,helping to identify potential candidates for further basic research and refine animal experimentation.According to previous studies,electroacupuncture at ST36 can activate the cholinergic anti-inflammatory pathway(CAP)to protect sepsis animals.But the mechanism of it on sepsis-induced ALI is still unclear and worth study.Objective1.Systematically review the evidence of acupuncture at ST36 for treating sepsis-induced ALI,summarizing characteristics of the studies and deriving refined design for further experimental research.2.Based on results of systematic review,the role of CAP in the protection effect of electroacupunture at ST36 on sepsis-induced ALI should be evaluated to provide a scientific basis for acupuncture therapy in sepsis-induced ALI.Methods1.We identified studies of acupuncture at ST36 in sepsis-induced ALI animal models from Pubmed,the Cochrane Central Register of Controlled Trials(CENTRAL)in The Cochrane Library,EMBASE,Chinese National Knowledge Infrastructure(CNKI),VIP Database,Wanfang Data,and Chinese Biomedical Literature Database(CBM)in English or in Chinese from the inception of each database up to May 31st 2019.By systematic reviewing the included studies,we summarized the results of the studies,analized the affecting factors of the results,and derived the refined design for our experimental research.2.We developed sepsis-induced ALI model in rats by intratracheal LPS instillation.In survival study,60 rats were randomly divided into 4 groups(n=15 in each group):sham group(SG),LPS+electroacupuncture group(EAG),LPS+anesthetic group(AG)and LPS+no anesthetic(NAG)group.Sepsis-induced ALI was performed by LPS instillation in EAG,AG and NAG,normal saline was instilled intratracheally instead in SG.Electroacupunture at ST36 was performed once a day for 3 days,30min per time(30min after model induction for the first time)in EAG.Anesthetic was given to AG whenever EAG received electroacupunture.No further intervention for NAG since model induction.The survival rates were evaluated daily for the 7-day period.3.We developed sepsis-induced ALI model in rats by intratracheal LPS instillation.In mechanism study,35 rats were randomly divided into 7 groups(n=5 in each group):sham group(SG),model group(MG),electroacupuncture group(EAG),PNU group(PG),MLA group(MG),PNU+electroacupuncture group(PEAG)and MLA+electroacupuncture group(MEAG).Normal saline was instilled intratracheally instead of LPS in SG.Electroacupunture at ST36 was performed once a day for 2 days,30min per time(30min after model induction for the first time)in EAG,PEAG and MEAG.PNU-282987(a a 7nAchR agonist)and Methyllycaconitine(a α 7nAchR antagonist)were given to PG,PEAG,MG and MEAG intraperitoneally respectively 30min after model induction.Tissue and samples were collected 24h after model induction.BALF were collected,centrifugated,the liquid supernatant was used for protein concentration dectection.The cell precipitation was re-suspended for counting white blood cells and neutropils.Lung tissues were collected for weighing and drying to caculate lung wet-to-dry ratios.Serum were isolated from blood samples to determine the protein concentration and the levels of IL-6 and IL-10.Pathological changes of lung tissue were observed by HE staining and scores were caculated.Western Blot was used to detect the protein level of NF-κB and α 7nAChR.Activity of ChAT and AChE of lung tissue were also examined.Results1.Results of systematic review:We retrieved 4417 studies from the 7 databases,from which we indentified 19 articles by inclusion and exclusion criteria.(1)Most of the studies(about 63%)were beyond the last five years.The efficacy of acupuncture at ST36 were not well evaluated enough.(2)Only 4 studies were using ST36 as monotherapy,while the rest were all using multiple acupoints for intervention.The efficacy of ST36 was not very clear.(3)89.5%of the 19 studies utilized pretreatment,while the rest 10.5%performed acupuncture right after model induction.Both were not comparable in clinical practice.(4)Research on mechanism were not thorough enough,there are still potential mechanism worthy exploration according to the literatures.(5)Quality of studies are poor.Publication Bias exists.Based on the results of the systematic review,we approve it is worthy of further experimental research on effects and mechanism of electroacupunture at ST36 on sepsis-induced ALI with more robust model and thorough evaluation.CAP might be a promising mechanism.2.Results of animal experiment:(1)Survival study:electroacupunture at ST36 improved survival rates of sepsis-induced ALI rats within 7 days,no matter it was compared with AG or NAG(P<0.05).(2)Physiological scores of lung tissue:compared with SG,the physiological scores of lung tissue in MG were significantly higher(P<0.01).Compared with MG,the physiological scores in EAG,PG and PEAG were significantly decreased.(P<0.01).(3)Wet-to-dry ratios of lung tissue:compared with SG,the wet-to-dry ratios of lung tissue in MG were significantly higher(P<0.01).Compared with MG,the wet-to-dry ratios of lung tissue in EAG were significantly decreased(P<0.05),while differences in the other groups were not statistically significant(P>0.05).(4)BALF cell counts:compared with SG,the count of white blood cells(WBC)and neutrophil(NEU)in BALF of MG were significantly higher(WBC,P<0.05;NEU,P<0.01).Compared with MG,the cell counts of WBC and NEU were relatively lower in EAG,but the differences were not statistically significantly(P>0.05).(5)Protein leakage of lung capillaries:compared with SG,the protein leakage of lung capillaries of MG were significantly higher(P<0.01).Compared with MG,the protein leakage of lung capillary were significantly lower in EAG(P<0.05),while differences in the other groups were not statistically significant(P>0.05).(6)Serum IL-6 and IL-10 levels:compared with SG,the levels of serum IL-6 and IL-10 were significantly higher(P<0.01).Compared with MG,the serum IL-6 levels were significantly lower in EAG(P<0.05),while differences in the other groups were not statistically significant(P>0.05).(7)Activity of ChAT in lung tissue:compared with SG,activity of ChAT in lung tissue was significantly lower(P<0.01).Compared with MG,the activity of ChAT in lung tissue was significantly lower in EAG(P<0.05).(8)Activity of AChE in lung tissue:compared with SG,activity of AChE in lung tissue was significantly lower(P<0.01).Compared with MG,the activity of AChE in lung tissue was relatively higher,but the difference was not statistically significant(P>0.05).(9)The protein expression level of NF-κB in lung tissue:compared with SG,the protein expression level of NF-κB in lung tissue was significantly higher P<0.05).Compared with MG,the protein expression level of NF-κB in lung tissue was significantly lower(P<0.05),while differences in the other groups were not statistically significant(P>0.05).(10)The protein expression level of a 7nAChR in lung tissue:there were no statistically signif icant differences in SG,MG and EAG(P>0.05),while the rest 4 groups were significant higher(P<0.05).Conclusion1.This is the first systematic review of acupuncture at ST36 for sepsis-induced ALI animal model of literatures in both English and Chinese.The present study indicated that acupuncture at ST36 might be useful in reducing sepsis-induce ALI,while the mechanism of it still need to be explored.CAP might be a potential mechanism.2.This study utilized LPS instilled intratracheally to induce sepsis-induced ALI rats,results revealed model successfully established.Compared with the model group,electroacupuncture at ST36 showed protective effect in reducing sepsis-induced ALI within 24h and improving survival rates in 7 days.3.By investigating levels of key factors in CAP,this study revealed that electroacupuncture at ST36 increased activity of ChAT in lung tissue,together with decreasing protein expression level of NF-κB and serum IL-6 level.Comparing with the utilization of α 7nAChR agonist and antagonist,this study indicated that electroacupuncture at ST36 doesn’t affect the expression ofα 7nAChR or the combination between α 7nAChR and its ligand,the mechanism mainly reled on stimulation on production and release of neurotransmitter.