Study On The Mechanism Of Immune Dysfunction In Sepsis Based On PD-1 Pathway And The Intervention Of Electroacupuncture

ObjectiveThis study includes two parts:clinical research and basic research.In this study we observed the mechanism of programmed cell death protein-1(PD-1)and the downstream pathway in the development of immune dysfunction in sepsis,and electroacupuncture treatment could improve the immune dysfunction of sepsis by regulating the PD-1 pathway,achieved the effect of alleviating the IFNlammatory response and improving the clinical efficacy.Methods1.Clinical studyThe sepsis patients admitted to the department of Intensive Care Medicine of the guangdong provincial hospital of Chinese Medicine from July 2019 to January 2021 were randomly divided into Western medicine treatment plus electroacupuncture group(EA group)and the west medicine treatment group(WM group).The WM group was treated with western medicine,The EA group was treated with western medicine and electroacupuncture of Zusanli(ST36),Guanyuan(CV4)and Qihai(CV6).The levels of T lymphocyte subsets(CD3T+,CD4+T,CD8+T,CD4+T/CD8+T),B cells,NK cells,soluble programmed cell death protein-1(sPD-1),interferon(IFN)-γ,interleukin(IL)-2,IL-4 and IL-10 in blood of patients in each group were compared before and after treatment.Changes of The white blood cell(WBC)count,neutrophil ratio(NE%)and neutrophil count(NE),lymphocyte ratio(LY%)and lymphocyte count(LY),neutrophil/lymphocyte ratio(NLR),C-reactive protein(CRP),tumor necrosis factor(TNF)-α and Lactic acid of patients in each group were compared before and after treatment.Acute physiology and chronic health evaluation(APACHE)-Ⅱ,Sequential organ failure assessment(SOFA)and proportion of patients recovered by 28 days were used to observe the efficacy of electroacupuncture in improving the clinical symptoms and prognosis of sepsis patients.The safety of electroacupuncture was judged by observing the clinical symptoms related to electroacupuncture,blood routine examination,liver and kidney functions.The above indicators were analyzed using the conventional intention-to-treat(ITT)analysis.To increase the reliability of the results,important indicators such as immune function were analyzed using the per-protocol(PP)analysis.We observe the correlation between IFNlammatory factors and APACHE-Ⅱ score through correlation analysis.We detected the intestinal flora before and after treatment in the two groups.2.Animal experimental study45 healthy male mice were randomly divided into sepsis model group(CLP group),electroacupuncture group(EA group),inhibitor control group(YZJ group)and sham operation group(Sham group).Sepsis model was made by cecal ligation and perforation(CLP).Mice in EA group was given electroacupuncture at Zusanli(ST36),Guanyuan(CV4)and Qihai(CV6)two days before modeling,and the sepsis model was made according to CLP.After modeling,electroacupuncture treatment was continued.Mice in YZJ group administered intraperitoneal injection of PD-1 inhibitor(100ul/piece)two days before modeling,and made the sepsis model according to CLP.After modeling,YZJ group continued to be given with PD-1 inhibitor.Mice In Sham group and CLP,normal saline was injected intraperitoneally 2 days before modeling,and the model was made according to sham operation or CLP.After modeling,saline was continued to be injected intraperitoneally.All mice were harvested after 3 days.Detection of the contents of TNF-α,CRP and sPD-1 in peripheral blood by ELISA.We observed the histopathological changes of mouse spleeny Hematoxylin eosin(HE)staining.Flow cytometry was used to detect the changes of T lymphocyte subsets(CD3+T,CD4+T,CD8+T,CD4+T/CD8+T),NK cell levels,and the expression of PD-1 on T lymphocytes and NK cells.Immunohistochemistry(IHC)was used to detect the protein levels of PD-1,SHP2,PI3K and phospho-Akt(pAkt)in mouse spleen tissues.The intestinal flora of mice in each group was detected by 16s rDNA method.Results1.Clinical study(1)Baseline comparisonA total of 64 patients,31 in EA group and 33 in the WM group.There were 4 patients who fell out.The data of the fallen out cases were supplemented with the single filling method.There was no statistical difference between the two groups in terms of gender,age,basic disease,primary IFNection site,damaged organs,APACHE-Ⅱ score,SOFA score,etc.(P>0.05).The two groups were comparable.(2)Clinical effect of electroacupunctureBefore treatment,there was no statistical difference in WBC,NE%,NE,LY%,LY and NLR between the two groups(P>0.05).Five days after treatment,WBC,NE%,NE,LY%and NLR of patients in the electroacupuncture group were significantly improved compared with WM group(P<0.05&P<0.01).The levels of NE,NE,LY%,LY and NLR of patients in EA group and NE%,NE and LY of patients in the WM group were improved compared with those before treatment(P<0.05&P<0.01).CRP,TNF-α,PCT and lactic acid levels of two groups of patients before treatment had no statistical difference(P>0.05).Five days after treatment,The levels of TNF-α of EA group and CRP,PCT and lactic acid of two groups decreased(P<0.01).The levelof CRP、TNF-α and lactic acid of patients in EA group was lower than those of WM group(P<0.05).Before treatment,there was no significant difference in APACHE-Ⅱ score and SOFA score between EA group and WM group(P>0.05).After five days of treatment,APACHE-Ⅱ score and SOFA score of EA group and WM group decreased significantly compared with those before treatment(P<0.01).The APACHE-Ⅱ score of EA group was lower than that of WM group(P<0.05).(3)Effect of electroacupuncture on immune function①Effect of electroacupuncture on T lymphocytes,NK cells and B lymphocytes in sepsis patientsThe levels of CD3+T,CD4+T,CD8+T,ratio of CD4+T/CD8+T,NK cells and B lymphocytes of patients in the two groups had no statistical difference before treatment(P>0.05).Five days after treatment,the levels of CD4+T,CD4+T/CD8+T ratio in the EA group were higher than those before treatment(P<0.01).The levels of CD3+T,CD4+T,CD4+T/CD8+T and NK cells of patients in the EA group were significantly higher than those in the WM group(P<0.05).The level of NK cells and B lymphocytes of patients in WM group decreased(P＜0.05).There was no significant change in B lymphocytes in EA group(P>0.05).② Effect of electroacupuncture on immune related cytokinesBefore treatment,the level of sPD-1,IFN-y,IL-2,IL-4,IL-10 and IFN-y/IL-4 of patients in EA group and WM group had no statistical difference(P＞0.05).Five days after treatment,the level of sPD-1 in EA group was significantly lower than that before treatment(P<0.05),and it is obviously lower than WM group(P<0.05).The level of IFN-γ and IFN-γ/IL-4 of EA group was significantly higher than those of WM group(P<0.05).In PP analysis,the level of IL-2 of patients in EA group was higher than that in WM group(P<0.05).However,The statistical difference of IL-2 level was not found in ITT analysis(P>0.05).There was no significant difference in IL-4 and IL-10 levels between EA group and WM group(P>0.05).(4)Effect of electroacupuncture on intestinal floraThere was no statistical difference in the richness index and diversity index between the two groups before treatment(P>0.05).The richness index(Observed index,Chao1 index)of EA group after treatment were higher than those before treatment((P＜0.05).After treatment,the levels of Firmicutes,Lactobacillus and Bacilli in EA group were higher than those in WM group(P<0.05).Proteobacteria,Enterobacteriaceae,Fusobacteriota and Staphylococcus decreased compared with WM group,but there is no statistical difference(P>0.05).(5)Correlation of indicatorssPD-1,NLR,CRP,lactic acid,TNF-α were positively correlated with APACHE-Ⅱscore.The level of IFN-γ was negatively correlated with APACHE-Ⅱ score.(6)28 day outcomeAfter 28 days of follow-up,the improvement rate was 67.7%and 60.6%(P>0.05).(7)Safety of electroacupunctureIn terms of safety,all patients involved did not suffer from needle fainting,needle bending,needle breakage,etc.There was no difference in hemoglobin,platelet,transaminase,creatinine and other indicators between the two groups after treatment(P>0.05).2.Animal experimental research(1)General situation after mold makingAfter CLP,the vital signs of mice in each group were unstable,the respiration was shallow and fast,and the response to external stimuli was reduced.The survival rate of CLP group was 53.8%,indicating that the CLP model was successful.(2)Immune and inflammatory factorsCompared with CLP group,the levels of TNF-α and sPD-1 in EA group and YZJ decreased,and the level of IFN-γ increased(P<0.05).(3)HE stainingHE staining showed that the spleen structure of mice in the sham operation group was normal.The spleen structure of mice in CLP group was scattered,the number of lymphocytes decreased.In EA group and YZJ group,the lymphocytes were slightly dense,with a small amount of chromatin condensed and deeply stained lymphocytes piled up,and the structure is similar to the sham operation group.(4)T lymphocyte subsets and NK cells levelCompared with Sham group,the levels of CD4+T,CD4+T/CD8+T and NK cells of mice in CLP group decreased(P<0.05).Compared with CLP group,the levels of CD3+T,CD4+T,CD4+T/CD8+T and NK cells of mice in EA group and YZJ group were significantly higher(P<0.05).(5)PD-1 expression on T lymphocytes and NK cellsCompared with Sham group,the PD-1 expression of CD3+T cells and NK cells in CLP group were significantly increased(P<0.05&P<0.01).Compared with CLP group,the expression of PD-1 of CD3+T cells and NK cells in EA group and YZJ group decreased significantly(P<0.05&P<0.01).(6)Protein expression of PD-l/SHO2/PI3K/pAktThe immunohistochemical results of spleen showed that the expression of PD-1 and SHP2 protein in CLP group was higher than that in Sham group,and the expression of PI3K and pAkt protein in CLP group were lower than that in Sham group.The expression of PD-1 and SHP2 protein of EA group and YZJ group were lower than those of CLP group,and the expression of PI3K and pAkt protein of EA group and YZJ group were higher than those of CLP group.(7)Intestinal floraThe results of intestinal flora showed that compared with Sham group,the Observed index and Chaol index in CLP group decreased(P<0.05),and the Observed index and Chaol index in the EA group increased(P<0.05)compared with CLP group.In terms of flora composition,the proportion of firmicutes in CLP was lower than those in Sham group,and the proportion of proteobacteria in CLP group was higher than those in Sham group.The flora composition of CLP was similar to those in YZJ group.Compared with CLP group,the proportion of firmicutes increased and proteobacteria decreased in EA group,and the flora composition was similar to those of Sham group.In terms of species differences in microbiota among groups,compared with Sham group,the levels of Proteobacteria,γ-proteobacteria and Enterobacteriaceae of CLP group were increased(P<0.01),while the levels of Bifidobacterium,Coriobacteria and Saccharomycetes were decreased(P<0.05).Compared with CLP group,the levels of Lactobacillales,Bifidobacteriales,Coriobacteriia and Bacilli were increased in EA group,there is statistical difference between Coriobasteriia and Bacilli(P<0.05).While the Proteobacteria y-proteobacteria,Enterobacteria levels of EA group decreased(P<0.05 or P<0.01).Biological correlation showed that the most characteristic predominant flora of sham and EA groups was lactobacillus,CLP group were Enterobacteriaceae and Proteobacteria,and YZJ group was Corynebacterium.From the species evolution diagram of the flora,it was seen that the intestinal flora of the mice in the EA group represented the microbiota that differed more from that of CLP group.ConclusionWhen sepsis occurred,PD-1 expression of T lymphocytes and NK cells is upregulated.The above conditions lead to changes in the PD-1/SHP2/PI3K/Akt signaling pathway,inducing apoptosis and functional inhibition of T lymphocytes and NK cells,resulting in the development of immune dysfunction in sepsis.It showed a decline in CD3+T,CD4+T,CD8+T;CD4+T/CD8+T ratios,NK cells.However,electroacupuncture at Zusanli(ST36),Guanyuan(CV4),and Qihai(CV6)improved immune dysfunction in sepsis by downregulating PD-1 expression on the cell surface,regulating immune inflammatory factors,inhibiting apoptosis of T lymphocytes and NK cells,and restoring their function.It showed an improved proportion of T subset lymphocytes and NK cells.Through the improvement of immune function,electroacupuncture treatment could reduce the levels of white blood cells,neutrophils,CRP and so on,to achieve the effect of reducing the inflammatory response and improving the clinical symptoms.However,for the mechanism of down-regulation of PD-1 expression by electroacupuncture,it may be achieved by regulating intestinal flora.Moreover,the results of clinical study suggestted that electroacupuncture is safe in treating immune dysfunction in sepsis.sPD-1,IFN-γ may have some value in the evaluation of clinical efficacy.