The Protective Effect And Mechanism Of Adipose-derived Mesenchymal Stem Cells On Acute Lung Injury Induced By Septic Rats

Sepsis should be defined as life-threatening organ dysfunction caused by a dysregulated host response to infection,and it is a common acute and serious disease clinically,which affecting millions of people around the world every year,and even up to a quarter of people die.Sepsis often causes acute lung injury(ALI)/acute respiratory distress syndrome(ARDS).ALI/ARDS is a common acute hypoxic respiratory failure with diffuse inflammatory injury of bilateral lung.Its pathological changes include gas exchange disorder,large infiltration of neutrophils,increased vascular permeability and lung parenchyma injury.The mortality rate remains high.Although some progress has been made in the treatment,the proportion of death in septic patients is still high.Therefore,it is necessary and urgent to develop a new treatment in clinical practice.Stem cells,especially adipose-derived mesenchymal stem cells(ADMSCs)could alleviate inflammation and oxidative stress,and could be used to treat diseases related to inflammation and tissue damage.Previous animal studies have found the therapeutic effect of mesenchymal stem cells(MSCs)in sepsis,however this conclusion is still controversial.In addition,three small-scale clinical phase I trials have proved the safety of MSCs in the treatment of sepsis currently.However there are few reports about MSCs in the treatment of septic ALI/ARDS,and thus we will make a specific discussion from four parts,in order to provide objective basic theory and strong evidence-based medicine evidence for MSCs in the treatment of sepsis.Part Ⅰ:The efficacy of MSCs therapy for sepsis:a meta-analysis of animal studiesObjectivePrevious animal studies have found that MSCs could reduce the mortality of sepsis,however this conclusion remains controversial.This section will systematically evaluate the efficacy of MSCs in the treatment of septic animals by meta-analysis.MethodsMedline,EMBASE,Cochrane CENTRAL,and Web of Science databases from inception to November 17,2019 were searched.According to the inclusion and exclusion criteria,we screened and evaluated the literature,and analyzed the efficacy of MSCs therapy on septic animals.The primary outcome was mortality.Two researchers independently screened the study,extracted data and assessed the risk of bias.The Inverse-Variance method with fixed-effect modeling was used to calculate pooled odds ratio(OR)and 95%confidence interval(CI).The quality of outcomes was evaluated using SYstematic Review Centre for Laboratory animal Experimentation(SYRCLE)bias risk tool.The heterogeneity between studies was evaluated by I2 and P values,the robustness of outcome was evaluated by sensitivity analysis,the publication bias was evaluated by Begg funnel and egger linear regression.Statistical analysis was performed with STATA 14.0.Results1.29 animal studies were finally included,totaling 1266 animals that including rats and mice in this meta-analysis.Meta-analysis showed that MSCs significantly reduced the mortality of sepsis(OR 0.29,95%CI 0.22-0.38,P<0.001),the heterogeneity between studies were low(I2=14.5%,P=0.248).Sensitivity analysis showed that the results were robust.The risk of bias was not clear.2.The animal species and model,MSCs source,administration dose,time and route after septic model constructed were used for subgroup analysis.The results as follows,administrated with MSCs significantly reduced the mortality of septic rats after septic model constructed in animal species(OR 0.39,95%CI 0.25-0.60,P<0.001);MSCs significantly reduced the mortality of sepsis induced by CLP in animal model(cecal puncture and ligation)(OR 0.29,95%CI 0.20-0.42,P<0.001);ADMSCs significantly reduced the mortality of sepsis(OR 0.43,95%CI 0.24-0.77,P<0.001);administrated with 1×106 MSCs after septic model constructed have the best effect on reducing the mortality of sepsis in MSCs administration dose(OR 0.24,95%CI 0.16-0.35,P<0.001);MSCs was administrated at 1 hour after septic model constructed have the best effect on reducing the mortality of sepsis in MSCs administration time(OR 0.28,95%CI 0.17-0.46,P<0.001);MSCs were injected by vein have the best effect on reducing the mortality of sepsis in MSCs administration route(OR 0.28,95%CI 0.21-0.38,P<0.001).SummaryMSCs treatment significantly reduced the mortality of sepsis,and intravenous administrated with 1×106 ADMSCs at one hour after CLP could reduce the mortality of septic rats.Therefore,the next step is necessary to explore the relevant mechanism of MSCs therapy for sepsis,so as to provide strong evidence-based medical evidence for future clinical trials of MSCs treatment in sepsis.Part Ⅱ:The construction of CLP-induced septic rats model and the protective effect and mechanism of sTNFR1 on ALI for this modelObjectiveTo investigate whether ADMSCs could secrete a large number of soluble tumor necrosis factor receptor 1(sTNFR1)to improve septic ALI.MethodsA total of 120 male adult Sprague-Dawley(SD)rats were separated into four groups:the sham control(SC),sepsis induced by CLP,CLP-ADMSCs and CLP-sTNFR1 siRNA groups.CLP-sTNFR1 siRNA groups and CLP-ADMSCs groups underwent CLP and then received 1×106 ADMSCs with and without knockdown of sTNFR1 via caudal vein injection at 1 h after CLP respectively.Rats were sacrificed at 3,6,24 and 48 h after the SC or CLP procedures.The levels of sTNFR1,tumor necrosis factor alpha(TNF-α),interleukin(IL)-6 and IL-10 in serum were detected by enzyme-linked immunosorbent assay(ELISA).The lung tissues were taken from the chest,and the pulmonary edema was measured by weight ratio of wet to dry(W/D).The pathological changes of lung tissues were detected by hematoxylin eosin(HE)staining.The changes in apoptosis of lung tissues were detected by TUNEL method.The protein expression level of TNF-α,nuclear factor kappa B(NF-κB),activator protein-1(AP-1)and p38 mitogen activated protein kinase(p38 MAPK)were detected by Western Blot.The mRNA level of TNF-α,NF-κB and AP-1 were detected by quantitative real-time polymerase chain reaction(RT-qPCR).Results5-ethylyl-2 deoxyuridine(EdU)-labeled ADMSCs extensively colonized the lungs at 6,24 and 72 h after ADMSCs injection.The inflammatory infiltration,edema and hemorrhage,the proportion of apoptotic cells of lung tissue were increased in the CLP group,however ADMSCs treatment improved these abnormalities,but this effect could be weakened by sTNFR1 siRNA treatment.The lung W/D weight ratios in the CLP group were higher than those in SC group,however ADMSCs ameliorated the W/D following CLP,and this effect was abolished by sTNFR1 siRNA treatment(all P<0.05).The levels of serum sTNFR1 and IL-10 were higher in the CLP-ADMSCs group and lower in the SC group than in other groups(all P<0.05);interestingly,these levels were higher in CLP and CLP-sTNFRl siRNA groups than in SC group(all P<0.05).Compared with SC group,TNF-α and IL-6 levels of CLP group increased significantly,and ADMSCs could alleviate these changes,but the effect was weakened by sTNFR1 siRNA treatment(all P<0.05).The result of Western Blot and RT-qPCR of lung showed that the expression of TNF-α,NF-κB,and AP-1 in CLP group was higher than that in SC group;ADMSCs treatment could reverse the change of CLP induced high pro-inflammatory factor expression,and which was eliminated by sTNFR1 siRNA treatment.Besides,the lung cell apoptosis and edema levels were consistent with IL-6 levels among all groups.SummaryADMSCs could reduce inflammation and ALI by secreting sTNFRl,which provides a potential mechanism for MSCs to treat sepsis.Part Ⅲ:The metabonomics research of ADMSCs in the treatment of CLP-induced septic ALI ratsObjectiveTo explore the therapeutic mechanism of ADMSCs on CLP-induced septic ALI rats by means of metabonomics.MethodsA total of 60 male adult SD rats were separated into three groups:SC,sepsis induced by CLP,and CLP-ADMSCs groups.CLP-ADMSCs groups underwent CLP and then received 1×106 ADMSCs with intravenously at 1 h after surgery.Rats were sacrificed at 24 h after the SC or CLP procedures.The histological evaluation of lungs was performed using HE,the serum levels of proinflammatory factors detected by ELIS A,lung and plasma were collected and analyzed by metabonomics analysis.Principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(PLS-DA)were used for discriminant analysis,and combine univariate statistical analysis to screen important differential metabolites.ResultsThe levels of TNF-α and IL-6 were significantly increased after CLP,but ADMSCs could significantly reduce the levels of these inflammatory factors(all P<0.05).The histological changes of lung were consistent with the level of IL-6.There were significant metabolic profile changes in plasma and lung tissue of septic rats at 24 hours after CLP compared with SC,which could be reversed by ADMSCs.22 metabolites were found in plasma,namely Creatinine,Betaine,L-Threonine,Niacinamide,Thymine,Creatine,Homocysteine,Acetylcholine,L-Methionine,Dopamine,L-Histidine,L-Phenylalanine,Pyridoxine,L-Tyrosine,Spermine,Deoxycytidine,Cytidine,Xanthosine,lysoPC(20:4),lysoPC(22:6),PC(38:6),Phenylpyruvic acid.11 metabolites were found in lung,namely L-Valine,Creatine,Acetylcholine,Glutamate,Xanthine,Tryptophan,Cytidine,lysoPC(18:3),lysoPC(22:4),PC(36:4),CDCA.We constructed the related metabolic network by the biological analysis of these biomarkers,and the retrieval of metabonomics related databases and literature,which suggest that septic rats mainly cause the abnormalities of amino acid metabolism,glycerophospholipids metabolism and other metabolic pathways,and which are closely related to energy consumption,inflammatory response,oxidative stress and so on,however ADMSCs could reverse above changes.SummaryIn septic rats,the metabolic pathways of amino acids and glycerophospholipids are disordered,however ADMSCs could ameliorate ALI by reversing this change.Part IV:The intestinal microecological research of ADMSCs in the treatment of CLP-induced septic ALI ratsObjectiveTo explore the therapeutic mechanism of ADMSCs on CLP-induced septic ALI rats by means of 16S rDNA.MethodsA total of 60 male adult SD rats were separated into three groups:SC,sepsis induced by CLP and CLP-ADMSCs;CLP-ADMSCs groups underwent CLP and then received 1×106 ADMSCs.Rats were sacrificed at 24 h after the SC or CLP procedures.To detect the effect of gut microbiota on septic ALI,the histological evaluation of lungs was performed using H&E staining,the serum levels of proinflammatory factors were detected by ELISA,and intestinal fecal samples were collected and analyzed by 16S rDNA sequencing,respectively.ResultsThe levels of TNF-α and IL-6 were significantly increased in CLP-induced septic rats,but ADMSCs could significantly reduce the levels of these inflammatory factors(all P<0.05).The histological changes of lung were consistent with the level of IL-6.ADMSCs significantly increased the diversity of gut microbiota in septic rats.The results of principal coordinate analysis(PCoA)showed that there was significant difference in gut microbiota between CLP-ADMSCs group and CLP group(P=0.001).In addition,the result of PCA were consistent with PCoA analysis(P=0.001).In septic rats,the proportion of Bacteroides related to energy consumption and Escherichia-Shigella related to lipopolysaccharide(LPS)production was increased;the proportion of Akkermansia related to the regulation of intestinal mucosal thickness and the maintenance of intestinal barrier function was decreased.The proportion of Firmicutes related to energy storage,such as Verrucomicrobia,was decreased.These changes of gut microbiota break the energy balance,aggravate the inflammatory reaction,cause intestinal barrier dysfunction and promote the translocation of intestinal bacteria.ADMSCs intervention could reverse the above changes of bacterial structure in septic rats,that is,increase beneficial bacteria,reduce the proportion of harmful bacteria,normalize the intestinal bacterial structure,and thus improve the septic ALI.SummaryADMSCs may improve ALI of septic rats by regulating gut microbiota.ConclusionsWe first evaluated and determined that MSCs could significantly reduce the mortality of septic animals that including rats and mice,and ADMSCs could reduce the mortality of septic rats.Secondly,CLP was used to establish septic rats model for basic research.We found that ADMSCs could block TNF-α effect by secreting sTNFR1 competitive binding TNF-α,so as to reduce NF-κB,AP-1,p38 MAPK and other factors,and ultimately reduce the inflammatory response.The main metabolic pathways of septic rats are amino acid and glycerophospholipid.The pathway is closely related to energy consumption,inflammatory reaction,oxidative stress,etc.The proportion of Bacteroides related to energy consumption and pathogenic bacteria related to LPS production in the intestine of septic rats was increased.The proportion of Ackermann bacteria related to the regulation of the thickness of intestinal mucosa and the maintenance of intestinal barrier function,and the thick walled bacteria related to energy storage,such as verruca Micrococcus,was increased.These changes in bacterial structure break the energy balance,aggravate the inflammatory response,cause intestinal barrier dysfunction and promote intestinal bacterial translocation,while ADMSCs could reverse the metabolic pathway abnormality and gut microbiota disorder of septic rats.In conclusion,ADMSCs could improve septic ALI through the above approaches.This study provides basic theoretical support for further research on the treatment of sepsis from the perspective of stem cell paracrine,the identification of early severity of septic ALI in clinical practice,and also provides strong evidence-based medicine evidence for the clinical trial of stem cell treatment in sepsis.It also has important reference significance for the study of sepsis from the perspective of regulating intestinal microecology.