Study On The Related Mechanism Of Stenotrophomonas Maltophilia OmpA-induced Cell Apoptosis And Inflammatory Response

Chapter 1:Related-study on adhesion and cytotoxicity of S.maltophilia outer membrane protein A to cellsObjective:To explore whether the S.maltophilia OmpA has adhesion and cytotoxicity to cells.Method:Firstly,this study obtained recombinant S.maltophilia OmpA(rOmpA)through the process of IPTG induction,ultrasonic disruption,and Ni column purification.In order to ensure the reliability of this study,Western blot was used to verify the identity of rOmpA.Then,this study used Alexa flour 647 fluorescent dye to label rOmpA,and detected the adhesion of rOmpA to human laryngeal carcinoma epithelial cells(HEp-2 cells)by flow cytometry.Finally,the release of LDH was used to detect the toxicity of rOmpA to HEp-2 cells,and the chemiluminescence and trypan blue staining were used to detect the effects of rOmpA on the viability and survival rate of HEp-2 cells respectively,thereby further reflecting the cytotoxicity of rOmpA..Results:Both rOmpA and the outer membrane protein of S.maltophilia standard strain K279a reacted specifically to rabbit anti-rOmpA serum.After stimulating HEp-2cells with rOmpA labeled with Alexa flour 647 fluorescent dye and the same concentration of BSA(negative control)for 8 hours,the fluorescence intensity of the cells stimulated by rOmpA increased significantly.The results of the LDH release experiment found that rOmpA can induce the release of LDH from HEp-2 cells,and the concentration of LDH in the cell culture medium was significantly increased compared with the negative control.Trypan blue staining found that the 30?g/m L rOmpA group and the apoptosis positive control group(0.1?M ST)had a significant decrease in cell viability,and the cell viability became stable around 8 h,and the curve became flat.Chemiluminescence detection revealed that the ATP concentration in HEp-2 cells of the 30?g/m L rOmpA group was significantly lower than the negative control group(equal volume of PBS),and the ATP concentration in the cells stabilized at about8 h,and the curve became flat.Chapter 2:Cellular experimental study on the mechanisms of S.maltophilia OmpA induced apoptosis and inflammatory responseObjective:To explore whether S.maltophilia OmpA can induce apoptosis and inflammatory response,and to clarify the related mechanisms.Method:Firstly,this study used flow cytometry to quantitatively analyze the apoptosis induced by rOmpA,and observed the morphological changes of HEp-2 cell apoptosis after rOmpA stimulation using DAPI nuclear staining and transmission electron microscopy respectively.Considering that mitochondria are important target organelles for apoptosis,our study used mitochondrial fluorescent probes to label the mitochondria of HEp-2 cells for observing the changes in the mitochondrial membrane potential of HEp-2 cells induced by rOmpA,and used fluorescence colorimetry to detect the opening of m PTP in HEp-2 cells.In addition,this study used confocal microscopy to detect nuclear translocation of TFAR19 in HEp-2 cells,which indirectly reflects the changes in mitochondrial membrane permeability.Western blot was used to analyze the expression of the mitochondrial pathway related pro-apoptotic protein Bax and anti-apoptotic protein Bcl-x L in HEp-2 cells.Western blot was also used to analyze the expression of the death receptor pathway related apoptotic protein Fas and Fas L in HEp-2 cells.In order to explore the effect of rOmpA on the generation of ROS and Ca²⁺concentration in HEp-2 cells,DCFH-DA probe and Fluo-3,AM fluorescent probe were used to detect the level of reactive oxygen species(ROS)in HEp-2 cells and changes in intracellular Ca²⁺concentration.Considering that Cyt-c and AIF entering the cytoplasm from mitochondria in the process of apoptosis mediated by the mitochondrial pathway,Western blot was used to detect the expression of AIF and Cyt-c in the mitochondria and cytoplasm of HEp-2 cells.Then,Western blot was used to detect the expression of caspase-3 and caspase-9 and the cleavage of PARP in HEp-2 cells to determine the activation of the caspase-3/-9 pathway.On this basis,the Ac-DEVD-p NA and Ac-LEHD-p NA probes were used to detect the changes in the substrates of caspase-3 and caspase-9 to determine the activities of caspase-3 and caspase-9 over time.In order to determine whether rOmpA induces apoptosis by localizing in mitochondria,this study used confocal microscopy to detect the subcellular localization of rOmpA in HEp-2 cells.As another important part of this study,ELISA was used to detect the secretion level of IL-6,IL-8 and TNF-?in HEp-2 cell culture fluid,thereby reflecting the induction of rOmpA in inflammatory response.Finally,direct ELISA and indirect ELISA were used to detect the expression of JNK signaling protein and phosphorylated JNK signaling protein(p-JNK)in HEp-2 cells respectively,to determine whether JNK protein activates during rOmpA-induced cell inflammatory response.Results:The results of flow cytometry showed that 30?g/m L rOmpA induced the apoptosis of HEp-2 cells,the proportion of early apoptotic cells reached 10.2%and the proportion of late apoptotic cells reached 46.3%in 30?g/m L rOmpA group,which was significantly higher than those in the control group.DAPI nuclear staining revealed that30?g/m L rOmpA induced nuclear pyknosis in HEp-2 cells.Transmission electron microscopy showed that chromatin condensation attached to the nuclear membrane in a cap shape after stimulation with 30?g/m L rOmpA for 8 h.The results of flow cytometry showed that the mitochondrial fluorescence of HEp-2 stimulated by 30?g/m L rOmpA was significantly lower than that of the control group,indicating that the mitochondrial membrane potential was depolarized.Fluorescence colorimetric results show that 30?g/m L rOmpA can induce the opening of m PTP.The results of confocal microscopy showed that 30?g/m L rOmpA induced nuclear translocation of TFAR19 protein in HEp-2 cells.Western blot results showed that 30?g/m L rOmpA induced the increasing expression of pro-apoptotic protein Bax and the decreasing expression of anti-apoptotic protein Bcl-x L in HEp-2 cells.The expression of Fas and Fas L proteins were not significantly different from those of the control group.Western blot results showed that 30?g/m L rOmpA decreased the expression of Cyt-c and AIF in the mitochondria,while increased their expression in the cytoplasm.The results of fluorescent detection showed that 30?g/m L rOmpA induced the production of ROS in HEp-2 cells and the increase of intracellular Ca²⁺concentration.Western blot results showed that 30?g/m L rOmpA induced the activation of caspase-3 and caspase-9 and the cleavage of PARP in HEp-2 cells.The time-varying curves of caspase-3 and caspase-9 showed that rOmpA aroused the activities of these two enzymes to show a trend of first increasing and then decreasing,which reached the peak at 8 h.The detection of rOmpA subcellular structure localization using confocal microscopy showed that the green fluorescence of labelled rOmpA was fused with the red fluorescence of labelled mitochondria,showing a yellow merged fluorescence,but did not merge with the blue fluorescence of labelled nucleus.The results of ELISA test for detecting the secretion level of pro-inflammatory cytokines showed that 30?g/m L rOmpA stimulated the release of IL-6,IL-8 and TNF-?from HEp-2 cells.The results of ELISA test also showed that 30?g/m L rOmpA induced the increase of the expression level of phosphorylated JNK signaling protein(p-JNK)in HEp-2 cells.Chapter 3:Animal experiment verification of studies on S.maltophilia OmpA-induced apoptosis and inflammatory responseObjective:To verify S.maltophilia OmpA-induced apoptosis and inflammatory response by animal experiments.Method:This study established an animal model of rOmpA-induced apoptosis,and used TUNEL staining to detect cell apoptosis in throat tissue based on this model,evaluating whether the model was successful.Western blotting was used to detect the expression of pro-apoptotic protein Bax and anti-apoptotic protein Bcl-x L in mice throat tissue.ELISA test was used to detect the levels of IL-6,IL-8 and TNF-?in mice oral lavage fluid and bronchoalveolar lavage fluid.Histopathological sections were made to observe the changes of inflammatory response in mouse lung tissue.ELISA test was used to detect the expression levels of JNK signal protein and phosphorylated JNK signal protein(p-JNK signal protein)in mice throat tissue homogenate.Results:TUNEL staining results showed that the percentage of apoptotic cells in the 300?g rOmpA group was significantly higher than that in the PBS negative control group.Compared with the PBS negative control group,the percentage of apoptotic cells in the 100?g rOmpA group increased to a certain extent,but the difference was not statistically significant.Western blot results showed that 300?g rOmpA stimulated the up-expression of pro-apoptotic protein Bax and down-expression of anti-apoptotic protein Bcl-x L in mice throat tissue.ELISA test results showed that the secretion levels of IL-6,IL-8 and TNF-?in the oral lavage fluid in the 300?g rOmpA group were higher than those in the negative control group,especially for IL-8 and TNF-?.The secretion levels of IL-6,IL-8 and TNF-?in the bronchoalveolar lavage fluid in the 300?g rOmpA group were increased to varying degrees compared with the negative control group,especially for IL-6 and TNF-?.The results of histopathological section observation showed that 300?g rOmpA induced the infiltration of inflammatory cells such as macrophages in the throat tissue of mice.ELISA test results showed that the expression of phosphorylated JNK signal protein in the throat tissue of the 300?g rOmpA group was significantly higher than that of the negative control group.Conclusion:This study confirmed that S.maltophilia OmpA could adhere to cells.In addition,it was also confirmed that OmpA had significant cytotoxicity,resulting in the decrease of viability and survival rate of HEp-2 cells.OmpA could induce cell apoptosis,leading to the typical changes on apoptotic cell morphology.This study confirmed that OmpA changed the permeability of the outer mitochondrial membrane while apoptosis.Studies on the expression of apoptosis-related proteins found that OmpA might induce apoptosis via the mitochondrial pathway.The changes of Cyt-c and AIF from the mitochondria into the cytoplasm further confirmed this conclusion.The generation of ROS in the cytoplasm and the increase of Ca²⁺concentration further confirmed the conclusion that OmpA induced changes in the permeability of the outer mitochondrial membrane,reflecting that this might be a potential factor leading to apoptosis.OmpA induced the activation of caspase-3 and caspase-9 in cells,which further confirmed the occurrence of apoptosis.The subcellular localization of OmpA confirmed that OmpA performed biological functions by localizing in the mitochondria.The detection of pro-inflammatory cytokines IL-6,IL-8 and TNF-?confirmed that OmpA could induce cell inflammatory response.The phosphorylation activation of JNK signaling protein in cells confirmed that OmpA might play a regulatory role in inducing inflammatory response,of which the specific mechanism needs further study.The mice animal model of OmpA-induced apoptosis was established and verified the conclusion that OmpA induced apoptosis by regulating the expression of pro-apoptotic protein Bax and anti-apoptotic protein Bcl-x L.At the same time,the detection of the pro-inflammatory cytokines IL-6,IL-8 and TNF-?in the oral lavage fluid and bronchoalveolar lavage fluid verified the conclusion that OmpA induced cellular inflammatory response,and histopathological observation further confirmed this view.The activation of JNK signaling protein was found in mice throat tissue homogenate,further confirming that JNK signaling protein might play an important role in the process of OmpA-inducd inflammatory response.