| Objective:Acute lung injury(ALI)/ acute respiratory distress syndrome(ARDS)is caused by severe infection,trauma,shock,poisoning,inhalation of harmful gases,acute pancreatitis,pathology and obstetrics,etc.Among them,the barrier function of endothelial cells(EC)is impaired,resulting in high permeability of blood vessels,and protein-rich fluid extravasation caused by pulmonary interstitial edema.It is the main cause of ALI and ARDS.Studies have shown that lipopolysaccharide(LPS)can induce the body to produce severe pneumonia and infection processes and produce a large number of inflammations.At present,LPS has been widely used in animal infection models of ALI.Hydrogen rich saline(HRS)is a very good antioxidant,which has anti-inflammatory,anti-oxidative stress and anti-apoptotic properties.It has been reported that different doses of hydrogen-saturated physiological saline can reduce acute lung injury in rats with inhalation injury through inhibition of cellular inflammatory response,and apoptosis,but the mechanism is unknown.Cell autophagy is a highly conserved biological process in which eukaryotic cells self-digest to obtain a chance of survival.The study found that rapamycin target protein(mTOR)mainly inhibits the entry of autophagy key factor--transcription factor EB into the nucleus.The activation of mTORC1 can inhibit nuclear translocation through phosphorylation of TFEB.This is a key molecule event that negatively regulates the occurrence of autophagy.It is unclear whether the relieving effect of HRS on ALI is related to autophagy.In this study,we established a model of LPS-induced acute lung injury in rats and pretreated with HRS to observe its protective effect on ALI and the effect on autophagy-related proteins;and to investigate whether the therapeutic effect of HRS is related to mTORC1 and mTORC1 / TFEB signaling pathway by in vivo and in vitro experiments,providing a theoretical basis for the mechanism of clinical acute lung injury and targeted therapy of drugs.Method: Part 1: In vivo experiments,SD rats were divided into 3 groups:(1)LPS-induced rat lung injury model group(LPS group);(2)hydrogen-rich water pretreatment group(HRS +LPS group),(3)control group(control group).First,prepare lung pathological sections of each group of rats,and observe the pathological changes of the lungs in each group of rats.TUNEL method was used to detect the apoptosis level of lung cells in each group.ELISA was used to detect alveolar lavage.Exam the changes of the expression of inflammatory factors IL-1β,IL-6,TNF-α and IL-10 in blood fluid,vascular endothelial function related factors ET-1,VEGF,SDF-1α and eNOS;Western Blot detect the expression of apoptosis-related proteins bcl2,Bax and Caspase3 in rat lungs.In vitro experiments: HPMEC subculture was divided into control group,LPS group,and HRS + LPS group.CCK8 was used to detect cell activity;flow cytometry was used to detect apoptosis;ELISA was used to detect inflammatory factors in the supernatant;Western Blot was used to detect changes in the expression of vascular endothelial function-related factors ET-1,VEGF,SDF-1α and eNOS.Part 2: In vivo experiment,add autophagy inhibitor groups on the basis of experiment 1: LPS + HRS + CQ group: Rats were injected intraperitoneally with hydrogen-rich water 5mg / kg daily before modeling,and 30 min before LPS injection,chloroquine was injected intraperitoneally;LPS + HRS + 3MA group: Rats were injected intraperitoneally with hydrogen-rich water 5mg / kg daily before modeling,30 min before LPS injection,3MA was injected intraperitoneally;HE staining was used to observe pathological changes;Western Blot and reverse transcription quantitative PCR(q RT-PCR)were used to detect the expression changes of lung autophagy-related proteins Beclin1,LC3-II,and P62 in each group.In vitro experiments: HPMEC cells were divided into control group,LPS group,HRS + LPS group,autophagy inhibitor group(HRS + LPS + CQ group),autophagy inhibitor group(HRS + LPS + 3MA group).CCK8 was used to detect cell activity in each group;flow cytometry was used to detect apoptosis in each group;ELISA was used to detect inflammatory factors in the supernatant;Western Blot and q RT-PCR were used to detect changes in expression of autophagy-related proteins Beclin1,LC3-II and P62.Part 3: Western Blot detect the changes in expression levels of mTORC1 / TFEB signaling pathway related proteins p-mTOR,p-S6,mTOR and S6 in each group of lungs;immunohistochemical detect changes of TFEB protein expression;in vitro experiments: HPMEC cell grouping: Control group,LPS group,HRS + LPS processing group,LPS +HRS + OSI-027 group: 10 ul / ml HRS solution was added 1h before LSP,mTOR inhibitor OSI-027 10 n mol / ml was added after LPS induction;HRS + LPS + MHY1485 group : Add 10 ul / ml HRS solution 1h before adding LSP,and add mTOR agonist MHY1485 for treatment after LPS induction.CCK8 was used to detect cell activity in each group;flow cytometry was used to detect cell apoptosis in each group;ELISA was used to detect HPMEC cells in each group.Exam the changes of inflammatory cytokines TNF-α,IL-6,IL-1β and IL-10 in serum;detect autophagy-related proteins mTOR,p-mTOR,Beclin1,LC3-II and P62 in each group of cells.Exam the expression of TFEB protein.Result: Part 1: In vivo experiments: Compared with the control group,HE staining showed lung bronchial wall thickening,pulmonary edema,and inflammatory cell infiltration in the lung tissue of rats in the LPS group;Compared with the LPS group,alveolar damage,inflammatory exudation and interstitial cell proliferation reduced in the HRS group;the levels of IL-1β,IL-6,and TNF-α in the alveolar lavage fluid of SD rats in the LPS group increased and the levels of IL-10 decreased(P <0.05).Compared with the LPS group,the contents of IL-1β,IL-6 and TNF-α decreased,and the contents of IL-10 were increased in the HRS group.There was a significant difference between the two groups(P <0.05).Lung apoptosis was observed by TUNEL.Compared with the control group,the apoptosis rate in the LPS group increased,and the apoptosis rate in the HRS group decreased.There was a statistical difference between the groups(P <0.05).Vascular function test results showed that compared with the control group,the content of ET-1,VEGF,and SDF-1α increased,and the content of eNOS decreased.Compared with the LPS group,the content of ET-1,VEGF,and SDF-1α decreased.The content of NOS increased,and there was a statistical difference between the groups(P <0.05).In vitro experiments: Compared with the control group,the apoptosis rate,IL-1β,IL-6,and TNF-α levels in the LPS group increased,cell activity decreased,and IL-10 content decreased.There was a statistical difference between the two groups(P <0.05);Compared with the LPS group,the HRS group had increased cell viability,decreased apoptosis rate,decreased IL-1β,IL-6,TNF-α content,and increased IL-10 content,and there was a significant difference between the two groups(P <0.05).Part 2: HRS + LPS group,pulmonary edema reduced,inflammatory cells reduced;in HRS + LPS + CQ group and HRS + LPS + 3MA group,there existed severe pulmonary edema,inflammatory cell infiltration.Compared with the control group,the expression of Beclin1,LC3-II protein increased,and the expression of P62 protein decreased in the LPS group.There was a statistical difference between the groups(P <0.05).Compared with the LPS group,autophagy was related to the LPS + HRS group.The expression levels of proteins Beclin1 and LC3-II were higher than those in LPS group,and the expression levels of P62 protein were lower than those in LPS group(P <0.05).Compared with HRS + LPS: LC3-II expression decreased in the LPS + HRS + 3MA group(P <0.05),P62 expression increased(p <0.05),Beclin1 expression decreased but not significantly;in LPS + HRS + CQ group,LC3-II and P62 increased significantly(P <0.05),and Beclin1 did not change significantly.In vitro experiments: Compared with the LPS group,HRS can increase the activity of HPMEC cells;however,the addition of autophagy inhibitors reduced the cell activity(P <0.05).Compared with the LPS group,the levels of IL-1β,IL-6,and TNF-α in the HRS group decreased,and the content of IL-10 increased.There were significant differences between the groups(P <0.05);compared with the HRS group,the levels of IL-1β,IL-6,TNF-α increased and IL-10 decreased in the LPS + HRS + CQ group and the LPS + HRS + 3MA group,there was a statistical difference between groups(P <0.05).Flow cytometry was used to detect the apoptosis of each group.As a result,the addition of HRS could inhibit the apoptosis caused by LPS,and the addition of autophagy inhibitors increased the apoptosis rate(P <0.05).Compared with the control group,the expression of Beclin1,LC3-II protein was increased,and the expression of P62 protein decreased in the LPS group.There was a statistical difference between the groups(P <0.05).Compared with the LPS group,autophagy was related to the LPS + HRS group.The expression levels of proteins Beclin1 and LC3-II were higher than those in LPS group,and the expression levels of P62 protein was lower than those in LPS group(P <0.05).Compared with HRS + LPS: LC3-II expression decreased in the LPS + HRS + 3MA group(p <0.05),P62 expression increased(p <0.05),Beclin1 expression decreased but not significantly;LPS + HRS + CQ group LC3-II and P62 increased significantly(p <0.05),and Beclin1 did not change significantly.Part 3: In vivo experiments: Western Blot detected mTORC1 / TFEB signaling pathway-related protein phosphorylation levels.Compared with the LPS group,the expression of p-mTOR and p-S6 proteins in the HRS group significantly reduced(P <0.05).The expression of TFEB was detected by immunohistochemistry.In the LPS group,a large amount of TFEB was expressed in the cytoplasm.After HRS was added,TFEB entered the nucleus and the nuclear expression significantly increased.In vitro experiments: HRS can increase cell viability.Cell activity decreased after adding mTORC1 agonist,and there was a statistical difference between groups(P <0.05).Compared with HRS group,cell activity significantly increased when mTORC1 inhibitor is added(P <0.05).Flow cytometry was used to detect the apoptosis of each group.Compared with HRS,the apoptosis rate in the HRS + LPS + MHY1485 group increased,and the apoptosis rate in the LPS + HRS + OSI-027 group was significantly reduced.There was a statistical difference between the groups(P < 0.05);Compared with the HRS group,the inflammatory factor test results showed that the expression levels of TNF-α,IL-6 and IL-1β in the HRS + LPS + MHY1485 group significantly increased,and the expression levels of IL-10 significantly decreased(P <0.05),HRS + OSI-027 group expression of cytokines TNF-α,IL-6 and IL-1β significantly decreased,IL-10 expression increased significantly(P <0.05)between groups.The comparison was statistically significant(P <0.05).Western Blot detected the expression of mTOR,p-mTOR,LC3-II,Beclin1,and P62 proteins in HPMEC.Compared with the HRS group,the expression of p-mTOR and P62 in the HRS + LPS + MHY1485 group increased,and the expression of LC3-II and Beclin1 decreased.The expression of p-mTOR and P62 decreased in the HRS + LPS + OSI-027 group,and the expression of LC3-II and Beclin1 increased.There was a statistical difference between the groups(P <0.05).The expression of TFEB was detected by immunofluorescence.Compared with the LPS group,the TFEB in the HRS group was mainly expressed in the nucleus.Compared with the HRS group,the TFEB expression in the nucleus of the HRS + LPS + MHY1485 group was reduced,and the expression of the HRS + LPS + OSI-027 group was increased.The difference was statistically significant(P <0.05).Conclusion: 1.Hydrogen-rich water improves endothelial function in endotoxin-induced acute lung injury rats;2.Hydrogen-rich water activates autophagy and protects rats with acute lung injury induced by endotoxin;3.Hydrogen-rich water can activate autophagy through the mTORC1 / TFEB signaling pathway,improve vascular endothelial function and reduce lung injury in rats with acute lung injury. |
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