| ObjectiveCigarette smoke is the main pathogenic factor of chronic obstructive pulmonary disease(COPD).Emphysema is one of the pathological changes of COPD caused by destruction of terminal alveolar wall.Inflammation,oxidative stress,protease imbalance and subsequent apoptosis of alveolar epithelial cells lead to the increase of alveolar space.Necroptosis is a genetic programmed cell death mechanism associated with inflammation and oxidative stress.Its function is to kill the infected or dysfunctional cells during some degenerative or inflammatory diseases.Some people think that the necroptosis may be related to the occurrence of emphysema.Cigarette smoke can activate the protein pathway related to necroptosis of lung epithelial cells,but the specific mechanism is not clear.Therefore,it is of great significance to study the role of necroptosis in the pathogenesis of emphysema.Theaflavin-3,3’-digallate(TF-3)is a kind of theaflavin in black tea.Some studies have shown that TF-3 can play an anti-inflammatory and antioxidant role,and has many biological activities such as reducing the incidence rate of coronary heart disease,improving bone density and preventing cancer.However,the preventive effect of TF-3 on cigarette smoke extract(CSE)induced emphysema remains unclear.Therefore,we hypothesized that TF-3attenuates cigarette smoke extract induced emphysema by inhibiting necroptosis.This study aims to provide potential therapeutic targets for the prevention of cigarette smoke induced emphysema.MethodsPart one: the mechanism of TF-3 in alleviating the injury of lung epithelial cells induced by CSE(1)BEAS-2B cells were stimulated with different concentrations of CSE(1%,5%,10%,20% and 50%)and different treatment time(2 h,4 h,6 h,8 h,10 h,12 h,24 h and48 h).Cell viability was detected with CCK-8 assay,and the cell injury model was established by selecting the appropriate time and concentration of CSE.(2)To determine whether TF-3 has cytotoxicity on lung epithelial cells,BEAS-2B cells were treated with different concentrations(5 μM,10 μM and 20 μM)of TF-3.The viability of lung epithelial cells was detected by CCK-8 assay.(3)Lactate dehydrogenase(LDH)is one of the indicators of cytotoxicity.The loss of cell membrane integrity will increase the release of LDH.LDH detection kit was used to detect the effect of TF-3 on LDH release of lung epithelial cells.(4)ROS was labeled with DCFH-DA,the pictures were observed by fluorescence microscope,and the fluorescence intensity was analyzed by Image J software.(5)Quantitative real-time polymerase chain reaction(q RT-PCR)was used to detect the m RNA levels of tumor necrosis factor-alpha(TNF-α),interleukin-6(IL-6)and interleukin-1 beta(IL-1β)in lung epithelial cells.(6)Western blot was used to detect the protein level of Caspase-3 in lung epithelial cells.In order to further clarify whether the occurrence of lung epithelial cells is necroptosis,lung epithelial cells were pretreated with necroptosis inhibitor Necrostatin-1(20 μM).Propidium iodide(PI)was used to label lung epithelial cells.The positive rate of PI was observed by fluorescence microscope.PI/Annexin-V was used to label lung epithelial cells,and the necroptotic rate of BEAS-2B was detected by flow cytometry.(7)To further explore the pathway mechanism of TF-3 inhibiting necroptosis of lung epithelial cells,after p38 MAPK inhibitor SB203580(0.5 μM)pretreatment,the protein levels of p38 MAPK,RIPK3 and MLKL were detected by Western blot.Part two: the mechanism of TF-3 in alleviating CSE induced emphysema in mice(1)To evaluate the modeling and therapeutic effect of TF-3,H&E staining sections of the lung tissue of mice were observed with microscope.(2)Malondialdehyde(MDA),superoxide dismutase(SOD)and glutathione(GSH)detection kits were used to detect the levels of oxidative stress related indicators in the lung tissue of mice,and evaluate the oxidative stress status of lung tissue.(3)Enzyme linked immunosorbent assay(ELISA)was used to detect the protein level of inflammatory cytokines(TNF-α and IL-1β)in lung tissue,and evaluate the inflammatory responses status of lung tissue.(4)In order to further explore the role of necroptosis in the pathogenesis of emphysema,Western blot was used to detect the protein levels of RIPK3 and MLKL.ResultsPart one: the mechanism of TF-3 in alleviating the injury of lung epithelial cells induced by CSE(1)CCK-8 results showed that BEAS-2B cell viability decreased with the increase of CSE concentration and treatment time.And 10% CSE treatment for 6h was selected as the suitable condition for cell injury modeling.(2)The viability of BEAS-2B cells did not decrease after being stimulated by TF-3 at different concentrations for 24 hours,which indicated that TF-3 had no toxic effect on BEAS-2B cells.In addition,CCK-8results showed that after TF-3 pretreatment for 24 hours and 10% CSE treatment for 6hours,TF-3(20 μM)pretreatment can significantly improve the cell viability after 10%CSE treatment.(3)Compared with the control group,10% CSE significantly increased LDH level,while TF-3(20 μM)pretreatment can effectively reduce LDH level,TF-3can reduce the toxic effect of CSE on lung epithelial cells.(4)The results of fluorescence microscopy showed that ROS production increased significantly in 10%CSE group,while TF-3(20 μM)pretreatment significantly reduced ROS production induced by 10% CSE.(5)q RT-PCR results showed that compared with the control group,10% CSE significantly increased the m RNA levels of inflammatory cytokines,and TF-3(20 μM)pretreatment could significantly reduce the inflammatory response of lung epithelial cells induced by 10% CSE.(6)Western blot showed that 10% CSE did not increase the apoptosis of BEAS-2B cells.And the results of immunofluorescence and flow cytometry showed that CSE increased the necroptosis of lung epithelial cells,while TF-3(20 μM)pretreatment could significantly reduce the necroptosis of lung epithelial cells.(7)Western blot showed that TF-3(5 μM,10 μM and 20 μM)and SB203580(0.5 μM)pretreatment significantly inhibited the phosphorylation of RIPK3 and MLKL induced by 10% CSE.TF-3(20 μM)pretreatment significantly inhibited the phosphorylation of p38 MAPK induced by 10% CSE.These results suggest that TF-3pretreatment can reduce the necroptosis of lung epithelial cells induced by 10% CSE by inhibiting p38MAPK/RIPK3/MLKL signaling pathway.Part two: the mechanism of TF-3 in alleviating CSE induced emphysema in mice(1)Compared with the normal group,the alveolar volume,alveolar wall rupture,mean linear intercept(MLI)and destructive index(DI)of lung tissue were significantly increased in CSE alone group,and the pathological characteristics of emphysema were formed in morphology.There was no significant difference in alveolar structure between normal group and TF-3 group.However,compared with CSE alone group,TF-3 treatment reduced the emphysema like pathological changes induced by CSE.The results showed that TF-3 could alleviate the mice emphysema induced by CSE.(2)Compared with the normal group,the MDA level was significantly increased in CSE alone group,while the SOD and GSH levels were significantly decreased.There was no significant difference in TF-3 treatment group.However,compared with CSE alone group,TF-3 treatment reversed the changes of oxidative stress indexes induced by CSE.The results showed that TF-3 reduced the oxidative stress in the lung tissue of mice with CSE induced emphysema.(3)Compared with the normal group,the protein level of inflammatory cytokines(TNF-α and IL-1β)in the CSE treated group was higher.And there was no significant difference in protein level of TNF-α and IL-1β between normal group and TF-3 group.However,TF-3 treatment reversed the protein level of TNF-αand IL-1β induced by CSE compared with CSE group.The results showed that TF-3reduced the inflammatory responses in CSE treated mice.(4)Compared with the normal group,the phosphorylation levels of RIPK3 and MLKL in CSE group were significantly increased,while the protein levels of RIPK3 and MLKL in TF-3 group were not significantly different.However,TF-3 treatment reversed the increased phosphorylation of RIPK3 and MLKL induced by CSE compared with CSE group.ConclusionIn conclusion,this study found that CSE had cytotoxicity on lung epithelial cells.The results showed that CSE could decrease the viability of lung epithelial cells,destroy the integrity of cell membrane,increase oxidative stress,inflammatory responses and necroptosis.However,we found that TF-3 attenuates CSE induced necroptosis of lung epithelial cells by inhibiting the activation of p38MAPK/RIPK3/MLKL signaling pathway.In vivo,we found that TF-3 could alleviate the pathological characteristics of mice emphysema induced by CSE.TF-3 reduced the oxidative stress and inflammatory responses in the lung tissue of mice,which were related to the inhibition of necroptosis pathway by TF-3.In the pathogenesis of emphysema,oxidative stress,inflammatory responses and necroptosis may be related in signaling pathways.Therefore,TF-3 may reduce the mice emphysema induced by CSE by inhibiting necroptosis.The significance of this study is to provide potential therapeutic targets for the prevention of cigarette smoke induced emphysema. |
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