The Effect And Mechanism Of Dexmedetomidine On Intestinal Ischemia-Reperfusion Injury In Rats Based On Mitophagy

Intestinal ischemia/reperfusion(I/R)injury is one of the common major organ injuries in surgery,which often occurs in pathological processes such as severe infection,shock,intestinal obstruction and cardiopulmonary bypass surgery.Intestinal I/R can cause intestinal mucosal barrier dysfunction,increased intestinal mucosal permeability,and translocation of intestinal bacteria and endotoxins into the bloodstream,causing severe inflammatory response and sepsis,which can further develop into multiple organ dysfunction syndrome(MODS).So far,although a lot of manpower and material resources have been invested in the research on the mechanism and protective measures of intestinal I/R injury,the research progress has been slow due to the limitations of the pathophysiology of intestinal I/R injury.Current studies suggest that the pathogenesis of intestinal I/R injury is closely related to factors such as inflammatory factors,reactive oxygen species release,apoptosis and autophagy.Based on this,this study has important research significance to explore new strategies for prevention and treatment of intestinal I/R injury from the perspective of autophagy.Dexmedetomidine is a sedative and analgesic drug widely used in clinical anesthesia and ICU in recent years.Studies have shown that in addition to certain anti-inflammatory and anti-apoptotic effects,dexmedetomidine also has certain effects on organ I/R injury.Dexmedetomidine may be a potential drug to prevent intestinal I/R injury,but its molecular mechanism is still unclear.However,most of the current research on the protective mechanism focuses on its anti-inflammatory and anti-apoptotic effects.Whether it plays a protective effect on intestinal I/R injury by regulating autophagy needs further research.Based on the above research background,this study intends to first observe the protective effect of dexmedetomidine on SD rats with intestinal I/R injury,and then observe the effect of dexmedetomidine on mitophagy of enteric glial cells in inflammatory environment in vitro.Finally,the possible molecular mechanism of dexmedetomidine’s anti-intestinal I/R injury effect by regulating mitophagy was explored in enteric glial cells model and SD rat intestinal I/R model.Part Ⅰ Dexmedetomidine improves intestinal ischemia-reperfusion injuryObjective: To investigate the protective effect of dexmedetomidine on intestinal ischemia-reperfusion.Methods: Rats were randomly divided into four groups: control group(Sham group),simple intestinal I/R injury group(I/R group),low-dose dexmedetomidine pretreatment + intestinal I/R injury group(Dex1 group)And high-dose dexmedetomidine pretreatment + intestinal I/R injury group(Dex2 group),10 in each group.In the control group,the abdominal cavity was incised but the superior mesenteric artery was not clipped after anesthesia,and the rats in the other three groups were anesthetized by clipping the superior mesenteric artery for 60 min after ischemia and reperfusion for 120 min to establish an intestinal I/R injury model.Before SMA clipping,the rats in the Dex1 group were pretreated by continuously pumping dexmedetomidine through the tail vein at a rate of 2.5 μg/kg/h for 1 h,and the rats in the Dex2 group were pretreated with 5.0 μg/kg/h.The rats in the I/R group were continuously pumped with normal saline at a rate of 1.5 ml/h through the tail vein for 1 h.After the experiment,the intestinal tissue was collected,and the wet/dry(W/D)ratio of the intestinal tissue was detected by conventional methods.The pathological changes of the intestinal mucosa were observed by HE staining,and the degree of damage was evaluated by Chiu’s score.The level of malondialdehyde(MDA)and the activity of superoxide dismutase(SOD)in rat intestinal tissue were measured to evaluate the oxidative stress injury.The expression levels of TNF-α and IL-6 in the serum of rats in each group were detected by ELISA.The expressions of GFAP and S-100β in the intestinal tissues of rats in each group were evaluated by immunohistochemistry and western blotting.The apoptosis of intestinal tissue of rats in each group was detected by TUNEL.Results: Compared with the Sham group,the rats in the I/R group had severe pathological damage,the Chiu’s score was significantly increased(P<0.05),the wet-dry weight ratio of the intestinal tissue was significantly increased(P<0.05),and the MDA concentration in the intestinal mucosa tissue was increased(P<0.05),the total SOD activity level in intestinal mucosal tissue was significantly decreased(P<0.05),the positive expression of GFAP and S-100β in EGCs of intestinal tissue increased,and the protein expression levels of GFAP and S-100β were significantly increased(P<0.05),and the apoptosis rate was significantly increased(P<0.05);Conclusion: Dexmedetomidine can inhibit intestinal oxidative stress,inflamm-ation,reduce apoptosis,thereby improving intestinal I/R injury.PartⅡ Dexmedetomidine attenuates mitochondrial damage and apoptosis of EGCs in inflammatory environment by promoting mitophagyObjective: To investigate the effect of dexmedetomidine on mitophagy of enteric glial cells in an inflammatory environment.Methods:In vitro models of inflammatory cells were constructed by stimulating EGCs with TNF-α(50 ng/m L)and IFN-γ(100 ng/m L)for 40 hours.They were divided into Untreated group,Stimulated group,Dex-treated group,and mitophagy inhibitor(3-MA)treated group,the Untreated group was the control group.The apoptosis level was detected by flow cytometry,hoechest33342 staining and mitochondrial membrane potential(MMP).The expression of SIRT3,PINK1,p53,LC3-II/I,p62,Atg5,Bax,Bcl-2,Caspase-3 and Cleaved-caspase-3 were detected by western blotting to evaluate mitophagy and apoptosis.The ultrastructural changes of EGCs were observed by transmission electron microscopy.Results: Compared with the Untreated group,the early apoptotic rate of EGCs cells in the Stimulated group was significantly increased(P<0.05),the mitochondrial membrane potential was significantly decreased(P<0.05),and Hoechst33342 staining showed nuclear condensation,deviation and cell number reduction.Transmission electron microscopy results showed that a few damaged mitochondria were seen in the EGCs of the Stimulated group.The results of western blotting showed that the ratio of LC3-II/LC3-I in EGCs cells in the Stimulated group was decreased(P<0.05),the expression of p62 was increased(P<0.05),the expression of Atg5 was decreased(P<0.05),and the expression of Bax was increased(P<0.05).,the expression of Bcl-2was decreased(P<0.05),the expression of Caspase-3 was decreased(P<0.05)(P<0.05),the expression of Cleaved Caspase-3 was increased(P<0.05);Compared with the stimulated group,the early apoptosis rate of EGCs cells in the Dex group was significantly decreased(P<0.05),the mitochondrial membrane potential was significantly increased(P<0.05),and the characteristic expression of apoptosis in Hoechst33342 staining was reduced.The results of transmission electron microscopy showed that obviously swollen mitochondria,mitochondrial dissolution,balloon-like degeneration and autophagosomes were observed in EGCs of Dex group.Western blotting results showed that the ratio of LC3-II/LC3-I in EGCs cells in Dex group was increased(P<0.05),the expression of p62 was decreased(P<0.05),the expression of Atg5 was increased(P<0.05),and the expression of Bax was decreased(P<0.05),the expression of Bcl-2 was increased(P<0.05),the expression of Caspase-3 was increased(P<0.05),the expression of Cleaved Caspase-3 was decreased(P<0.05);Indicating that dexmedetomidine can reduce the inflammatory environment mitochondrial damage and apoptosis in EGCs.Compared with the Dex group,the early apoptosis rate of EGCs cells in the autophagy inhibitor 3-MA treatment group was significantly increased(P<0.05),and the mitochondrial membrane potential was significantly decreased(P<0.05).Hoechst33342 staining showed decreased cell numbers and apoptotic bodies.Transmission electron microscopy results showed that no autophagosomes were found in EGCs of 3-MA group.The results of western blotting showed that the ratio of LC3-II/LC3-I in EGCs cells in 3-MA group was decreased(P<0.05),the expression of p62 was increased(P<0.05),the expression of Atg5 was decreased(P<0.05),and the expression of Bax was increased(P<0.05),the expression of Bcl-2 was decreased(P<0.05),the expression of Caspase-3 was decreased(P<0.05),and the expression of Cleaved Caspase-3 was increased(P<0.05).Conclusion: Dexmedetomidine can regulate the expression of mitophagyrelated proteins and promote mitophagy to reduce mitochondrial damage and apoptosis of EGCs in inflammatory environment.Part Ⅲ Dexmedetomidine reduces mitochondrial damage and apoptosis of EGCs in inflammatory environment by promoting mitophagy through SIRT3-mediated PINK1/HDAC3/p53 pathwayObjective: To explore the regulatory mechanism of dexmedetomidine reducing mitochondrial damage and apoptosis of EGCs in inflammatory environment by promoting mitophagy through PINK1/HDAC3/p53 pathway mediated by SIRT3.Methods: In vitro models of inflammatory cells were constructed by stimulating EGCs with TNF-α(50 ng/m L)and IFN-γ(100 ng/m L)for 40 hours.In the first step,EGCs cells were divided into Stimulated group,Dex-treated group,and SIRT3inhibitor(3-TYP)-treated group.The apoptosis level was detected by flow cytometry and MMP,and the autophagy-related protein(LC3-II/I,Atg5 and p62)and expression of apoptosis-related proteins(Bax,Bcl-2,caspase-3 and Cleaved-caspase-3).To observe the role of SIRT3 in the process of dexmedetomidine promoting mitophagy and reducing mitochondrial damage and apoptosis of EGCs under inflammatory environment.n the second step,EGCs cells were divided into control group,sh-NC treatment group,sh-PINK1 treatment group,and HDAC3inhibitor(RGFP966)treatment group.The interaction between PINK1,HDAC3 and p53 was verified by co-immunoprecipitation.By silencing the PINK1 gene and intervening with HDAC3 inhibitors,the expression levels of PINK1,HDAC3,p-HDAC3 and p53 were detected by western blotting to verify the regulatory relationship between PINK1,HDAC3 and p53.Finally,by silencing the PINK1 gene and intervening with HDAC3 and p53 inhibitors,the apoptosis level was detected by flow cytometry and MMP,and the autophagy-related proteins(LC3-II/I,Atg5 and p62)were detected by western blotting.and the expression of apoptosis-related proteins(Bax,Bcl-2,caspase-3 and Cleaved-caspase-3).To demonstrate the role of SIRT3-mediated PINK1/HDAC3/p53 pathway in dexmedeto-midine promoting mitophagy and reducing mitochondrial damage and apoptosis in EGCs under inflammatory environment.Results:(1)Compared with the Stimulated group,the changes of flow cytome-try,mitochondrial membrane potential,transmission electron microscopy and Western blot results in EGCs cells in the Dex group were consistent with those in the second part of the paper.Compared with the Dex group,the early apoptosis rate of EGCs cells in the SIRT3 inhibitor 3-TYP treatment group was significantly increased(P<0.05),and the mitochondrial membrane potential was significantly decreased(P<0.05).The results of transmission electron microscopy showed that no autophago-somes were found in the EGCs of the 3-TYP group.The results of Western blotting showed that the expression of SIRT3 was significantly decreased(P<0.05),the expression of PINK1 was significantly decreased(P<0.05),the expression of p53 was significantly increased(P<0.05),and the ratio of LC3-II/LC3 I in EGCs cells in3-TYP group was significantly decreased(P<0.05),p62 expression increased(P<0.05),Atg5 expression decreased(P<0.05),Bax expression increased(P<0.05),Bcl-2 expression decreased(P<0.05),the expression of Caspase-3 was decreased(P<0.05),and the expression of Cleaved Caspase-3 was increased(P<0.05),These results suggest that dexmedetomidine regulates mitophagy by regulating the expression of SIRT3 and PINK1,and inhibits mitochondrial damage and apoptosis in EGCs under inflammatory environment.(2)The results of co-immunoprecipitation showed that PINK1 and HDAC3 protein and HDAC3 and p53 protein were bound to each other in the immune complex.Compared with the empty control group,the expression of PINK1 and p-HDAC3 in the sh-PINK1 group were significantly decreased(P<0.05),while the expression of p53 was significantly increased(P<0.05),and silencing PINK1 could weaken the binding of HDAC3 to p53.Compared with the sh-PINK1 group,the expression of HDAC3 in EGCs co-treated with sh-PINK1 and the HDAC3 inhibitor RGFP966 was significantly decreased(P<0.05),while the expression of p53 was significantly increased(P<0.05).Compared with the control group,the expression of HDAC3 in the RGFP966 group was significantly decreased(P<0.05),and the expression of p53 was significantly increased(P<0.05).The above results indicate that PINK1,HDAC3 and p53 are combined with each other,and PINK1 reduces the expression of p53 by promoting the phosphorylation of HDAC3.(3)Compared with the sh-NC group,there was no significant difference in the early apoptosis rate,mitochondrial membrane potential,p53,LC3-1,LC3-II,p62,Atg5,Bax,Bcl-2,Caspase-3,Cleaved-Caspase-3,GAPDH protein expression and transmi-ssion electron microscopy results of EGCs in the sh-PINK1 group.Compared with the sh-NC group,the expression of PINK in EGCs of the sh-PINK1 group was significantly decreased(P<0.05).Compared with the sh-PINK1 group,the early apoptosis rate of EGCs in the PFT-α group was significantly decreased(P<0.05),and the mitochondrial membrane potential was significantly increased(P<0.05).Compared with the sh PINK1 group,the expressions of PINK1,p53,p62,Bax and Cleaved Caspase-3 in the PFT-α group were significantly decreased(P<0.05),the ratio of LC3-II/LC3-I,the expressions of Atg5,Bcl-2 and Caspase-3 Significantly increased(P<0.05).The results of transmission electron microscopy showed that mitochondria in the p53 inhibitor PFT-α treatment group had obvious morphological abnormalities and autophagosomes.These results suggest that PINK1 attenuates mitochondrial damage and apoptosis in EGCs under inflammatory environment by inhibiting p53.(4)Compared with the Stimulated group,the trend of flow cytometry,mitochondrial membrane potential,Hoechst33342 staining,transmission electron microscopy and Western blotting results in EGCs cells in Dex group was consistent with the second part of the paper.Compared with the Dex group,the early apoptosis rate of EGCs cells in the HDAC3 inhibitor RGFP966 treatment group was significantly increased(P<0.05),and the mitochondrial membrane potential was significantly decreased(P<0.05).Hoechst33342 staining results showed that the number of cells was reduced,and the nucleus was condensed and shifted.Transmission electron microscopy results showed that damaged mitochondria were occasionally found in EGCs of RGFP966 group,but no autophagosomes were found.The results of Western blotting showed that the expressions of SIRT3 and PINK1 in EGCs cells in the RGFP966 group were slightly decreased(P>0.05),and the expressions of p53,p62,Bax,and Cleaved Caspase-3 were significantly increased(P<0.05),and the ratio of LC3-II/I,the expressions of Atg5,Bcl-2,HDAC3,p-HDAC3 and Caspase-3 were decreased(P<0.05);These results suggest that dexmedetomidine alleviates the inflammatory environment through SIRT3-mediated PINK1/HDAC3/p53 pathway mitochondrial autophagy and apoptosis of EGCs.Conclusion: Dexmedetomidine can regulate the expression of mitophagyrelated proteins through the PINK1/HDAC3/p53 pathway mediated by SIRT3,and promote mitophagy to reduce mitochondrial damage and apoptosis in EGCs under inflammatory environment.Part IV Dexmedetomidine attenuates intestinal ischemia-reperfusion injury by promoting mitophagy through SIRT3-mediated PINK1/HDAC3/p53 pathwayObjective: To explore the regulatory mechanism of dexmedetomidine to reduce intestinal ischemia-reperfusion injury by promoting mitophagy through SIRT3-mediated PINK1/HDAC3/p53 pathway.Methods: According to the first part of this paper,the rat model of intestinal I/R injury was constructed,and the rats were divided into control group(Sham group),simple intestinal I/R injury group(I/R group),and dexmedetomidine pretreatment +intestinal I/R injury group.(Dex group),and RGFP966+dexmedetomidine pretreatment + intestinal I/R injury group(RGFP966 group)4 groups.The wet/dry(W/D)ratio of intestinal tissue was detected by conventional methods,and the pathological changes of intestinal mucosa of rats in each group were observed under light microscope,and the degree of injury was evaluated by Chiu’s score.The level of malondialdehyde(MDA)and the activity of superoxide dismutase(SOD)in rat intestinal tissue were measured to evaluate the oxidative stress injury.The expression levels of TNF-α and IL-6 in the serum of rats in each group were detected by ELISA.The expressions of GFAP and S-100β in the intestinal tissues of rats in each group were evaluated by immunohistochemistry and western blotting.The apoptosis of intestinal tissue of rats in each group was detected by TUNEL.The expression of SIRT3,PINK1,p53,LC3-II/I,p62,Atg5,Bax,Bcl-2,Caspase-3 and Cleavedcaspase-3 were detected by Western blotting.Results: Compared with the Sham group,the rats in the I/R group had severe pathological damage,the Chiu’s score was significantly increased(P<0.05),the wet-dry weight ratio of the intestinal tissue was significantly increased(P<0.05),and the MDA concentration in the intestinal mucosa tissue was increased(P<0.05),the total SOD activity level in intestinal mucosa tissue was significantly decreased(P<0.05),and the apoptosis rate was significantly increased(P<0.05);Compared with the Sham group,the expressions of GFAP,S-100β,Bax,p53,HDAC3,CleavedCaspase-3 and p62 in the intestinal tissue of the rats in the I/R group were significan-tly increased(P<0.05).The expressions of SIRT3,PINK1,Bcl-2,p-HDAC3,Caspase-3,Atg5 and the ratio of LC3-II/I were significantly decreased(P<0.05).Compared with the I/R group,the pathological damage of the rats in the Dex treatment group was improved.,Chiu’s score was significantly decreased(P<0.05),the wet-dry weight ratio of intestinal tissue was significantly decreased(P<0.05),the concentration of MDA in intestinal mucosal tissue was decreased(P<0.05),and the total SOD activity level in intestinal mucosal tissue was significantly increased(P<0.05),the apoptosis rate was significantly decreased(P<0.05);Compared with the I/R group,the protein expressions of GFAP,S-100β,Bax,p53,HDAC3,Cleaved-Caspase-3 and p62 in the intestinal tissue of the Dex treated rats were significantly decreased(P<0.05).Compared with the I/R group,the protein expressions of SIRT3,PINK1,Bcl-2,p-HDAC3,Caspase-3,Atg5 and the ratio of LC3II/LC3 I in the intestinal tissue of the Dex group were significantly increased(P<0.05);compared with the Dex group,RGFP966 group rats suffered severe pathological damage,Chiu’s score increased significantly(P<0.05),intestinal tissue wet to dry weight ratio was significantly increased(P<0.05),MDA concentration in intestinal mucosa tissue increased(P<0.05),intestinal mucosal tissue The total SOD activity level in the tissue was significantly decreased(P<0.05),and the apoptosis rate was significantly increased(P<0.05);Compared with the Dex group,the protein expressions of Bcl-2,HDAC3,p-HDAC3,Caspase-3,Atg5 and the ratio of LC3II/LC3 I in the intestinal tissue of the RGFP966 group were significantly decreased(P<0.05).The expression of SIRT3 protein in mouse intestinal tissue was slightly increased,and the expression of PINK1 protein was slightly decreased,the difference was not statistically significant(P>0.05).Conclusion: Dexmedetomidine can regulate the expression of mitophagyrelated proteins through SIRT3-mediated PINK1/HDAC3/p53 pathway,and promote mitophagy to reduce ischemia-reperfusion injury.