| Background and aims:Acute pancreatitis（AP）is an uncontrolled inflammatory response of pancreatic tissue caused by cholelithiasis,alcoholism,and other factors,severe cases can progress to multiple organ dysfunction syndrome（MODS）.Heat shock factor 1（HSF1）is an important transcription factor that regulates the expression of heat shock protein under the stimulation of various stress factors.The heat shock response mediated by HSF1 is an endogenous damage resistance mechanism in the long-term evolution of biological organisms.Our previous studies have shown that the expression of HSF1 in pancreatic tissue of AP mice is significantly reduced,and resveratrol can improve AP by increasing the expression of HSF1,suggesting that HSF1is involved in the occurrence and development of AP,but its underlying mechanism still needs further study.Mitophagy is a kind of selective autophagy.In this process,damaged mitochondria are specifically recognized and wrapped by the autophagosome and then fused with the lysosome for degradation,thus maintains mitochondrial homeostasis.Inhibition of PINK1/PARK2-mediated mitophagy has been proven to be involved in the occurrence and development of a variety of diseases.Recent studies have shown that PINK1/PARK2-mediated mitophagy can reducing the inflammatory response during AP through inhibiting the activation of NOD-like receptor thermal protein domain associated protein 3（NLRP3）inflammasome.However,the regulatory mechanism of PINK1/PARK2-mediated mitophagy during AP remains unclear.Through bioinformatics analysis,we found that the promoter regions of PINK1 and PARK2 genes contain multiple complete heat shock elements（HSE）.It is speculated that HSF1 can promote mitophagy and improve AP by regulating the transcription of PINK1/PARK2 in pancreatic acinus cells.This study aims to reveal the protective effect and mechanism of HSF1 on AP from the perspective of mitochondrial autophagy,provide new ideas for the diagnosis,treatment and prognosis prediction of AP.Experimental methods:（1）To explore the role of HSF1 in AP.Hsf1-/-and Hsf1+/+mice were selected as research subjects,and the AP mouse model was established by intraperitoneal injection of L-arginine or cerulein respectively.The detection indexes and methods were as follows:（1）The 7-day survival rate of mice;（2）Pancreatic tissue was stained with HE,and pathological score was performed.（3）Serum amylase,lactate dehydrogenase,alanine aminotransferase,aspartate aminotransferase,creatine kinase isoenzyme,blood urea nitrogen and creatinine levels were detected by biochemical analyzer.（4）The content of myeloperoxidase in pancreatic tissue was determined by enzyme-linked immunosorbent assay（ELISA）.（5）The m RNA expression of tumor necrosis factor（TNF）,interleukin 6,and interleukin 1B in pancreatic tissue caused by real-time fluorescence quantitative polymerase chain reaction（RT-q PCR）.（2）To investigate the effect of HSF1 on mitophagy during acute pancreatitis.Firstly,Hsf1-/-and Hsf1+/+mice were selected as research subjects,and the necrotic AP mouse model was established by intraperitoneal injection of L-arginine.Pancreatic tissues were collected at 24,48 and 72h after the last injection of l-arginine.The detection indexes and methods were as follows:（1）The expressions of PINK1,PARK2,MAP1LC3B and SQSTM1 were detected by western blotting.（2）The ultrastructure of pancreatic mitochondria was observed by transmission electron microscopy.（3）The changes of mitochondrial membrane potential were detected by JC-1 kit.Then,rat pancreatic acinar cell line AR42J was selected as research subjects.（1）AR42J cells were treated by different concentrations of L-arginine for different time,cell viability was detected by cell counting kit 8（CCK8）,and the expression of HSF1 was detected by western blotting.（2）HSF1 was overexpressed in AR42J cell through transfection with pc DNA3.1-HSF1 plasmid,then AR42J cells were treated with 10 mg/ml L-arginine for 24h.CCK8 was used to detect cell viability,the expressions of PINK1,PARK2,MAP1LC3B and SQSTM1 in cells and the release of high mobility group box 1（HMGB1）in supernatant were detected by western blots.The production of reactive oxygen species（ROS）and mitochondrial membrane potential were detected through DCFH-DA and JC-1 staining,m RNA expression of Il1b,Il6,Tnf and Il10 was detected by RT-q PCR.（3）To investigate the role of PARK2 in HSF1-reguated mitophagy during AP.Rat pancreatic acinar cell line AR42J was selected as research subjects.（1）PARK2 was overexpressed in AR42J cells through transfection with p LVX-PARK2 plasmid,then AR42J cells were treated with 10mg/ml L-arginine for 24h.CCK8 was used to detect cell viability.Western blotting was used to detect the expression of PINK1,PARK2,MAP1LC3B,SQSTM1 and the release of HMGB1 in the supernatant.ROS production and mitochondrial membrane potential were detected through DCFH-DA and JC-1 staining,m RNA expression of Il1b,Il6,Tnf and Il10 was detected by RT-q PCR.（2）AR42J cells was transfected with pc DNA3.1-HSF1 plasmid to overexpress HSF1,and then it was treated by specific PARK2 small interfering RNA（si RNA）to downregulated the expression of PARK2.After L-arginine treatment,the cell viability was detected by CCK8,the expressions of PINK1,PARK2,MAP1LC3B and SQSTM1 and the release of HMGB1 in the supernatant were detected by western blotting.ROS production and mitochondrial membrane potential were detected through DCFH-DA and JC-1 staining,m RNA expression of Il1b,Il6,Tnf and Il10 was detected by RT-q PCR.（4）JARSPAR database was used to predict probable HSF1 binding sites existed in the promoter of PARK2.Then electrophoretic mobility shift assay（EMSA）was used to verify the binding site in the promoter of PARK2 that can bind to HSF1.Dual luciferase reporter assay was performed to evaluate the effect of HSF1 on the transcription activities of PARK2 promoter.The interaction between HSF1 and PARK2 protein was determined by co-immunoprecipitation.Results:（1）The 7-day survival rates of L-arginine-treated Hsf1+/+and Hsf1-/-mice were 75%and 25%,respectively.The 7-day survival rates of cerulein-treated Hsf1+/+and Hsf1-/-mice were 100%and 70%,respectively.The 7-day survival rates of Hsf1-/-mice were significantly lower than those of Hsf1+/+mice.Compared with control mice,the serum amylase,lactate dehydrogenase,glutamic-pyruvic transaminase,glutamic-oxalacetic transaminase,creatine kinase isoenzyme,blood urea nitrogen and creatinine levels and the content of myeloperoxidase in pancreas of Hsf1+/+mice were significantly increased after treatment with L-arginine and cerulein,and the above indexes of Hsf1-/-mice treated with L-arginine and cerulein were significantly higher than those of Hsf1+/+mice.After intraperitoneal injection of L-arginine,the pancreatic acini and leaf space in pancreatic tissue of Hsf1+/+mice became wider,the inflammatory cell infiltration and hemorrhage,necrosis area was as high as 30%,and the pancreatic tissue of cerulein-induced Hsf1+/+mice appeared different degree of pancreatic acinar cell edema,acini and leaf space broadening,accompanied by inflammatory cells infiltration.The above pathological changes of Hsf1-/-mice treated with L-arginine or cerulein were more serious than those of Hsf1+/+mice,and the pathological scores at different time points were significantly higher than those of Hsf1+/+mice at the same time points.The m RNA expressions of Il1b,Tnf and Il6 in pancreatic tissue of L-arginine treated Hsf1+/+mice were significantly higher than those of control mice,and.The above gene expressions of L-arginine treated Hsf1-/-mice were significantly higher than Hsf1+/+mice.（2）Compared with Hsf1+/+control mice,the expression of PINK1 in L-arginine-treated Hsf1+/+mice was slightly decreased,the protein expression of PARK2 and SQSTM1,the proportion of MAP1LC3B II/I and mitochondrial membrane potential were significantly decreased.The above indexes of Hsf1-/-mice treated with L-arginine were significantly lower than those of Hsf1+/+mice.The results of transmission electron microscopy（TEM）showed that L-arginine treated Hsf1+/+mice appeared obvious mitochondrial autophagosomes,while Hsf1+/+mice showed increased mitochondrial autophagosomes.L-arginine decreased the viability of AR42J cells,the protein expression of HSF1,PARK2,SQSTM1 and the ratio of MAP1LC3B II/I in a time-and dose-dependent manner,it slightly decreased the expression of PINK1.Overexpression of HSF1 could significantly reverse L-arginine-decreased protein expression of PARK2 and SQSTM1,the ratio of MAP1LC3B II/I in AR42J cells,restored mitochondrial membrane potential,decreased ROS production,m RNA levels of Il1b,Il6,Tnf,Il10,and Nlrp3,HMGB1 release and elevated the cell viability of AR42J cells.（3）Overexpression of PARK2 significantly reversed L-arginine-decreased protein expression of PARK2 and SQSTM1,the ratio of MAP1LC3B II/I in AR42J cells,restored mitochondrial membrane potential,decreased ROS production,m RNA levels of Il1b,Il6,Tnf,Il10,and Nlrp3,HMGB1 release and elevated the cell viability of AR42J cells.Downregulation PARK2 using specific si RNA abrogated the effect of HSF1 overexpression on the protein expression of PARK2 and SQSTM1,the proportion of MAP1LC3B II/I,mitochondrial membrane potential,production of ROS,m RNA levels of Il1b,Il6,Tnf,Il10,and Nlrp3,HMGB1 release and the cell viability of AR42J cells.（4）There was no direct interaction between HSF1 and PARK2 protein,but overexpression of HSF1 could significantly increase the m RNA level of Park2 in AR42J cells,and the m RNA level of pancreatic tissue of Hsf1-/-mice was significantly lower than that of Hsf1+/+mice.EMSA assay showed that HSF1 could bind to the heat shock element that located-942～-928bp in the promoter of PARK2,and dual luciferase reporter assay showed that HSF1 could bind to PARK2 promoter and increase the transcription activities of PARK2 promoter in HEK293 cells.Conclusion:HSF1 protects against acute pancreatitis through activating PARK2-mediated mitophagy in the acinar cells.As a transcription factor,HSF1 can promote the transcription of PARK2 through directly binding to the HSE in its promoter of PARK2,activating PARK2-mediated mitophagy,reducing the production of ROS,thus inhibiting NLRP3inflammasome-mediated inflammatory response in the pancreatic tissue.Figures:36,Tables:10,References:108... |
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