BACKGROUND The incidence rate of acute pancreatitis(AP), which is a pathophysiological process with complex etiology, is increasing globally. miR-125b-5p, a bidirectional regulatory miRNA, is speculated to exhibit ant...BACKGROUND The incidence rate of acute pancreatitis(AP), which is a pathophysiological process with complex etiology, is increasing globally. miR-125b-5p, a bidirectional regulatory miRNA, is speculated to exhibit anti-tumor activity. However,exosome-derived miR-125b-5p in AP has not been reported.AIM To elucidate the molecular mechanism of exosome-derived miR-125b-5p promoting AP exacerbation from the perspective of the interaction between immune cells and acinar cells.METHODS Exosomes derived from AR42J cells were isolated and extracted in active and inactive states by an exosome extraction kit, and were verified via transmission electron microscopy, nanoparticle tracking analysis, and western blotting. RNA sequencing assay technology was used to screen differentially expressed miRNAs in active and inactive AR42J cell lines, and bioinformatics analysis was used to predict downstream target genes of miR-125b-5p. The expression level of miR-125b-5p and insulin-like growth factor 2(IGF2) in the activated AR42J cell line and AP pancreatic tissue were detected by quantitative real-time polymerase chain reaction and western blots. The changes in the pancreatic inflammatory response in a rat AP model were detected by histopathological methods. Western Blot was used to detect the expression of IGF2, PI3K/AKT signaling pathway proteins, and apoptosis and necrosis related proteins.RESULTS miR-125b-5p expression was upregulated in the activated AR42J cell line and AP pancreatic tissue,while that of IGF2 was downregulated. In vitro experiments confirmed that miR-125b-5p could promote the death of activated AR42J cells by inducing cell cycle arrest and apoptosis. In addition,miR-125b-5p was found to act on macrophages to promote M1 type polarization and inhibit M2type polarization, resulting in a massive release of inflammatory factors and reactive oxygen species accumulation. Further research found that miR-125b-5p could inhibit the expression of IGF2 in the PI3K/AKT signaling pathway. Additionally, in vivo experiments revealed that miR-125b-5p can promote the progression of AP in a rat model.CONCLUSION miR-125b-5p acts on IGF2 in the PI3K/AKT signaling pathway and promotes M1 type polarization and inhibits M2 type polarization of macrophage by inhibiting IGF2 expression, resulting in a large release of pro-inflammatory factors and an inflammatory cascade amplification effect, thus aggravating AP.展开更多
Background:Pancreatic stellate cells(PSCs)activation plays a critical role in the development of chronic pancreatitis.Previous studies confirmed that thromboxane A2 receptor(TxA2r)was overexpressed in activated PSCs i...Background:Pancreatic stellate cells(PSCs)activation plays a critical role in the development of chronic pancreatitis.Previous studies confirmed that thromboxane A2 receptor(TxA2r)was overexpressed in activated PSCs in rats.The purpose of this study was to investigate the role of TxA2r in the activation of PSCs induced by 8-epi-prostaglandin F2α(8-epi-PGF2α).Methods:TxA2r expression in both quiescent and activated PSCs was detected by immunocytochemistry and immunoblot assay.Isolated PSCs were treated with 8-epi-PGF2α(10^-6,10^-7,10^-8 mol/L)for 48 h,and SQ29548(10^-4,10^-6,and 10^-7 mol/L),a TxA2r-specific antagonist,for 48 h,respectively,to identify the drug concentration with the best biological effect and the least cytotoxicity.Then isolated PSCs were treated with SQ29548(10^-4 mol/L)for 2 h,followed by 10^-7 mol/L 8-epi-PGF2α for 48 h.Real-time polymerase chain reaction was performed to detect the messenger RNA(mRNA)levels of a-smooth muscle actin(a-SMA)and collagen I.Comparisons between the groups were performed using Student’s t test.Results:TxA2r was up-regulated in activated PSCs in vitro compared with quiescent PSCs(all P<0.001).Compared with the control group,different concentrations of 8-epi-PGF2α significantly increased mRNA levels of a-SMA(10^-6 mol/L:2.23±0.18 vs.1.00±0.07,t=10.70,P<0.001;10^-7 mol/L:2.91±0.29 vs.1.01±0.08,t=10.83,P<0.001;10^-8 mol/L,1.67±0.07 vs.1.00±0.08,t=11.40,P<0.001)and collagen I(10^-6 mol/L:2.68±0.09 vs.1.00±0.07,t=24.94,P<0.001;10^-7 mol/L:2.12±0.29 vs.1.01±0.12,t=6.08,P<0.001;10^-8 mol/L:1.46±0.15 vs.1.00±0.05,t=4.93,P=0.008).However,different concentrations of SQ29548 all significantly reduced the expression of collagen I(10^-4 mol/L:0.55±0.07 vs.1.00±0.07,t=10.47,P<0.001;10^-6 mol/L:0.56±0.10 vs.1.00±0.07,t=6.185,P<0.001;10^-7 mol/L:0.27±0.04 vs.1.00±0.07,t=15.41,P<0.001)and a-SMA(10^-4 mol/L:0.06±0.01 vs.1.00±0.11,t=15.17,P<0.001;10^-6 mol/L:0.28±0.03 vs.1.00±0.11,t=11.29,P<0.001;10^-7 mol/L:0.14±0.04 vs.1.00±0.11,t=12.86,P<0.001).After being treated with SQ29548(10^-4 mol/L)and then 8-epi-PGF2α(10^-7 mol/L),the mRNA levels of a-SMA(0.20±0.08 vs.1.00±0.00,t=17.46,P<0.001)and collagen I(0.69±0.13 vs.1.00±0.00,t=4.20,P=0.014)in PSCs were significantly lower than those of the control group.Conclusions:The results show that 8-epi-PGF2α promoted PSCs activation,while SQ29548 inhibited PSCs activation induced by 8-epi-PGF2α.The result indicated that TxA2r plays an important role during PSC activation and collagen synthesis induced by 8-epi-PGF2α in vitro.This receptor may provide a potential target for more effective antioxidant therapy for pancreatic fibrosis.展开更多
基金Supported by The Beijing Municipal Science and Technology Commission,Capital Research and Demonstration Application of Clinical Diagnosis and Treatment Technology,No. Z191100006619038 and No. Z171100001017077The Capital Health Research and Development of Special,No. 2020-1-2012
文摘BACKGROUND The incidence rate of acute pancreatitis(AP), which is a pathophysiological process with complex etiology, is increasing globally. miR-125b-5p, a bidirectional regulatory miRNA, is speculated to exhibit anti-tumor activity. However,exosome-derived miR-125b-5p in AP has not been reported.AIM To elucidate the molecular mechanism of exosome-derived miR-125b-5p promoting AP exacerbation from the perspective of the interaction between immune cells and acinar cells.METHODS Exosomes derived from AR42J cells were isolated and extracted in active and inactive states by an exosome extraction kit, and were verified via transmission electron microscopy, nanoparticle tracking analysis, and western blotting. RNA sequencing assay technology was used to screen differentially expressed miRNAs in active and inactive AR42J cell lines, and bioinformatics analysis was used to predict downstream target genes of miR-125b-5p. The expression level of miR-125b-5p and insulin-like growth factor 2(IGF2) in the activated AR42J cell line and AP pancreatic tissue were detected by quantitative real-time polymerase chain reaction and western blots. The changes in the pancreatic inflammatory response in a rat AP model were detected by histopathological methods. Western Blot was used to detect the expression of IGF2, PI3K/AKT signaling pathway proteins, and apoptosis and necrosis related proteins.RESULTS miR-125b-5p expression was upregulated in the activated AR42J cell line and AP pancreatic tissue,while that of IGF2 was downregulated. In vitro experiments confirmed that miR-125b-5p could promote the death of activated AR42J cells by inducing cell cycle arrest and apoptosis. In addition,miR-125b-5p was found to act on macrophages to promote M1 type polarization and inhibit M2type polarization, resulting in a massive release of inflammatory factors and reactive oxygen species accumulation. Further research found that miR-125b-5p could inhibit the expression of IGF2 in the PI3K/AKT signaling pathway. Additionally, in vivo experiments revealed that miR-125b-5p can promote the progression of AP in a rat model.CONCLUSION miR-125b-5p acts on IGF2 in the PI3K/AKT signaling pathway and promotes M1 type polarization and inhibits M2 type polarization of macrophage by inhibiting IGF2 expression, resulting in a large release of pro-inflammatory factors and an inflammatory cascade amplification effect, thus aggravating AP.
基金This study was supported by the grant from the National Natural Science Foundation Youth Fund(No.81200325).
文摘Background:Pancreatic stellate cells(PSCs)activation plays a critical role in the development of chronic pancreatitis.Previous studies confirmed that thromboxane A2 receptor(TxA2r)was overexpressed in activated PSCs in rats.The purpose of this study was to investigate the role of TxA2r in the activation of PSCs induced by 8-epi-prostaglandin F2α(8-epi-PGF2α).Methods:TxA2r expression in both quiescent and activated PSCs was detected by immunocytochemistry and immunoblot assay.Isolated PSCs were treated with 8-epi-PGF2α(10^-6,10^-7,10^-8 mol/L)for 48 h,and SQ29548(10^-4,10^-6,and 10^-7 mol/L),a TxA2r-specific antagonist,for 48 h,respectively,to identify the drug concentration with the best biological effect and the least cytotoxicity.Then isolated PSCs were treated with SQ29548(10^-4 mol/L)for 2 h,followed by 10^-7 mol/L 8-epi-PGF2α for 48 h.Real-time polymerase chain reaction was performed to detect the messenger RNA(mRNA)levels of a-smooth muscle actin(a-SMA)and collagen I.Comparisons between the groups were performed using Student’s t test.Results:TxA2r was up-regulated in activated PSCs in vitro compared with quiescent PSCs(all P<0.001).Compared with the control group,different concentrations of 8-epi-PGF2α significantly increased mRNA levels of a-SMA(10^-6 mol/L:2.23±0.18 vs.1.00±0.07,t=10.70,P<0.001;10^-7 mol/L:2.91±0.29 vs.1.01±0.08,t=10.83,P<0.001;10^-8 mol/L,1.67±0.07 vs.1.00±0.08,t=11.40,P<0.001)and collagen I(10^-6 mol/L:2.68±0.09 vs.1.00±0.07,t=24.94,P<0.001;10^-7 mol/L:2.12±0.29 vs.1.01±0.12,t=6.08,P<0.001;10^-8 mol/L:1.46±0.15 vs.1.00±0.05,t=4.93,P=0.008).However,different concentrations of SQ29548 all significantly reduced the expression of collagen I(10^-4 mol/L:0.55±0.07 vs.1.00±0.07,t=10.47,P<0.001;10^-6 mol/L:0.56±0.10 vs.1.00±0.07,t=6.185,P<0.001;10^-7 mol/L:0.27±0.04 vs.1.00±0.07,t=15.41,P<0.001)and a-SMA(10^-4 mol/L:0.06±0.01 vs.1.00±0.11,t=15.17,P<0.001;10^-6 mol/L:0.28±0.03 vs.1.00±0.11,t=11.29,P<0.001;10^-7 mol/L:0.14±0.04 vs.1.00±0.11,t=12.86,P<0.001).After being treated with SQ29548(10^-4 mol/L)and then 8-epi-PGF2α(10^-7 mol/L),the mRNA levels of a-SMA(0.20±0.08 vs.1.00±0.00,t=17.46,P<0.001)and collagen I(0.69±0.13 vs.1.00±0.00,t=4.20,P=0.014)in PSCs were significantly lower than those of the control group.Conclusions:The results show that 8-epi-PGF2α promoted PSCs activation,while SQ29548 inhibited PSCs activation induced by 8-epi-PGF2α.The result indicated that TxA2r plays an important role during PSC activation and collagen synthesis induced by 8-epi-PGF2α in vitro.This receptor may provide a potential target for more effective antioxidant therapy for pancreatic fibrosis.