The Effect And Mechanism Of GGPPS In The Pancreatic Fibrosis Of Chronic Pancreatitis

Part Ⅰ Correlation study between GGPPS expression and pancreatic fibrosis and pancreatic stellate cell activation in chronic pancreatitisBackground: Pancreatogenic diabetes mellitus is a type of diabetes secondary to pancreatic exocrine disease with impaired glucose regulation,also known as type 3c diabetes(T3c DM).The most identified cause of T3 c DM is chronic pancreatitis(CP).The pathological features of CP mainly include ductal dilatation,acinus atrophy,diffuse interstitial fibrosis and somewhat enlarged islets in the late CP.Activated pancreatic stellate cells(PSC)play a central role in fibrogenesis associated with CP.Previous studies demonstrated that statin has beneficial effect on some organ fibrosis mainly through inhibition of hydroxy-methyl-glutarylcoenzyme A(HMG-Co A)reductase activity and downstream proteins prenylation.However,the clinical application of statins for anti-fibrosis is still controversial,mainly due to the lack of effective concentration in vivo and multiple sites of action resulting in adverse reactions such as hepatotoxicity and myotoxicity.Geranylgeranyl diphosphate synthase(GGPPS)was an important branch enzyme in the mevalonate pathway,catalyzing the synthesis of geranylgeranyl pyrophosphate(GGPP)from farnesyl pyrophosphate(FPP)and accurately regulating the isoprene modification of small G proteins in cells.Therefore,GGPPS may be associated with pancreatic fibrosis and PSC activation in CP.Objectives: To investigate the expression of GGPPS in human CP clinical pancreatic tissue samples and CP mouse pancreatic tissue,as well as primary PSC.Methods: In this study,clinical and basic methods were combined including clinically collected pancreatic tissue samples from CP group and control group and experimentally constructed CP mouse model by continuous intraperitoneal injection of cerulein,a classical modeling method.The expression of GGPPS in CP and activated PSC was evaluated by immunostaining,western blot and biochemical assay in pancreatic tissue and primary cells.Results: 1.Clinically,compared with the control group,GGPPS expression was increased in the fibrotic pancreatic tissue of the CP group.2.GGPPS was highly expressed in the activated PSC in the CP group compared to the control group in clinically.3.During the process of establishing mouse CP animal model,compared with the control group,the weight gain of mice in CP group gradually slowed down,with a significant difference starting from the third week.4.There was no significant change in fasting blood glucose in CP mice compared with the control group.5.In CP mouse model,the serum amylase showed a decreasing trend compared with the control group,but there was no statistical difference.6.In the CP mouse model,GGPPS expression was increased in the fibrotic pancreatic tissue compared with the control group.7.And GGPPS expression was increased in primary PSC compared with control group.Conclusions: GGPPS was highly expressed in CP and activated PSC,suggesting that GGPPS may be involved in the activation of PSC and fibrosis progression of CP.Part Ⅱ Interventional study of knockdown GGPPS in vivo by VitA-lip-siRNA on pancreatic fibrosis in chronic pancreatitisBackground: Chronic pancreatitis(CP)is a progressive fibrosis of the pancreas characterized by irreversible destruction of pancreatic exocrine,extensive fibrosis,and later changes in islet area.Activated pancreatic stellate cell(PSC)are the main effector cells of pancreatic fibrosis in CP.Thus,targeting activated PSC has gradually become the main direction for the treatment of pancreatic fibrosis.In the first part,it’s found that GGPPS expression was increased in pancreatic tissue of CP and activated PSC,which suggested that GGPPS may participate in the activation of PSC or pancreatic fibrogenesis of CP,but it still need to confirm through further in vivo study.Thus,it will provide a powerful experimental basis for targeting PSC to knock down GGPPS to alleviate the fibrosis degree of CP.Objectives: To explore whether VitA-lip-siRNA-GGPPS can target PSC to knock down GGPPS and alleviate CP fibrosis by inhibiting the activation of PSC.Methods: In this part,based on CP mouse model established by repeated and continuous intraperitoneal injection of cerulein,VitA-lip-siRNA-GGPPS or NC were injected by tail intravenous to interfere GGPPS in vivo.Pancreatic fibrosis in CP and activation of primary PSC were evaluated by immunostaining,western blot and biochemical assay.Results: 1.Compared with the control group injected with VitA-lip-siRNA-NC,GGPPS in the pancreatic tissue of the intervention group injected with VitA-lip-siRNA-GGPPS could be knocked down after 1d of the first injection,and the expression of GGPPS in the pancreatic tissue returned to the normal level after 2 days.2.Compared with the control group injected with VitA-lip-siRNA-NC,there were no significant differences in the pancreatic weight,the ratio of pancreatic weight to body weight of mice and serum amylase in the intervention group injected with VitA-lip-siRNA-GGPPS,but the serum amylase tended to increase.3.Compared with the control group injected with VitA-lip-siRNA-NC,the pancreatic tissue in the intervention group injected with VitA-lip-siRNA-GGPPS showed more intactstructure and significantly reduced fibrous deposition;immunohistochemical staining showed decreased expression of GGPPS in pancreatic tissue;western blot showed that GGPPS,α-SMA and Col I were significantly decreased,but FN was not significantly decreased,and amylase was increased.4.Compared with the control group injected with VitA-lip-siRNA-NC,the expression of GGPPS,a-SMA and Col I in the intervention group injected with VitA-lip-siRNA-GGPPS in the primary PSC was decreased,and FN was not significant.Conclusions: Specifically knocking down GGPPS in PSC by VitA-lip-siRNA can improve pancreatic fibrosis and amylase deficiency caused by CP.Part Ⅲ Mechanism researches of GGPPS involved in TGF-β1-induced activation of pancreatic stellate cellsBackground: Geranylgeranyl diphosphate synthase(GGPPS)is a key branch enzyme of the mevalonate pathway,which affects the occurrence of a variety of diseases by regulating the isoprene of small G proteins in cells.Previous studies have confirmed that GGPPS expression is increased in pancreatic tissues with chronic pancreatitis(CP)and in activated pancreatic stellate cell(PSC);in vivo targeted PSC knockdown of GGPPS can alleviate pancreatic fibrosis and CP induced amylase deficiency.In conclusion,the results suggest that GGPPS is involved in PSC activation during CP,but the specific mechanism of GGPPS involved in PSC activation is still unclear.Objectives: To investigate the expression and related mechanism of GGPPS in TGF-β1-induced activation of PSC and the mechanism of GGPPS in PSC activation.Methods: In the cell model of TGF-β1 induced PSC activation,siRNA transfection,adenovirus infections were conducted to knock down and overexpress GGPPS,respectively.Then western blot detection,real-time fluorescent quantitative PCR,phalloidin staining and protein isoprene detection were used to identify PSC activation and GGPPS expression to confirm the relationship between TGF-β1-induced PSC activation and GGPPS expression and the mechanism of GGPPS involvement in PSC activation.Activation of the TGF-β/Smad,MEK/ERK1/2 signaling pathway in PSC were blocked by SB431542,U0126,respectively.And adenovirus infection Egr-1 in PSCwas used to overexpress Egr-1.The mechanism of GGPPS expression in TGF-β1-induced activation of PSC was detected by western blot.Results: 1.GGPPS expression was increased during TGF-β1-induced PSC activation in a concentration-and time-dependent manner.In this study,7.5ng/m L TGF-β1 was treated with PSC for 24 h as a cell model of TGF-β1-induced PSC activation.2.Compared with the control group treated by TGF-β1 after siRNA-NC transfection,the expression of GGPPS,α-SMA,Col I and FN decreased in the experimental group treated by TGF-β1 after siRNA-GGPPS transfection.3.Phalloidin staining showed that compared with the siRNA-NC group,the morphology of PSC cell in siRNA-GGPPS group was nearly spherical with less polygonal shape.Compared with the siRNA-NC group,PSC in the siRNA-NC+TGF-β1 group showed obvious polygonal and stellar shape.Compared with the siRNA-NC+TGF-β1 group,the cells in the siRNA-GGPPS +TGF-β1 group were still round and the polygonal tentacles disappeared.4.q RT-PCR and cell fluorescence were used to select the appropriate adenovirus amount of 4u L for overexpression of GGPPS.5.PSC was infected with adenovirus overexpressing GGPPS,and the results showed that GGPPS,α-SMA,Col I,and FN were increased in the group infected with the overexpressing GGPPS adenovirus compared with the control group infected with the GFP-containing adenovirus.6.The suitable MOI of lentiviruses(GL407NC2,Y10360,Y10361 and Y10362)to infect PSC was 80,and the appropriate concentration of puromycin was 4ug/m L.On this basis,the lentivirus that can effectively knock down GGPPS is Y10362 and the stable PSC with interfering GGPPS was constructed by using this lentivirus.7.Based on the PSC stable interfering GGPPS,the isoprenoid protein detection showed that Rho A in the membrane was significantly decreased,and the ratio of membrane/cytoplasm was also significantly decreased in the Interfering-GGPPS+TGF-β1 group,compared with NC+TGF-β1 group.8.The TGF-β/Smad signaling pathway was blocked by SB431542.The results showed that with the increase of the concentration of SB431542 inhibitor,the inhibitory effect increased gradually,Col I and FN decreased gradually,and α-SMA and GGPPS did not change significantly.In addition,the activation of ERK1/2 in the non-Smad signaling pathway was gradually increased during the blocking of Smad signaling pathway.9.The MEK/ERK signaling pathway was blocked by U0126.The results showed that with the increase of U0126 inhibitor concentration,the inhibitory effect was gradually increased,and α-SMA,Col I,FN and GGPPS were gradually decreased.In addition,during the blocking of MEK/ERK1/2 signaling pathway,Egr-1,the GGPPS regulator,also decreased gradually.10.Further prolonging the time of TGF-β1 induction showed that the expression of α-SMA and GGPPS was always decreased with the prolonging of TGF-β1 induction time,and EGR-1 was still decreased during this process.11.The results of Egr-1 overexpression showed that Egr-1,α-SMA and GGPPS expression were increased in overexpressed-EGR-1+U0126+TGF-β1 group compared with that in adenovirus-GFP +U0126+TGF-β1 group,while Col I and FN were still decreased.Conclusions: In vitro cell experiments confirmed that GGPPS expression was increased in TGF-β1-induced PSC activated cell model.The knockdown and overexpression of GGPPS confirmed that GGPPS were involved in TGF-β1-induced activation of PSC.TGF-β1 can increase the expression of GGPPS in activated PSC through ERK1/2/ Egr-1 signaling pathway,and GGPPS with high expression can regulate Rho A membrane translocation and participate in the activation of PSC.Part Ⅳ Effect of GGPPS expression by activated pancreatic stellate cell on pancreatic acinar cellBackground: Chronic pancreatitis(CP)is a continuous progressive disease characterized by atrophy of pancreatic acinar cell(PAC)and diffuse fibrosis.The main clinical outcome of long-term CP is nutritional deficiency caused by decreased pancreatic protease and impaired glucose regulation caused by pancreatic islet dysfunction.The results of previous studies showed that serum amylase decreased in CP mouse model,and amylase decreased significantly in pancreatic tissue protein;on the basis of CP model,Geranylgeranyl diphosphate synthase(GGPPS)knockdown by targeting pancreatic stellate cell(PSC)resulted in the increase of serum amylase,and the level of amylase in pancreatic tissue protein was significantly increased.Activated PSC is the key effector cell of pancreatic fibrosis in CP,and the continuous activation of PSC is the main source of the development of pancreatic fibrosis.However,the effect of activated PSC on the expression of amylase in PAC still needs to be further confirmed by in vitro experiments.Objectives: To investigate the effect of PSC activation on the synthesis and secretion of amylase in PAC and knockdown GGPPS in PSC on the production and secretion of amylase in PAC.Methods: The cell model of TGF-β1 induced PSC activation,adenovirus infections with overexpression GGPPS in PSC and lentivirus infections with downregulation GGPPS in PSC were co-cultured simulatively with PAC,respectively.The PSC activation and GGPPS expression,the amount of amylase and acinar cell viability were detected by western blot,starch-iodine colorimetry and Cell Counting Kit-8(CCK-8)to investigate the effect of PSC activation on the synthesis and secretion of amylase in PAC and knockdown GGPPS to ameliorate PSC activation on the effect of PAC.Results: 1.The cell model of TGF-β1 induced PSC activation was co-cultured simulatively with PAC,and the results showed that:(1)Amylase in medium: Amylase in medium of PAC in TGF-β1-induced PSC activation group was significantly reduced compared to that in the group without TGF-β1-induced PSC activation group.(2)Cellular amylase: Cellular amylase was significantly reduced in PAC in the TGF-β1-induced PSC activation group compared to the group without TGF-β1-induced PSC activation.(3)Cell viability: The cell viability of PAC in the TGF-β1-induced PSC activation group was significantly reduced compared with that in the non-TGF-β1-induced PSC activation group.2.Knockdown GGPPS changes the activation of PSC on effect of PAC,the results showed that:(1)Amylase in medium: Compared with overexpression GGPPS group,amylase in medium of PAC in GGPPS knockdown group increased significantly after 72 h.(2)Cellular amylase: Compared with overexpression GGPPS group,cellular amylase in PAC of GGPPS knockdown group increased significantly after 72 h.(3)Cell viability: Compared with overexpression GGPPS group,the cell viability of PAC in GGPPS knockdown group was significantly increased.Conclusions: Activated PSC is involved in regulating the synthesis and secretion of amylase in PAC,and knockdown GGPPS to change the activation of PSC can further improve the synthesis and secretion of amylase in PAC,but the specific mechanism still needs to be further studied.