Font Size: a A A

ATF4/TRIB3 Pathway Induces Mitochondrial Stress To Promote Activation Of Hepatic Stellate Cells

Posted on:2024-03-22Degree:MasterType:Thesis
Country:ChinaCandidate:M P YeFull Text:PDF
GTID:2544307082466484Subject:Pharmacology
Abstract/Summary:PDF Full Text Request
Objective:The activation of hepatic stellate cells(HSCs)is central to the pathogenesis of liver fibrosis,which directly leads to fibrotic pathological changes in the hepatic tissue.Mitochondrial stress exacerbates inflammatory diseases by inducing pathogenic shifts in normal cells,but the role of mitochondrial stress in HSC activation remains to be elucidated.In this study,we investigated the effect of mitochondrial stress on HSC activation in vitro.The purpose of this study is to investigate whether transcription activator 4(ATF4)can induce mitochondrial stress by up-regulating Tribble homologue 3(TRIB3),thus activating HSC.Method:(1)In vitro experiment,the HSC-T6 cell model activated by 10 ng/m L PDGF-BB was first established,and then cultured in complete medium for 24 hours.The expressions ofα-SMA,ATF4,Collagen-1,Cyclin D1 and PCNA were detected by Western blot and RT-q PCR.;Flow cytometry was used to detect the distribution of cell cycle,and EDU staining showed the number of cell proliferation.(2)To explore the effect of ATF4 on the activation and proliferation of HSC-T6,overexpression of ATF4by pc DNA3.1 ATF4 transfection into HSC-T6 cells and silencing of ATF4 by si RNA technology,and detection of Collagen-1,α-The expression of SMA,Cyclin D1,PCNA,and cell cycle distribution,EDU staining showed the number of cell proliferation to explore the activation and proliferation of HSC-T6 cells by ATF4.(3)To explore the effect of ATF4 on mitochondrial stress,in HSC-T6 cells treated with ATF4 si RNA and pc DNA3.1 ATF4,the expression of mitochondrial stress-related markers HSP60,Clp P and LONP1 was detected,the mitochondrial morphology in HSC-T6 cells was detected by transmission electron microscopy,intracellular ROS levels were measured by flow cytometry,mitochondrial ROS levels and mitochondrial membrane potential were measured by laser confocal method,and the intracellular ATP content was detected.To explore the impact on mitochondrial function.(4)To explore the relationship between mitochondrial stress and activated proliferation of HSC-T6 cells,HSC-T6 cells were treated with azaramide,an inhibitor of UPRmt,and Erso,an activator,to detect the effect on their activation and proliferation.(5)To explore the relationship between ATF4 and TRIB3,observe the expression of TRIB3 by silencing and overexpressing ATF4,and verify the relationship between ATF4 and TRIB3 by CHIP-PCR.(6)The effects of silencing and overexpression of TRIB3 on HSC-T6activation,proliferation and mitochondrial function were examined.Results:(1)ATF4 is highly expressed in activated HSC-T6 cells.(2)ATF4 silencing can inhibit the activation and proliferation of HSC-T6,which was mainly reflected in the decreased expressions of colinea-1,α-SMA,Cyclin D1 and PCNA,and inhibited the transition of HSC-T6 cells from G0/G1 to S phase;the number of EDU-positive cells decreased.Overexpression of ATF4 promoted its activation and proliferation.(3)The treatment of the silencing of ATF4 improved mitochondrial stress,which was mainly manifested in the reduction of the expression of HSP60,Clp P and LONP1,the reduction of mitochondrial swelling,crista rupture,vacuolation and other injuries,the reduction of the increase of ROS levels in cells and mitochondria,the improvement of the increased membrane potential,and the reduction of ATP content;Overexpression of ATF4 promoted mitochondrial stress response.(4)Azoramide,UPRmtinhibitor suppressed activation and proliferation of HSC-T6 cells,and Erso,the activator,promoted the activation and proliferation of HSC-T6 cells.(5)Silencing ATF4down-regulated the expression of TRIB3,overexpression of ATF4 up-regulated the expression of TRIB3,and Ch IP-PCR results showed that ATF4 directly bound to a specific region of the TRIB3 promoter(903--762bp).(6)After silencing TRIB3 in HSC-T6 cells,the expressions of Collagen-1,α-SMA,Cyclin D1,PCNA,HSP60,Clp P and LONP1 were all decreased,while the overexpression of TRIB3 was the opposite result.Conclusion:(1)ATF4 participates in regulating mitochondrial stress response in HSCs.(2)Mitochondrial stress induced by ATF4 induces activation of HSCs.(3)TRIB3,as a downstream target gene of ATF4,participates in the activation of hepatic stellate cells induced by mitochondrial stress promoted by ATF4.
Keywords/Search Tags:hepatic fibrosis, hepatic stellate cells, mitochondrial stress, transcriptional activator 4, tribble homolog 3
PDF Full Text Request
Related items