Study On The Mechanism Of SENP1 Regulating Liver Sinusoidal Endothelial Cells To Promote Liver Regeneration By Relying On HIF-1 α Signal Pathway

Objective:The ability of the liver to recover and regenerate after partial hepatectomy(PHx)is unique,but small-for-size syndrome(SFSS)is still unavoidable.Liver sinusoidal endothelial cells(LSECs)are the gatekeepers of the liver and are in contact with the outside world.LSECs can promote and regulate not only the bidirectional transfer of substrates between blood and liver parenchyma,forming a blood-hepatocyte barrier,but also the production of various paracrine and autocrine cytokines,such as HGF,to regulate hepatocyte proliferation and form new blood vessels in the process of regeneration.Therefore,LSECs plays a very important role in the process of liver regeneration.But the specific molecular mechanism behind it is still unknown.Recent studies have shown that the process of liver regeneration after hepatectomy is carried out in a state of hypoxia,which can promote liver regeneration.Then LSECs,which have a key role in liver regeneration,must also play a role in promoting liver regeneration in hypoxic microenvironments.However,when hepatic sinusoids are hypoxic and ischemic,LSECs will suffer hypoxic damage,and in parallel,hepatic sinusoids will also experience pathophysiological processes of ischemia and reperfusion.Therefore,LSECs will also suffer from hypoxia-reoxygenation injury(H-R).After LSECs are injured,their unique fenestrations structure will disappear,and thus transform into a dedifferentiated state,and eventually lose the effect of promoting liver regeneration.So after hepatectomy,how LSECs can adapt to the hypoxic environment,reduce its hypoxia and reoxygenation damage,and continue to play its normal function of promoting liver regeneration,and the specific molecular mechanism behind this is still inconclusive.In the early stage,we found that the expression of SENP1,HIF-1 a and VEGF in hypoxia cultured LSECs was significantly higher than that in the control group.Recent studies have reported that SENP1 can promote the stability of HIF-1 a and up-regulate its expression,thus acting on downstream VEGF,and finally promote tumor proliferation and angiogenesis.Is there a similar process in the process of liver regeneration?According to this,we speculate that after PHx,LSECs in hypoxic state may up-regulate the expression of VEGF by regulating the signal axis of SENP1/HIF-1 α,and then promote the secretion of HGF and other target proteins,thus promoting hepatocyte and hepatic vascular regeneration.In this study,LSEC was used as the target cell,SENP1 as the cut-in point,and 70%hepatectomy mice as the model.Through in vivo and in vitro experiments,we explored the mechanism of SENP1 regulating LSECs to promote liver regeneration by relying on HIF-1 a signal pathway,so as to provide theoretical basis for clinical prevention of liver regeneration disorders.Methods:1.Firstly,the mouse model of 70%hepatectomy was established to determine the expression of SENP1 in the process of liver regeneration.RT-PCR and WB experiments confirmed that the expression of SENP1 was up-regulated in the process of liver regeneration.Then the expression of SENP1 in liver tissue was down-regulated by adeno-associated virus AAV8-Senp1-RNAi,and the liver regeneration rate was determined to confirm that SENP1 can promote liver regeneration.At the same time,the pathological changes of liver tissue were detected by liver histopathology,the expression of Ki67 and vWF in liver tissue was detected by immunohistochemistry,the apoptosis of liver tissue was detected by TUNEL method,and the effect of SENP1 on liver tissue injury and hepatocyte proliferation was observed.Finally,we can decipher the effect of SENP1 on the liver regeneration process after PHx.2.The primary LSECs of mice were cultured in hypoxia to determine the expression of SENP1 in LSECs cultured in hypoxia.RT-PCR and WB experiments confirmed that the expression of SENP1 was up-regulated in LSECs hypoxia culture.Then the changes of fenestration and proliferation ability of LSECs were observed by scanning electron microscope,CCK8 and EdU detection.Finally,si-RNA silencing SENP1,HIF-1 α,RT-PCR and WB were used to detect the expression of downstream gene VEGF,and scanning electron microscope and EdU were used to detect the fenestration and proliferation ability of LSECs.From this,we can clarify the effect of SENP1 on the characteristic fenestration and proliferation ability of LSECs through HIF-1α signal.3.To establish a model of hypoxia-reoxygenation injury of mice primary LSECs in vitro,and to observe the expression of LSECs after H-R.RT-PCR and WB experiments confirmed that the expression of SENP1 was up-regulated after hypoxia-reoxygenation in LSECs.Then the fenestration,proliferation and apoptosis of LSECs were observed by scanning electron microscope,CCK8 and flow cytometry.Finally,SENP1 expression was silenced by si-RNA,and HIF-1α signal was activated by HIF-1α agonist.RT-PCR and WB were used to detect the expression of HIF-1α,HO-1,apoptosis-related proteins cleaved-Caspase3,Bax and Bcl2,ELISA was used to detect the expression of VEGF,IL-6 and TNF-α,and then the fenestration,proliferation and apoptosis of LSECs were observed by scanning electron microscope,CCK8 and flow cytometry.It can be confirmed that SENP1 attenuates hypoxia-reoxygenation injury of LSECs through HIF-1α signal.4.In vitro experiment:SENP1 overexpression plasmid was transfected into mouse primary LSECs.And the expression of SENP1 in LSECs was detected by WB and RT-PCR.The fenestration and proliferation activity were detected by scanning electron microscope and CCK8.The expression of HIF-1α and Cyclin D1 was detected by WB,and the changes of cell cycle were detected by flow cytometry.Then the LSECs overexpressing SENP1 was co-cultured with hepatocytes,and the proliferation activity of liver was observed by EdU,and the expression of VEGF and HGF was detected by ELISA.In vivo experiment:AAV8-Senp1-RNAi was transfected into mouse liver to down-regulate the expression of SENP1,and DMOG activated HIF-1α signal,then the expression of LYVE-1,a specific marker of hepatic sinusoidal vessels,was observed by immunofluorescence,the expression of SENP1,HIF-1α,VEGFR2,Idl and WNT2 in liver tissue was detected by WB,and the expression of VEGF and HGF was detected by RT-PCR.Therefore,it is clear that SENP1 through HIF-1α acts on the VEGF/VEGFR2/Idl signal axis to regulate LSECs to promote liver regeneration.Results:1.The mouse model of 70%hepatectomy was successfully established.Liver regeneration began on the 1st day after hepatectomy,and the expression of SENP1 in liver tissue was significantly up-regulated on the 1st and 3rd day after hepatectomy.However,after silencing SENP1 with AAV8-Senp1-RNAi,the 5-day survival rate decreased,the transaminase increased significantly,and the liver regeneration rate decreased significantly;HE staining showed that the edema of liver cells was aggravated,the tissue structure was disordered,and the storage of lipid droplets slowed down;immunohistochemistry showed that the expression of Ki-67 and vWF in liver tissue decreased;TUNEL showed that hepatocyte apoptosis index increased.These results suggest that SENP1 may promote liver regeneration by reducing liver tissue injury and promoting hepatocyte proliferation and vascular proliferation.2.When LSECs were cultured in hypoxia,scanning electron microscopy showed that the fenestrae lasted longer,CCK8 and Edu showed that the proliferation activity was enhanced,and RT-PCR and WB showed that the expression of SENP1,HIF-1αand VEGF was up-regulated.However,after the use of si-SENP1 or si-HIF-1α,WB and ELISA showed that the expression levels of SENP1,HIF-1α and VEGF in LSECs decreased,and the fenestrae maintenance time became shorter and the proliferation activity decreased.It is suggested that during hypoxia culture,LSECs may promote the fenestration maintenance and proliferation ability of LSECs through SENP1/HIF-1α/VEGF signal.3.After hypoxia-reoxygenation culture of LSECs,the expression of SENP1 was up-regulated by RT-PCR and WB,and the damage of fenestrae was aggravated by scanning electron microscope,CCK8 showed impaired proliferative activity,and flow cytometry showed that its apoptosis was aggravated.After silencing SENP1 with siRNA,the proliferation activity of LSECs increased,the fenestrae damage increased,and the apoptosis rate increased,and the expression of SENP1,HIF-1α,HO-1 and Bcl-2 was down-regulated by WB,while the expression of apoptosis-related proteins cleaved-Caspase3 and Bax was up-regulated.ELISA detection showed that the level of VEGF in the supernatant decreased,and the level of IL-6 and TNF-α increased.After the use of HIF-1α signal pathway agonist(Rescue group),the situation was reversed.These results suggest that SENP1 attenuates the apoptosis rate and fenestrae damage of LSECs after H-R through HIF-1α signal pathway.4.The proliferation activity of LSECs with overexpression of SENP1 was increased and the fenestration was maintained better;WB detection showed that the expression of SENP1,HIF-1 a and CyclinD1 was up-regulated,and flow cytometry showed that the proportion of cells in S phase increased.After LSECs overexpressing SENP1 were co-cultured with hepatocytes,the proliferation ability of hepatocytes was significantly increased by EdU assay,and the levels of VEGF and HGF in the supernatant of hepatocytes were significantly increased by ELISA assay,but the above conditions were reversed after the use of SENP1 inhibitors or HIF-1 a inhibitors.In vivo experiment,after silencing SENP1,the expression of LYVE-1 in liver tissue was down-regulated by immunofluorescence,and the expression of Ki-67 was down-regulated by immunohistochemistry.The expression of SENP1,HIF-1 α,Idl and WNT2 was down-regulated by WB,and the expression of VEGF and HGF was down-regulated by RT-PCR after hepatectomy.However,the above situation was reversed after the use of proline hydroxylase inhibitor(DMOG),a HIF-1 a signal activator.It is suggested that SENP1 may regulate the VEGF-VEGFR2-Idl-HGF signal axis in LSECs through HIF-1 a to promote liver regeneration after partial hepatectomy.Conclusions:1.SENP1 can promote liver regeneration after partial hepatectomy by reducing liver tissue injury,promoting hepatocyte proliferation and angiogenesis;2.LSECs cultured under hypoxic conditions may maintain fenestrae and promote proliferation through the SENP1/HIF-1α/VEGF signaling axis,thereby adapting to the hypoxic environment;3.SENP1 can reduce LSECs apoptosis rate and maintain fenestrae through HIF-lα signal,thus reducing the hypoxia-reoxygenation injury of LSECs;4.SENP1 may promote liver regeneration after partial hepatectomy by regulating VEGF-VEGFR2-Idl-HGF signal axis in LSECs through HIF-1 α.