Endoplasmic Reticulum Stress Mechanism Of Estrogen Improving Lymphatic Constriction Following Hemorrhagic Shock

Lymph circulation disturbance is one of important components of the theory of shock microcirculation dysfunction,and is closely associated with the prognosis of shock.Previous studies have indicated that exogenous 17β-estradiol(E2)significantly increased the contractility of mesenteric lymphatic vessels of rats with hemorrhagic shock,and endoplasmic reticulum stress(ERS)agonists suppressed this protective effect of E2,the role of ERS playing in estrogen improving the contractility of lymphatic vessels of rats with hemorrhagic shock needs to be further clarified.Therefore,to determine whether E2 can suppress ERS of shock lymphatic vessels,this study observed the influence of E2 on the expression of ERS marker protein in mesenteric lymphatic tissues of shock rat using a hemorrhagic shock model.Since the contraction of lymphatic smooth muscle cells(LSMCs)is the main internal driving force of lymph motion,intact contractile function of LSMCs is the basis of lymph flows transport.To this end,using LSMCs subjected to hypoxia/reoxygenation(H/R)to simulate the hemorrhage/resuscitation process in vivo,this study established a tunicamycin(TM)-induced ERS cell model,and observed the role and mechanism of ERS in E2 and its receptors(ERs)modulating of LSMCs contraction.Firstly,twelve male Wistar rats were randomly divided into 4 groups(n=3): sham(Sham),sham plus E2(Sham+E2),shock(Shock)and shock plus E2(Shock+E2).After hemorrhagic shock was established,the whole intestinal loop was resected.Mesenteric lymphatic vessels were separated from the intestinal loop with a stereoscope and immersed in lysis buffer to extract proteins.Western Blot method was used to detemine the expression of ERS marker protein glucose regulated protein 78 k Da(GRP78).The results showed that there was no significant difference in the expression of GRP78 in lymphatic tissues between sham and Sham+E2 groups.The expression of the marker in shock group was significantly higher than that in sham group,while the expression of GRP78 in lymphatic tissue of shock+E2 group was significantly lower than that of shock group.These results showed that E2 replacement can significantly reduce the lymphatic ERS caused by hemorrhagic shock.Secondly,after primary culture of LSMCs established and identified,immunofluorescence and Western Blot were used to detect the expression of estrogen receptor in LSMCs.The results showed that the primary cultured rat LSMCs was positive in the expression of α-SMA protein and had a purity of 99.4%.Moreover,LSMCs of rat was positive in the expression of estrogen receptors ERα and GPR30,and was weakly positive in the expression of ERβ.These results indicated that the primary culture of LSMCs was successfully established and was feasible to be used for the subsequent experiments.Thirdly,the LSMCs culture was passaged to about fifth generations and was used to establish H/R model in vitro.The experiment was divided into control group(Control),estrogen control group(E2),H/R group(H/R),E2 treatment group(H/R+E2),ERα agonist PPT treatment group(H/R+PPT),ERβ agonist DPN treatment group(H/R+DPN),GPR30 agonist G1 treatment group(H/R+G1),H/R combined with E2 and ER α,βinhibitor ICI182780 group(H/R+E2+ICI),H/R combined with E2 and GPR30 inhibitor G15 group(H/R+E2+G15).The viability of cells in each group was detected by CCK-8 method,and the contractile ability of cells in each group was observed using a Transwell culture system.The expression of GRP78 protein was detected by Western Blot.The results of cell viability showed that the viability of LSMCs decreased significantly after challenged by H/R.E2,DPN,PPT and G1 treatment significantly increased the viability of H/R LSMCs,while ICI and G15 significantly inhibited the effect of E2 on LSMCs viability.The study of cell contractility showed that E2 significantly improved the contractility of LSMCs insulted by H/R.The experimental results of DPN,PPT,G1 treatment on LSMCs contractility after H/R episode,ICI and G15 on E2 improving LSMCs contractility after H/R were being processed.The results of Western Blot showed that,when LSMCs was chelleged by H/R,there was no significant changes in GRP78,a marker protein reflecting the presence of ERS in LSMCs,at 3 h the end of reoxygenation after different time of hypoxia,the change of the marker wasn’t observed although E2 treatment.The experimental results showed that H/R decreased the viability and contractility of LSMCs,but did not cause ERS;E2 increase the viability and contractility of LSMCs challenged by H/R,which was realized by ERs.Finally,the LSMCs culture was passaged to about fifth generation and was used to establish TM inducing ERS model in vitro.The experiment was divided into Control,E2,TM,TM+E2,TM+PPT,TM+DPN,TM+G1,TM+E2+ICI and TM+E2+G15 groups to detect cell viability,cell contractility and the expression of ERS-related proteins GRP78,ATF6,IRE1 and PERK.The results showed that after TM treatment,the viability and contractility of LSMCs decreased significantly,while the expression of ATF6,GRP78 and IRE1 increased significantly.E2 treatment significantly increased the viability and contractility of LSMC cells after TM treatment,and decreased the expression of ATF6,GRP78 and IRE1.DPN,PPT and G1 treatment increased cell viability and contractility,significantly decreased the ATF6 and GRP78 expression of LSMC after TM treatment,and G1 decreased the IRE1 expression of LSMC after TM treatment.ICI and G15 significantly inhibited the effect of E2 on the viability and contractility of LSMC and the decrease of protein expression of ATF6,GRP78 and IRE1 after TM treatment.The results showed that E2 decreased the ERS of LSMCs induced by TM through ERs,and then increased the cell viability and contractility of LSMCs.Based on the above results,E2 treatment reduced the ERS of the mesenteric lymphatic tissue in hemorrhagic shock rats;H/R failed to cause ERS in LSMCs,but decreased the viability and contractility of LSMCs.E2 increased the viability and contractility of LSMCs after H/R through ERs.TM treatment could induce ERS in LSMCs and decrease cell viability and contractility.E2 decreased the ERS of LSMCs through ERs and increased the cell viability and contractility of LSMCs.In conclusion,the effect of estrogen on lymphatic constriction of rats with hemorrhagic shock is associated with the inhibition of ERS.This paper enriches the basic theory of lymphatic circulatory dysfunction after hemorrhagic shock,and targeting E2 provides a new idea for clinical prevention and treatment of severe hemorrhagic shock.