The Effect Of Endoplasmic Reticulum Stress On EP1 Receptor And EP3 Receptor Induced By TGF-?1 In Mice Mesangial Cells And Its Associated Mechanism

Objective To explore the effects and mechanisms of the prostaglandin E2(PGE2) receptor E-prostanoid 1(EP1) and the prostaglandin E2(PGE2) receptor E-prostanoid 3(EP3) on endoplasmic reticulum stress induced by TGF-?1 in mouse mesangial cell.Methods in vitro1. Primary cultured EP1-/-,EP3-/- and WT mice glomerular mesangial cells.2 grouping:EP1 grouping(1)EP1-/- mice glomerular mesangial cells grouping(1)WT control group,(2)WT +TGF-?1 group,(3)EP1-/- control group,(4)EP1-/-+TGF-?1 group;( 2) EP1 agonist mice glomerular mesangial cells grouping(1)WT control group,(2)WT +TGF-?1 group,(3)EP1 agonist control group,(4)EP1 agonist +TGF-?1 group;(3)SC-19220 mice glomerular mesangial cells grouping(1)WT control group,(2)WT +TGF-?1 group,(3)SC-19220 control group,(4)SC-19220 +TGF-?1 group?EP3 grouping(1)EP3-/- mice glomerular mesangial cells grouping(1)WT control group,(2)WT +TGF-?1 group,(3)EP3-/- control group,(4)EP3-/-+TGF-?1 group;( 2) Sulprostone mice glomerular mesangial cells grouping(1)WT control group,(2)WT +TGF-?1 group,(3)Sulprostone control group,(4)Sulprostone +TGF-?1 group;(3)L-798106 mice glomerular mesangial cells grouping(1)WT control group,(2)WT +TGF-?1 group,(3)L-798106 control group,(4)L-798106 +TGF-?1 group.3.Expression of CTGF, LN,FN, GRP78, TRPC1 protein and m RNA was examined by Western blotting and RT-PCR, ERK1/2 or phospho-ERK1/2 was measured by Western blotting as well.4.The proliferation degree of mesangial cell by MTT.5. The cell apoptosis was measured by flow cytometry.In vivo1. The set up of 5/6 nephrectomize model.2.The serum levels of blood urea nitrogen(BUN), serum creatinine(SCr),and urine osmolality in rats were measured.3. Immunohistochemical staining were used to detect degrees of accumulation and glomerular sclerosis with associated fibrosis indexes.Results in vitro1.Identification EP1-/- and EP3-/- mice DNA by PCR and Western blot.2. TGF-?1 significantly upregulated the expression of FN ? CTGF ?GRP78?TRPC1 m RNA and protein. Expression of phospho-ERK1/2 protein was increased(P < 0.05). The values of EP1-/- +TGF-?1 groups were significantly lower than those of WT +TGF-?1 groups on GRP78, TRPC1 detected by protein and m RNA(P<0.05). Expression of phospho-ERK1/2 protein was decreased(P<0.05); EP1 agonist significantly up-regulationed above changes and their activities(P<0.05). All the effects of EP1 agonist were high-dependent; SC-19220 significantly attenuated above changes and their activities(P<0.05). All the effects of SC-19220 were dose-dependent.3. TGF-?1 significantly upregulated the expression of GRP78 ? TRPC1 m RNA and protein. Expression of phospho-ERK1/2 protein was increased(P <0.05). The values of EP3-/- +TGF-?1 groups were significantly lower than those of WT +TGF-?1 groups on GRP78, TRPC1 detected by protein and m RNA(P<0.05). Expression of phospho-ERK1/2 protein was decreased(P < 0.05);Sulprostone significantly up-regulationed above changes and their activities(P <0.05). All the effects of Sulprostone were high-dependent; L-798106 significantly attenuated above changes and their activities(P < 0.05). All the effects of L-798106 were dose-dependent.4. Compared with the control group, TGF-?1 significantly stimulated mesangial cells proliferation(P < 0.05); compared with the WT+TGF-?1 group,EP3-/- significantly decreased mesangial cells proliferation(P < 0.05).5. Compared with the control group, TGF-?1 significantly stimulated mesangial cells apoptosis(P < 0.05); compared with the WT+TGF-?1 group, EP1-/- ?EP3-/- ignificantly decreased mesangial cells apoptosis(P < 0.05).In vivo1.Compared to the respective sham group, 5/6 nephrectomy model group had higher levels of serum creatinine and BUN but lower urine osmolality.whereas the changes of EP1-/- ? EP3-/- 5/6 nephrectomy modwl group were significantly lower than those in the EP1+/+ ? EP3+/+ 5/6 nephrectomy model group( P<0.05).2. In 5/6 nephrectomy model kidneys, the interstitial fibrosis including tubular atrophy, loss and dilation, inflammatory cell infiltration and interstitial matrix deposition was prominent,whereas the changes of EP1-/- 5/6 nephrectomy group were significantly lower than those in EP1+/+5/6 nephrectomy group;similarly, the changes of EP3-/- 5/6 nephrectomy group were significantly lower than those in EP3+/+5/6 nephrectomy group.3. After receiving 5/6 nephroectomy for 8 weeks, Compared with the WT nx group, glomerular sclerosis and the desity of CTGF?GRP78 relieved in EP1-/-?EP3-/- nx group with staining( P<0.05).Conclusion TGF-?1 induced cell proliferation and extracellular matrix accumulation, increasing the synthesis of intracellular FN ? CTGF, restrain the expression of EP1 receptor and EP3 receptor could decreased the protein expression of FN ? CTGF ? GRP78 and TRPC1 induced by TGF-?1,then reducing mesangial cell damage. And through the 5/6 nephroectomy, further confirmed our study in vitro results.Presumably the relevant mechanisms may be related to the inhibition of endoplasmic reticulum stress.