Studies On The Effect On Cell Injury Of Pancreatic Acinar Cell By Erythropoietin

[Background] Acute pancreatitis(AP) is a disease with complex etiology, but its’pathogenesis is not yet completely clear. There are about20%to30%patients with AP inChina may aggravate into severe acute pancreatitis (SAP), while，it can easily trended intomultiple organ failure. The fatality rate of all patients is between5%and10%. Currently,the main clinical treatments for AP are symptomatic treatment.There is no specifictreatments for this disease. Nowadays, erythropoietin (EPO) is used in clinic more andmore, furthermore, studies have shown that EPO, to a certain extent, can attenuate lunginjury in lipopolysaccharide treated rats.[Objective] To explore the effects of erythropoietin on LPS-induced cell injury ofpancreatic acinar cell[Methods] Pancreatic acinar cells (PAC) of SD rats were isolated by improved collagenasemethod and cultured in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12(DMEM/F12) medium.Observation of the change of PAC induce by differentconcentrations of LPS (5mg/L,10mg/L, and20mg/L,40mg/L). And the intervention thePAC with different concentration of EPO (3U/L,6U/L,12U/L, and24U/L) ordexamethasone (DEX)(50μg/L,100μg/L,200μg/L, and400μg/L),while DEX Groupas the control intervention group. Observation of the morphology changes of PAC of allgroups, Measurinig the cell concentration with counting plate,while, observation of the rateof PAC purity with methylene blue staining.Measuring the cell viability in each group withCCK-8kit.Measuring the concentration of amylase (AMS) in culture supernatant with theway of iodine colorimetry.To detect concentration of malondialdehyde (MDA) in culturesupernatant with MDA Kit. To detect the level of superoxide dismutase(SOD) activity in culture supernatants with SOD kit. The concentration of tumor necrosis factor-alpha(TNF-α), interleukin-1β (IL-1β) was detected by enzyme-linked immunosorbent assay(ELISA) kit.[Results]①There were (1±0.23)×107cells isolated from each SD rat. The freshlyisolated PAC vitality was (96±2.8)%,with purity above90%.②There is no obviousexpression of EPOR on PAC.③the vitality of freshly isolated PAC was relatively stablewithin6h, while, significantly decreased at12h.④the cell viability in LPS group decreasedin6h, after adding EPO with dose of6U/L,it decreased significantly. The cell viabilitieswere no significant difference among all group at3h and12h, but in EPO interventiongroup,The cell viability increased with the increasing concentration of EPO.⑤There wereno significantly difference in concentration of AMS among all groups at3h. Theconcentration of AMS in LPS group was significantly lower than it in the NS group at6h (P<0.05), The concentration of AMS in EPO intervention group was significantly lower thanit in the NS group at12h (P <0.05).⑥Compared with NS group, the MDA level in LPSgroup was lower, while, it in DEX intervention group was lower than it in LPS group (P<0.05)at3h. The MDA level in EPO pretreatment group was higher than it in LPS group (P<0.05)at3h. The MDA levels in EPO pretreatment group and DEX intervention groupwere higher than it in LPS group (P <0.05)at6h,but the MDA level in EPO pretreatmentgroup was higher than it in DEX intervention group(P <0.05). The MDA level in NS groupwas highest of all groups.,while the MDA level in EPO pretreatment group was lowest ofall groups.⑦There were no no significant difference (P>0.05)in SOD concentrationamong all groups at3h and12h. But at6h, compared with LPS group,the SOD level in theEPO pretreatment group, the EPO intervention group and DEX intervention group wasincreased (P <0.05), The SOD level in EPO pre-intervention group was highest.⑧The leveof TNF-α in DEX group was significantly lower than that in NS group (P <0.05)at3h;while, compared with LPS group,it in EPO treatment group and DEX treated group wassignificantly lower (P <0.05),especially for it in DEX treated group. Compared with NSgroup, the TNF-α level in the LPS group decreased significantly (P <0.05) at6h,while,theTNF-α level in the EPO treatment group, DEX treatment group, DEX group and the EPOgroup was increased significantly (P <0.05). Compared with NS group, the TNF-α levelsin DEX group, LPS group were significantly increased (P <0.05)at12h,but the TNF-α levelin DEX treated group was lower than it in LPS group (P <0.05). the TNF-α level in In LPSgroup were significantly lower at6h and then increased at12h,⑨The IL-1β level in LPS group was higher than it in the NS group (P <0.05)at3h, but as time goes on,the IL-1βlevel decreased gradually (P <0.05). Compared with the EPO group，the IL-1β level inDEX group reduced more significantly. The IL-1β level in the EPO group wassignificantly higher than it in the NS group at6h. Compared with the EPO group, IL-1βlevel in the LPS and EPO pretreatment group decreased at6h. There were no significantdifference in IL-1β level among all group at12h.[Conclusion]①The nature freshly isolated PAC is relatively stable to meet building cellmodel.②There is no or a very small amount of expression of EPOR on PAC.③LPS caninhibit secretory function of PAC,while,the EPO intervention may be extended LPSinhibition of secretory function of the PAC④EPO may down-regulated the level ofinflammatory factor in the early stage of cell injury, up-regulated the level of inflammatoryfactor in intermediate stage⑤The effects of EPO on LPS-induced cell injury of pancreaticacinar cell may not depend on EPOR.