Effect Of EPO Preconditioning On Hepatic Microcirculation In A Rat Model Of Limbs Ischemia Reperfusion Injury

Objective To observe the protective effect of EPO pretreatment on hepatic and hepaticmicrocirculation after establishing the rat hind limb Ischemia-Reperfusion Injury(LIRI)model. To investigate the changes in vivo rats hepatic blood flow,the different eNOSlevels in hepatic tissue,the extent of oxidative stress injury and the changes of hepatictissue following the ischemia-reperfusion injury. To discuss the correlation between thisseries of changes and the EPO protective effect on hepatic microcirculation and further toshow the EPO pretreatment protective effect on hepatic microcirculation in the LIRIpreliminarily.Methods Forty-five adult male Sprague-Dawley rats were used for LIRI model in thisstudy. The rats were randomly divided into three groups as described(n=15): Controlgroup; IR group; EPO pretreatment group(EPO+IR). Methods of treating ischemia4hours and then reperfusion2hours in rats were adopted in this study. EPO was given toEPO+IR group at a dose of3000U/kg30minutes before the beginning of repersusionby peritoneum injection and the same dose of NS were given to Control group and IRgroup. Changes in each group of rats in vivo hepatic microcirculation blood flow wereobserved using laser Doppler; Rats hepatic tissue morphology changes were measuredafter HE staining with an optical microscope; The level of AST、ALT in serum and thelevels of SOD and MDA in serum and hepatic tissue were detected. The expression ofeNOS in hepatic tissue was investigated using Immunohistochemical staining. Theexpression level of hepatic eNOS in hepatic tissue was detected using Western blot.Results1）The changes in the hepatic microcirculation blood flow observed by laserdoppler have shown in figure. Compared with Control group,IR group significantlydecreased and EPO+IR group also reduced to a certain extent while higher than IR group.This results showed EPO pretreatment have improved the hepatic microcirculation bloodflow following the LIRI.2）HE staining have observed changes in the rats hepatictissue:the hepatic tissue morphology of Control group is normal. But the hepatic lobulesstructural degraded、the hepatic sinusoid narrowing、the hepatic sinusoid varyingdegrees of central venous congestion in part of hepatic tissue、the inflammatory cellinfiltration etc. have found in IR group. Every pathology changes in EPO+IR group is weaker than IR group.3）Compared with Control group,IR group and EPO+IR grouphave increased significantly(P＜0.01). Meanwhile, compared with IR group,the serumlevel of ALT、AST in EPO+IR group have decreased obviously (P＜0.01). The resultshave shown the hepatic function were protected in a certain extent in EPO+IR group.4）The SOD vitality of hepatic tissue and serum significantly decreased (P＜0.01) in IRgroup and EPO+IR group while the MDA content obviously increased(P＜0.05) ascompared with Control group. But the SOD vitality in EPO+IR group increased higherthan IR group.Moreover, the MDA extent in EPO+IR group decreased higher than IRgroup (P＜0.05). These showed that EPO pretreatment mitigated lipid peroxidation.5）Hepatic immunohistochemical staining have shown that both IR group and EPO+IRgroup found eNOS protein granules stained brown（P＜0.01）while EPO+IR group wasmore obvious.6）Western blot experimental found that the expressing level of eNOSprotein in EPO+IR group was the highest meanwhile IR group did thesecond.Moreover,both EPO+IR group and IR group were higher than Control groupwhile it had predominantly statistical significance. These results have shown that EPOfurther improved the expressing and activating of eNOS in hepatic tissue following theLIRI.Discussion The reasons why EPO pretreatment can alleviate the rats LIRI hepatic injurymay be related to the following points: reduce hepatic microcirculation injury after IR;inhibit oxidative stress reactions in liver tissue; increase eNOS activation and expressionin hepatic tissue.EPO pretreatment can reduce hepatic tissue pathology changes with theLIRI happening in rats.To relatively increase the hepatic microcirculation blood flow byimproving the SOD vitality,alleviating oxidative stress by decreasing the MDAcontents,promoting the eNOS activation and increasing the P-eNOS expression. Thusthese processing paly a protective role in hepatic microcirculation.