| Research Backgroud:Ischemia reperfusion(I/R)injury is a common clinical pathological phenomenon,which can seriously affect the prognosis of patients.Remote ischemic preconditioning(RIPC)is an organ protection method that regulates the tolerance of target organs to I/R injury through neural and humoral pathways through transient ischemic and reperfusion preconditioning of one or more distant organs.Limb RIPC is one of the most simple and feasible RIPC methods at present,but its best mode of action for organ protection and mechanism of action in liver I/R injury are not completely clear,resulting in its limited clinical application.Research purpose:This study intends to evaluate the effects of different ischemic preconditioning methods on liver protection in rats by using the rat liver I/R injury model,identify the most effective RIPC methods of limbs,explore the possible mechanism of RIPC on liver protection,and finally further verify the effect of RIPC on liver protection in elderly rats.Experimental methods:1.Study on the optimization of hepatic protection mode of RIPC in unilateral limb:Male SD rats were randomly divided into Sham operation group,I/R group,IPC group,RIPC group immediately before hepatic ischemia.Distal limb ischemic preconditioning(RIPC ’)group 6 hours before hepatic ischemia.The classical rat model of 70% liver heat I/R was used,in which the left middle lobe of the liver was blocked by a noninvasive vascular clamp and reperfusion was performed after 60 minutes of ischemia.In Sham group,only laparotomy and vascular separation were performed without clipping ischemia.I/R group received classical liver heat I/R;Hepatic ischemia and reperfusion were performed 10 min before hepatic I/R in IPC group.In RIPC group,unilateral lower limbs of rats were performed immediately before liver I/R:(1)single cycle of 10 min ischemia-10 min reperfusion(RIPC-1×10),(2)3 cycles of 10 min ischemia-10 min reperfusion(RIPC-3×10),(3)4 cycles of 5min ischemia-5min reperfusion(RIPC-4×5)and(4)6 cycles of 4min Ischemia-4min reperfusion(RIPC-6×4);In the RIPC ’group,the same operation was performed 6 hours before liver I/R.Venous blood and liver tissue samples were collected at 2,6 and 24 h after liver reperfusion,and tissue and serum enzyme assessment were performed to compare the liver protection effect of RIPC in different forms of unilateral limb during liver I/R,and to determine the best way of liver protection.2.Study on the difference in protective effect and mechanism between unilateral and bilateral limb RIPC: SD rats were randomly divided into Sham group,I/R group,unilateral lower limb RIPC control(C-RIPC)group,unilateral lower limb RIPC(RIPC)group and bilateral lower limb RIPC(D-RIPC)group.The rat 70% liver heat I/R model was adopted,and the unilateral or bilateral lower limbs of rats were pretreated immediately before I/R by using the optimal RIPC method of unilateral limb identified in part I.To observe the effects of reperfusion on liver cell necrosis,apoptosis,serum enzyme,tissue cyclic adenosine phosphate(c AMP)concentration,reactive oxygen species(ROS)level and mitochondrial calcium capacity(CRC)at 2,6 and 24 h.The expression of er K1/2,P-ERK1/2,GSK-3β and P-GSK-3 β in liver tissues was evaluated by Western blot.3.Study on protective effect of unilateral limb RIPC on liver I/R injury in elderly rats: The aged SD rats were randomly divided into Sham group,I/R group,IPC group and RIPC group.The animals were treated with 70% liver heat I/R model and the best RIPC mode of unilateral limb.Liver injury and apoptosis were evaluated by serum enzymology and histopathology at 6h after reperfusion,the effects of RIPC on ultrastructure of liver cells were observed by transmission electron microscopy,and the levels of inflammatory factors and oxidative stress in liver tissues were determined by ELISA.4.Statistical analysis: SPSS 20.0 software was used for statistical processing,and Shapiro-Wilk test was used for normality test.If the data were not normally distributed,the experimental results were described by median and interquartile Range(IQR).Mann-whitney U test was used for comparison.If the data were normally distributed,the experimental results were expressed as mean ± standard deviation(`x ±s).Student’s T-test was used to analyze the differences between the two groups of data,ANOVA was used to compare the data between groups,and Graphpad Prism 8.0 software was used to plot.P <0.05 was considered statistically significant,P <0.01 considered that the difference was statistically significant.Research results:1.Optimization of hepatic protection methods of unilateral limb RIPC:(1)H-E and TUNEL staining results showed that compared with Sham group,the proportion of hepatic cell necrosis and positive percentage of apoptotic cell count in 2,6 and 24 H I/R reperfusion groups were significantly increased;Compared with I/R group,liver cell necrosis degree and percentage of apoptotic cell count in RIPC group and RIPC’group were significantly decreased at each time point of reperfusion.Compared with RIPC ’group,the degree of liver cell necrosis and apoptosis was significantly reduced in RIPC group at each time point of reperfusion,and the proportion of liver cell necrosis and apoptosis was the lowest in RIPC 4×5 group(i.e.,4 cycles of 5min ischemia &5min reperfusion).(2)Serum enzymology results showed that compared with Sham group,serum ALT and AST levels in 2,6 and 24 h I/R groups were significantly increased;Compared with I/R group,serum ALT and AST of RIPC and RIPC ’groups were significantly decreased at each time point of reperfusion.Compared with RIPC ’group,serum ALT and AST levels in RIPC group were significantly decreased,and the serum ALT and AST levels in RIPC 4×5 group were the lowest.2.Differences in the protective effects and mechanisms of RIPC in unilateral and bilateral limbs: Compared with I/R group,RIPC and D-RIPC groups at 2,6 and 24 hours of reperfusion significantly decreased the levels of liver cell necrosis and apoptosis,as well as ALT,AST and LDH in serum.Compared with RIPC group,liver cell necrosis,apoptosis and serum ALT and AST levels in d-ripc group were significantly decreased at 2h after reperfusion,but there was no statistical difference in these indexes between the two groups at 6 and 24 h after reperfusion(P > 0.05).The ultrastructure of liver cells observed by transmission electron microscopy showed that compared with I/R group,the swelling degree of endoplasmic reticulum and mitochondria of liver cells in RIPC and D-RIPC groups was significantly reduced,and the ultrastructure damage was reduced.The results of mechanism study showed that compared with I/R group,the levels of CRC and c AMP in liver tissues in RIPC and DRIPC groups were significantly increased,the levels of TNF-α,IL-1β,ROS and MDA in liver tissues and serum were significantly decreased,and the levels of SOD and CAT in liver tissues were increased.Compared with I/R group,the expression of Bax and Bcl-2 in RIPC group was decreased,and the expression of P-ERK and P-GSK-3 βprotein was increased.3.Study on the protective effect of unilateral limb RIPC on liver I/R injury in elderly rats: compared with I/R group,liver cell necrosis,apoptosis and serum ALT and AST levels in RIPC group were significantly reduced after 6h reperfusion.The results of transmission electron microscopy showed that mitochondria and endoplasmic reticulum swelling of liver cells in RIPC group were lighter,and the ultrastructural damage of liver cells was reduced.Compared with the I/R group,the expression of TNF-α and IL-1β in RIPC group was significantly decreased,the expression of IL-10 was increased,the levels of ROS and MDA were decreased,and the expression of CREB was increased.Research conclusion:1.In rats with 70% liver heat I/R injury,different forms of UNILATERAL limb RIPC can significantly inhibit liver cell necrosis and apoptosis,and alleviate liver injury,and the best protective effect is achieved by unilateral limb ischemia for 5min,reperfusion for 5min,and circulation for 4 times.2.Both unilateral and bilateral RIPC can reduce liver reperfusion injury in rats by inhibiting visceral cell inflammation,oxidative stress and mitochondrial damage.The specific mechanism may be related to the regulation of camp-ERK1/2-Bax/Bcl-2pathway and camp-GSK-3 β-MPTP axis by RIPC.At the early stage of reperfusion(2h),bilateral limb RIPC had more significant liver protection than unilateral limb RIPC,but there was no significant difference between the two at the middle and late stage of reperfusion(6 and 24h),further suggesting that RIPC mode of unilateral limb ischemia for 5min,reperfusion for 5min,and circulation for 4 times is the optimal RIPC method for alleviating liver I/R injury in rats.3.In elderly rats,RIPC pattern of unilateral limb ischemia for 5min,reperfusion for 5min and circulation for 4 times can significantly inhibit hepatocyte necrosis and apoptosis during I/R,and the mechanism may be related to limb RIPC regulation of inflammatory factors,antioxidant stress and mitochondrial protection. |
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