| Objective: To explore whether the water extract and alcohol extract of Eucommia ulmoides have protective effect on rat Hepatic ischemia-reperfusion injury(HIRI).If there is a protective effect,which extract method has better protective effect,and analyze its possible causes and effects mechanism.Methods:(1)64 male SPF rats(180g ± 20g)were randomly divided into 8 groups,rats each respectively.They were sham group,I/R group,eucommia ulmoides water extract high,medium and low dose group,eucommia alcohol extract low,medium and high dose group.The Eucommia ulmoides extract groups were pretreated with corresponding doses of Eucommia ulmoides extract for 10 days.The three different dosages of water extract and alcohol extract were 20 g crude drug/kg,40 g crude drug/kg,80 g crude drug/kg.The sham operation group and the HIRI group were pretreated with the same dose of normal saline for 10 days.Except the sham operation group,liver ischemia-reperfusion injury models were established on the 11 th day after gavage.Ischemia time was 1h,reperfusion time was 4h.Detected the expression levels of tumor necrosis factor-a(TNF-a),interleukin-1b(IL-1b),malondialdehyde(MDA)and superoxide dismutase(SOD)in rat liver tissues,and HE staining was used to observe liver pathological changes,and serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels were measured at the same time.(2)Forty male SPF rats(180g ± 20g)were randomly divided into 5 groups of 8 rats each.They were sham operation group,HIRI group,Eucommia ulmoides polysaccharide low,medium and high dose groups.The dosage of Eucommia ulmoides polysaccharide was 80mg/kg,160mg/kg and 320mg/kg,respectively.Pretreatment and establishment of liver ischemia-reperfusion injury model are the same as before.HE staining to observe the pathological changes of liver tissue and to determine the serum high mobility group protein B1(HMGB1),ALT,AST,TNF-a,IL-1b levels,detected the production of reactive oxygen species(ROS),MDA and SOD in liver tissue and HMGB1,Toll-like receptor 4(TLR-4)m RNA and protein expression levels and interferon regulatory factor 1(IRF-1),nuclear factor kB-p65(P65),phosphorylated p65(P-p65),nuclear factor kB(NF-kB)inhibits the expression of peptone(IkB-a),phosphorylated IkB-a(P-IkB-a)Level.Results: 1.Protective effect of Eucommia ulmoides extracts with different extraction methods on HIRI in rats.(1)Liver structure and function: Compared with the sham operation group,the liver cord in the HIRI group was significantly disordered,a large number of inflammatory cell infiltration,and serum ALT and AST levels were significantly higher than those in the sham operation group.Compared with the HIRI group,the liver cord structure of rats in the Eucommia extract pretreatment group was more regular,and the inflammatory cell infiltration was significantly reduced,and the expression levels of ALT and AST were reduced.(2)Determination of oxidative stress and inflammatory response levels: Compared with the sham operation group,the levels of MDA,IL-1b and TNF-a in the liver tissue homogenate of the HIRI group were significantly increased,while the SOD activity was significantly decreased(P<0.05),the pretreatment group of Eucommia ulmoides extract can inhibit the increase of MDA,IL-1b,TNF-a and the decrease of SOD caused by HIRI.The high dose of Eucommia water extract(80g crude drug/kg)has the most obvious effect(P< 0.05).2.The protective effect of Eucommia ulmoides polysaccharide on HIRI in rats.(1)Liver structure and function: Obvious hepatic cord disorder,inflammatory cell infiltration and hepatocyte necrosis were seen in the HIRI group.The serum ALT and AST levels were significantly higher than those in the sham operation group.The area of necrosis,hepatic cord disorder and inflammatory cell infiltration of rats in the Eucommia polysaccharide pretreatment group were significantly reduced compared with the HIRI group,and the levels of ALT and AST were significantly reduced.Especially the high-dose Eucommia polysaccharide group improved liver structure and function damage most significantly.(2)Determination of oxidative stress and inflammatory response levels: Compared with the sham operation group,the MDA content of liver tissue homogenate,serum IL-1b and TNF-a levels in the HIRI group were significantly increased,while the SOD level was significantly reduced(P<0.05);The three pretreatment groups of Eucommia ulmoides polysaccharides can reduce the levels of MDA,IL-1b,and TNF-a,and increase the level of SOD.The high-dose Eucommia polysaccharide group(320mg/kg)has the most significant effect(P<0.05).(3)Detection of relevant indicators of aseptic inflammatory response mechanism: The high-dose Eucommia ulmoides polysaccharide can significantly reduce the release of HMGB1 in peripheral blood and the increase of ROS and IRF-1 level in liver tissue.In addition,high-dose Eucommia ulmoides polysaccharide pretreatment can significantly reduce the increase of HMGB1 and TLR-4 m RNA and protein expression caused by HIRI,reduce the expression levels of My D88 and p65 proteins,inhibit the production of P-p65 protein,and inhibit IkB-a Protein degradation and PIkB-a production(P<0.05),inhibit HMGB1/TLR-4/NF-kB signaling pathway.Conclusion: 1.Different doses of Eucommia ulmoides water extract and alcohol extract have protective effects on HIRI,and the Eucommia lmoides water extract high-dose group(80g crude drug/kg)has the best protection.2.During the occurrence of HIRI,Eucommia ulmoides polysaccharide can play an antioxidant role by reducing ROS levels and increasing SOD levels,thereby reducing MDA production;it can also reduce IRF-1 expression,thereby reducing HMGB1 expression and release,and inhibiting TLR-4/NF-?B pathway activation,so as to play an anti-inflammatory role,ultimately protect HIRI.Determination of TNF-? and IL-1? in serum,and observation of the inhibitory effect of Eucommia ulmoides polysaccharides on aseptic inflammation induced by HIRI. |
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