The Effect Of Isoliquiritin Pretreatment On The Inflammatory Response And TLR4/NF-κB Pathway Of Liver Ischemia-reperfusion Injury In Rats

ObjectiveTo explore the effect of isoliquiritin pretreatment on the inflammatory response and TLR4/NF-κB pathway of liver ischemia-reperfusion injury in rats.MethodsAfter 40 SPF male SD rats were adaptively reared for 7 days,they were randomly divided into 4 groups according to the random number table method,namely group A(sham operation group),group B(model group),and group C(50 mg/kg isolicorice Supplement intervention group),D group(200mg/kg isoliquiritin intervention group),10 rats in each group.Three days before the operation,groups C and D were given50mg/kg and 200mg/kg isoliquiritin by gavage respectively.Groups A and B were given the same amount of normal saline for gavage,and the four groups were given 1ml/100 g.Gavage once a day for 3 consecutive days.In group A,the hepatic hilum was dissected after laparotomy,and the left and middle liver pedicles were freed without further treatment.In groups B,C,and D,after exposing the hilum and freeing the left and middle liver pedicles,clamp the left and middle hepatic blood vessels of the liver pedicle with small non-damaged vascular clips.After 60 minutes,the vascular clips were released and reperfused 6 hour.After the completion of perfusion,the changes of serum transaminase(ALT,AST)and inflammatory factors(TNF-α,IL-1β)in each group of rats were detected;the oxidative stress-related substance of each group of rat liver tissue malondialdehyde(MDA)was detected And the content of superoxide dismutase(SOD);the morphological changes of rat liver tissues in each group were examined under HE staining microscope;the expression of TLR4 and NF-κB protein in liver tissues of each group was detected by WB method.Results1.Compared with group A,ALT,AST,TNF-α and IL-1β of group B,C and D were all significantly increased(P<0.01),among which group B rat serum ALT(175.49±8.27 Kar U),AST(188.37±9.90 Kar U),TNF-α(49.23±5.58pg/ml)and IL-1β(50.92±20.83pg/ml)and group A ALT(76.42±7.83 Kar U),AST(109.93 ±8.73 Kar U),TNF-α(26.83±3.64pg/ml)and IL-1β(23.85±2.25pg/ml)were significantly higher(P<0.01),ALT in group C(141.51±12.84 Kar U)),AST(154.21±9.48 Kar U),TNF-α(42.00±4.77pg/ml),IL-1β(39.06±3.48pg/ml)were significantly lower than those in group B(P<0.01),group D ALT(116.10±9.46 Kar U),AST(130.64±8.78 Kar U),TNF-α(32.44±4.16pg/ml),IL-1β(32.98±2.81pg/ml)were significantly reduced compared with group C(P<0.05).2.Compared with group A,SOD of groups B,C,and D was significantly lower(P<0.01),and MDA was significantly increased(P<0.01).Among them,group B rats liver tissue SOD(138.76 ±7.28U/mgprot),MDA(5.69±0.25nmol/mgprot)Compared with group A,SOD(273.87±11.55U/mgprot)was significantly reduced(P<0.01),and MDA(2.16±0.14nmol/mgprot)was significantly increased(P<0.01);compared with group B Compared with group C,SOD(188.72±10.84U/mgprot)was significantly increased,and MDA(3.80 ±0.20nmol/mgprot)was significantly decreased,with significant differences(P<0.01);compared with group C,SOD of group D(227.07± 8.99)U/mgprot)was significantly increased,MDA(3.07 ±0.13nmol/mgprot)was significantly decreased,which was statistically significant(P<0.05).3.Pathological observation and scoring results of HE liver tissue staining:(1)Pathological observation: The structure and morphology of the liver lobules in group A were roughly the same as usual,and the liver cells had no obvious damage and necrosis.Group B: The structure of liver lobules is vague and unclear,the central vein and sinusoids are obviously enlarged and congested,and the liver cells are damaged and necrotic to varying degrees.Group C: The degree of swelling,degeneration and necrosis of liver cells was significantly reduced.Group D: Compared with group C,hepatocyte swelling and inflammatory cell infiltration were significantly reduced.(2)Histopathological score: The pathological scores of groups B,C,and D were significantly higher than those of group A(P<0.01).The scores of groups C and D were significantly lower than those of group B(P<0.01).4.WB detected the expression of TLR4 and NF-κB in the liver tissues of rats in each group:(1)The TLR4 protein expression levels of the three groups of B,C,and D were 0.534±0.023 times,0.356±0.025 times and 0.534 times of the internal control β-actin protein,respectively.248±0.025 times,of which the B and C groups were significantly higher than the 0.174±0.039 times of the A group(P<0.01),and the D group was slightly higher than the A group without statistical significance(P=0.183>0.05).The expression levels of groups C and D were significantly lower than those of group B(P<0.01).(2)The expression levels of NF-κB protein in the three groups B,C and D were 0.602±0.022 times,0.516±0.015 times and 0.378±0.021 times that of the internal control β-actin protein,respectively,which were significantly higher than those of group A 0.238±0.024 times(P<0.01),the expression levels of C and D groups were significantly lower than those of B group(P<0.01).ConclusionIsoliquiritin can significantly inhibit the HIRI inflammatory response in rats,and its mechanism may be related to down-regulating the expression of TLR4/NF-κB signal transduction pathway proteins,and the high dose(200mg/kg)is better than the low dose(50mg/kg).