| Liver is an important organ of human body,which has physiological functions such as metabolism,secretion,excretion and detoxification.Many factors can lead to liver injury,the most common factor is virus infection.In recent years,with the increasing frequency of social interaction,alcoholic liver injury has an upward trend.In addition,drug-induced liver injury such as non-steroidal anti-inflammatory drugs(NSAIDs)is also more common.At present,clinical treatment of acute liver injury should first control the inducement or cause of disease,and then carry out liver protection,enzyme reduction and antiviral treatment.Commonly used drugs are glycyrrhizin amine,reduced glutathione,glucocorticoid,lamivudine,etc,but their toxic side effects are greater,among which glycyrrhizin amine can cause pseudoaldosteronism,electrolyte changes,etc.Reduced glutathione can be induced.It can cause digestive tract symptoms and rashes,and lamivudine can cause anemia,erythrocyte regeneration disorder,thrombocytopenia,etc.Therefore,the search for effective,low toxicity and wide application of therapeutic drugs and methods has become a hot research topic.Baicalin has the pharmacological activities of scavenging free radicals,antioxidant and anti-inflammatory.It is mainly used to treat hepatitis,acute biliary tract infection and other diseases in clinic,but its application is limited by its insolubility in water.Magnesium salts of baicalin are the original form of Baicalin in Scutellaria baicalensis.Their water solubility and stability are excellent.Professor Liu Cuizhe,Institute of Traditional Chinese Medicine,Chengde Medical College,has mastered the process of extracting magnesium baicalin from Scutellaria baicalensis and preparing magnesium baicalin from commercial baicalin,and has applied for national invention patent.Scutellaria baicalensis is abundant in resources in China.If we can establish stable animal models similar to clinical acute liver injury and explore the protective effect of magnesium baicalin,it will provide experimental basis for the study of the pathogenesis of acute liver injury and the popularization and application of magnesium baicalin.Objective:To investigate the protective effect of magnesium baicalin pretreatment on acute liver injury induced by lipopolysaccharide(LPS)combined with D-galactosamine(D-GalN)and its mechanism,and to provide experimental basis for the development and clinical application of magnesium baicalin.Methods:1 Determination of the Optimum Modeling dose and sampling time of LPSand D-GalN1.1 Determine the optimum dosage and sampling time for LPS and D-GalNmouldingConsidering the first-order interaction of each factor,the experiment was designed according to L9(34)orthogonal table by SPSS software.The three levels of LPS(factor A)were 20 ?g/kg,30 ?g/kg,40 ?g/kg,the three levels of D-GalN(factor B)were 200 mg/kg,300 mg/kg,400 mg/kg,and the three levels of sampling time(factor C)were 3 h,6 h and 12 h.After combination,they were divided into 9 groups,A2B3C1 group,A3B2C1 group,A1B1C1 group,A3B3C2 group,A2B1C2 group,A1B2C2 group,A3B1C3 group,A1B3C3 group and A2B2C3 group and a normal group.Male SD rats were fed adaptively for 7 days.According to their body weight,they were divided into 10 rats in each group by random block method.After fasting for 12 hours,the model group was given corresponding drugs(now available)by body weight for intraperitoneal injection,and the normal group was given normal saline of equal volume.Normal diet and free drinking water.Ob served the general situation of rats in each group.1.2 Detection of IndicatorsThe liver index was calculated,the serum ALT and AST levels were detected by automatic biochemical analyzer and the pathological changes of liver tissues were observed by HE staining to determine the optimum conditions for the establishment of the model.2 Protective effect of magnesium baicalin pretreatment on LPS/D-GalNinduced acute liver injury in rats2.1 Grouping and administrationThe rats were randomly divided into 7 groups with 9 rats in each group.They were normal control group,model group,magnesium isoglycyrrhizinate(MAIG)group(18mg/kg),baicalin magnesium high(BA-Mg)(100mg/kg),medium(50mg/kg),low(25mg/kg)dose group,baicalin group(equal to the mole number of Baicalin in the medium dose group of Baicalin Magnesium).The normal group and the model group were given equivalent saline via caudal vein,and the other drug groups were given corresponding drugs by tail vein injection once a day for 7 days.The optimal dosage combination was determined,that is,20 ?g/kg LPS combined with 400 mg/kg D-GalN intraperitoneal injection to prepare acute liver injury model.The general situation of rats during the whole experimental process was observed and the samples were taken 12 hours after the establishment of the model.The pathological changes of HE staining and the higher levels of transaminase AST and ALT in the normal group were used to judge the success of the model.2.2 Indicators of detectionLiver index was calculated by weighing rat's body weight and liver weight.ALT and AST in serum were detected by colorimetry.pathological changes of liver tissue were observed by HE staining.MDA in serum and liver tissue was detected by TBA colorimetry.SOD in serum and liver tissue was detected by purine oxidase method.IFN-,IL-lbeta,IL-6,TNF-a in serum were detected by ELISA.IL-6 and TNF-a mRNA expression were detected by RT-PCR.2.3 Statistical analysis methodSPSS 19.0 statistical software analysis,a=0.05 as the test level.To determine the optimum modeling conditions,the contents of ALT and AST in the serum of each experimental group were analyzed by orthogonal experiment and visual analysis.Other measurement data were compared by LSD or Games-Howell test with one-way ANOVA,and the results were expressed by x±s.Results:1 Determine the best dosage and sampling time combination for modeling1.1 General situation of rats in each groupDuring the experiment,the mental state of the normal group was good.The mental state of the SD rats in A1B3C3 and A2B2C3 groups was depressed and their activities decreased after the model was established.The mental state of the other groups was acceptable.1.2 Liver indexThere was no significant difference in liver index between model group and normal group(P>0.05).The average liver index of A1B3C3 group was the highest.1.3 Pathological changes of liver tissue in each groupHE staining showed that the structure of hepatic lobules in the normal group was regular,hepatocytes were stranded and centered around the central vein,radiated around,and there was no necrosis of hepatocytes and infiltration of inflammatory cells.The structure of hepatic lobules in A1B3C3 group was disordered,hepatocytes became punctate necrosis,nuclei disappeared,a large number of inflammatory cells infiltrated,and hepatocyte necrosis and inflammatory cell infiltration were mild in other groups.1.4 Analysis results of serum transaminase changesThe orthogonal experimental visual analysis shows that the range of ALT is as follows:RA(1299.79)>RB(1215.81)>RC(1112.03),and the range of AST was RA(2250.57)>RB(2094.58)>RC(1861.2),that is,factor A had the greatest influence on the results of transaminase and factor C had the least influence on the results of transaminase.ALT and AST were the highest in A1B3C3 group,respectively,1470.44,1453.54,1308.04,2582.85,2555.85 and 2285.6.According to the general situation of rats,the contents of ALT,AST and the pathological results of HE staining,the best combination was determined that it is 20 ?g/kg LPS combined with 400mg/kg D-GalN get the material in 12 hours.In other words A1B3C3 was the best combination.2 Protective effect of magnesium baicalin on LPS/D-GalN-induced acute liver injury in rats2.1 General situation of ratsDuring the whole experiment,the weight of rats in each group increased.About 3 days after administration,the urine of rats in each group of Baicalin Magnesium and Baicalin was yellowing,but no abnormality was found in other groups.After the establishment of the model group and the drug groups,the rats in the model group were mentally depressed,slowly responding,reduced movement and decreased appetite,especially in the model group.2.2 Liver Index ResultsCompared with the normal group,the liver index of the model group increased(P<0.05).Compared with the model group,the liver index of each drug group decreased(P<0.05).There was no significant difference between magnesium baicalin group and magnesium isoglycyrrhizinate group(P>0.05).2.3 Changes of serum transaminase ALT and AST in each groupThe level of ALT in serum of model group was higher than that in normal group(P<0.05).Compared with the model group,the serum ALT content of each dose group of Baicalin Magnesium and magnesium isoglycyrrhizinate group was decreased(P<0.05).Compared with the magnesium isoglycyrrhizinate group,the serum ALT content in the high and middle dose groups of baicalin magnesium decreased(P<0.05),while that in the low dose group increased(P<0.05).There was no significant difference in serum ALT content between different dose groups of Baicalin Magnesium and baicalin group(P>0.05).Compared with the normal group,the serum AST level in the model group increased(P<0.05).Compared with model group,the content of AST in serum of Magnesium isoglycyrrhizinate group,high and middle dose group of Baicalin Magnesium was lower(P<0.05),and there was no difference in low dose group and baicalin group compared with model group(P>0.05).Compared with magnesium isoglycyrrhizinate group,the content of AST in serum of high and low dose group was higher than that of magnesium isoglycyrrhizinate group(P<0.05),whil there was no significant difference between midle dose group and magnesium isoglycyrrhizinate group(P>0.05).There was no significant difference in serum AST between different dose groups and baicalin group(P>0.05).2.4 Histopathological changes of liver stained with HE in each groupIn the normal control group,the structure of hepatic lobules was regular centered around the central vein,radiated around,without compression,rupture,necrosis and infiltration of inflammatory cells.The normal structure of hepatic lobules was destroyed,the strands of hepatocytes were disordered,the sinuses of hepatic blood became wider,the normal morphology of hepatocyte nuclei was disappeared,hepatocytes were diffusely multiple punctate and fragmentary necrosis,and inflammatory cells infiltrated in the necrotic foci.The degree of hepatocyte damage in the magnesium baicalin group was less than that in the model group,and the cytoplasm staining of hepatocytes was uneven,and hepatic sinuses were widened in the low dose group.The cells were divided into small patches with relatively complete nuclei and a little necrosis foci in some visual fields.The morphological structure of hepatocytes in the magnesium isoglycyrrhizinate group was not significantly different from that in the normal group except for a small amount of inflammatory cells infiltration.The structure of hepatic lobules in the baicalin group was basically intact,and some hepatocytes were destroyed with some patches of necrosis foci.2.5 Changes of MDA and SOD in each group2.5.1 MDA resultsThe content of MDA in serum of model group was higher than that in normal group(P<0.05).The content of MDA in serum of each dose group,baicalin group and magnesium isoglycyrrhizinate group was significantly lower than that in model group(P<0.05).There was no significant difference in serum MDA content between the magnesium isoglycyrrhizinate group and the high dose group(P>0.05),but it was lower than that in the medium and low dose group(P<0.05).Compared with baicalin group,there was no significant difference in serum MDA content compared with high dose group,whil it was higher in middle and low dose group.The content of MDA in liver homogenate of model group was significantly higher than that in normal group(P<0.05).Compared with model group,MDA content in liver homogenate of high dose group and magnesium isoglycyrrhizinate group decreased significantly(P<0.05),but there was no difference between middle and low dose group(P>0.05).There was no difference of MDA content in liver homogenate of high and middle dose groups compared with magnesium isoglycyrrhizinate group(P>0.05),while the low dose group was significantly higher than the magnesium isoglycyrrhizinate group(P<0.05).The content of MDA in liver homogenate of high dose group was significantly lower than that in baicalin group(P<0.05),but there was no difference between middle and low dose group compared with baicalin group(P>0.05).2.5.2 SOD resultsCompared with the normal group,the serum SOD level in the model group was significantly lower(P<0.05).The serum SOD levels in the different dosage groups of Baicalin Magnesium,baicalin group and magnesium isoglycyrrhizinate group were significantly higher than those in the model group(P<0.05).There was no difference in serum SOD content between the high and middle dose groups of Baicalin Magnesium compared with magnesium isoglycyrrhizinate group(P>0.05),while the low dose group was significantly lower than that in magnesium isoglycyrrhizinate group(P<0.05).Compared with the baicalin group,the serum SOD content in the high dose group was significantly higher(P<0.05),while the serum SOD content in the low dose group was significantly lower(P<0.05)than that in the baicalin group,and there was no difference between the middle dose group and the baicalin group(P>0.05).Compared with the normal group,the content of SOD in the liver homogenate of the model group decreased significantly(P<0.05).Compared with model group,SOD content in liver homogenate of high dose group and magnesium isoglycyrrhizinate group increased significantly(P<0.05),but there was no difference between middle and low dose group and model group(P>0.05).Compared with the magnesium isoglycyrrhizinate group,the SOD content in liver homogenate of the medium dose group of baicalin magnesium was significantly lower(P<0.05),and there was no difference between the high and low dose group compared with the magnesium isoglycyrrhizinate group(P>0.05).The content of SOD in liver homogenate of high dose group was significantly higher than that of baicalin group(P<0.05),but there was no difference between middle and low dose group and baicalin group(P>0.05).2.6 Changes of serum cytokines in each group measured by ELISA2.6.1 Changes of IL-6 content in serumThe level of IL-6 in serum of model group was higher than that in normal group(P<0.05).Compared with the model group,the levels of IL-6 in the serum of the high and middle dose group,the magnesium isoglycyrrhizinate group and the baicalin group were significantly lower(P<0.05),while there was no difference between the low dose group and the model group(P>0.05).Compared with the magnesium isoglycyrrhizinate group,the serum IL-6 level in the middle and low dose groups of Baicalin Magnesium were increased(P<0.05),while there was no difference between the high dose group of Baicalin Magnesium and the magnesium isoglycyrrhizinate group(P>0.05).Compared with baicalin group,the content of IL-6 in serum of high dose group was decreased(P<0.05),but there was no difference between middle dose group and low dose group(P>0.05).2.6.2 Changes of IL-1? content in serumThe serum level of cytokine IL-1beta in model group was significantly higher than that in normal group(P<0.05).Compared with the model group,there was no difference between model group and the low dose group of Baicalin Magnesium.(P>0.05),the levels of IL-lbeta in serum of the high and middle dose group of Baicalin Magnesium,the magnesium isoglycyrrhizinate group and the baicalin group were significantly lower than modle group(P<0.05).Compared with the magnesium isoglycyrrhizinate group,the serum IL-lbeta content in the middle and low dose groups of Baicalin Magnesium was higher(P<0.05),and there was no difference between the high dose group and the magnesium isoglycyrrhizinate group(P>0.05).Compared with baicalin group,the content of IL-lbeta in serum of high and medium dose groups of Baicalin Magnesium was significantly lower(P<0.05),while there was no difference between low dose group and Baicalin group(P>0.05).2.6.3 Changes of TNF-? content in serumThe content of TNF-a in serum of model group was significantly higher than that in normal group(P<0.05).The content of TNF-a in serum of each dose group of Baicalin Magnesium,magnesium isoglycyrrhizinate group and baicalin group showed a decreasing trend compared with model group,but there were no significant difference(P>0.05).There was no significant difference in serum TNF-? content of different dose groups of magnesium baicalin,magnesium isoglycyrrhizinate group compared with baicalin group(P>0.05).2.6.4 Changes of IFN-? content in serumThe serum IFN-? level in model group was significantly higher than that in normal group(P<0.05).The levels of IFN-? in serum of magnesium baicalin group,magnesium isoglycyrrhizinate group and baicalin group were significantly lower than that in model group(P<0.05).Compared with the magnesium isoglycyrrhizinate group,the serum IFN-? levels in the middle and low dose groups of baicalin magnesium were significantly increased(P<0.05),while there was no difference between the high dose group and the magnesium isoglycyrrhizinate group(P>0.05).The serum IFN-? levels in the middle and low dose groups of Baicalin Magnesium were significantly higher than that in the Baicalin group(P<0.05),whil there was no difference between the high dose group and the Baicalin group(P>0.05).2.7 Detection of cytokine mRNA expression in each group by RT-PCR2.7.1 PCR detection results of TNF-? in each groupThe expression of TNF-a in the model group was significantly higher than that in the normal group(P<0.05).There was no difference in the expression of TNF-? in different dose groups of Baicalin Magnesium,Baicalin group compared with model group(P>0.05).The expression of TNF-a in the magnesium isoglycyrrhizinate group was significantly lower than that in the model group(P<0.05).The expression of TNF-a in all the magnesium baicalin groups were significantly higher than that in the magnesium isoglycyrrhizinate group(P<0.05).There was no difference in the expression of TNF-a between the magnesium baicalin group and the baicalin group(P>0.05).2.7.2 PCR Detection results of IL-6 in each groupCompared with the normal group,IL-6 expression in liver tissue of model group increased significantly(P<0.05).Compared with the model group,the expression of IL-6 mRNA in different doses of magnesium baicalin,magnesium isoglycyrrhizinate and baicalin groups decreased significantly(P<0.05).Compared with the magnesium isoglycyrrhizinate group,the expression of IL-6 mRNA in each dose of magnesium baicalin group tended to increase,and the difference between the low dose group and the magnesium isoglycyrrhizinate group was statistically significant(P<0.05).There was no significant difference between the different dose groups of Baicalin Magnesium and Baicalin(P>0.05).Conclusion:1.20 ?g/kg LPS combined with 400mg/kg D-GalN get the material in 12 hours,was the best combination to induce acute liver injury in rats.2.Magnesium baicalin pretreatment has protective effect on acute liver injury induced by LPS combined with D-GalN,which may be related to the regulation of inflammatory cytokine production and antioxidant stress. |
PDF Full Text Request