The Therapeutic Effect And Mechanism Of Baicalin Magnesium On Non-Alcoholic Steatohepatitis In Rats

Non-alcoholic steatohepatitis（NASH）is an important link in the transformation of simple fatty liver to end-stage liver disease such as liver fibrosis,cirrhosis,primary liver cancer and liver failure.Currently,the morbidity and mortality of NASH are increasing worldwide.Clinical pharmacological treatment for NASH mostly adopts drugs that regulate metabolism,inhibit inflammatory response,reduce cellular damage and stabilize cellular membranes,which have a long course,high side effects and less than ideal efficacy.Scutellaria baicalensis Georgi is one of the commonly used herbal medicines to dispel dampness and reduce yellowing.Its main active ingredient baicalin has anti-inflammatory,antioxidant,anti-lipid deposition and hepatoprotective effects,etc.In clinical practice,baicalin tablets/capsules are used for the auxiliary treatment of acute and chronic hepatitis and prolonged hepatitis.However,baicalin exhibits poor solubility and bioavailability,limiting its clinical application.To solve the existing problems,we isolated baicalin magnesium with good aqueous solubility from the water extract of Scutellaria baicalensis Georgi,it was shown that baicalin magnesium was the original form of baicalin present in Scutellaria baicalensis Georgi,relating research has been granted invention patents in China,the United States and the European Union.Pharmacological studies have demonstrated that baicalin magnesium may exert therapeutic effects on carbon tetrachloride（CCl₄）and lipopolysaccharide（LPS）combined with d-galactose-induced acute liver injury（ALI）by mediating inflammation and oxidative stress,but it is unclear whether baicalin magnesium can treat NASH.Objective:In this experiment,a high-fat diet（HFD）was adopted to elicit and establish a rat NASH model,and to evaluate the influence of various factors on the efficacy of baicalin magnesium in treating NASH rats.This experiment investigated the therapeutic effect and mechanism of baicalin magnesium on NASH rats,a reliable theoretical and experimental basis for the clinical treatment of NASH with baicalin magnesium based on the nod like receptor family pyrin domain containing 3（NLRP3）/cysteinyl aspartate specific proteinase（Caspase）-1/interleukin（IL）-1βsignaling pathway.Methods:1.NASH was induced through a HFD,and rats were assigned at random into 6 groups:control,model,and baicalin magnesium group（25,50,100,150 mg/kg）,which administered by tail vein injection to investigate the impact of modeling time,administration time and dose on the liver index,biochemical index and pathological results.2.HFD was used to induce modeling,and 48 SD rats were randomly divided into control group,baicalin magnesium 50 mg/kg group,baicalin magnesium 150 mg/kg group,baicalin group（146.4 mg/kg）and magnesium sulfate group（Mg SO₄,19.7 mg/kg）.The doses of baicalin and magnesium sulfate（Mg SO₄）group were equimolar to the baicalin magnesium（150mg/kg）group.The control group and the other groups were fed conventional and high-fat diets for 8 weeks,separately.Treated by tail vein injection in rats for 2 weeks from the 9th week to observe the general status of the rats.Following dosage,specimens were taken from rats with serum and liver tissue collected for further investigation.3.The liver index was counted by measurement of body and liver wet weight.Liver tissues were stained with HE and oil red O to visualize the changes in the lesions,and a NAS was performed.Alanine transaminase（ALT）,glutamic transaminase（AST）,triglycerides（TG）,total cholesterol（TC）,high-density lipoprotein（HDL-C）in serum were measured by automated biochemistry.The kits were used to determine myeloperoxidase（MPO）and superoxide dismutase（SOD）activities,and malondialdehyde（MDA）levels in serum,as well as IL-6,IL-10 and IL-1βlevels in liver tissue homogenates.4.Protein and gene expression levels of tumor necrosis factor（TNF）-α,NLRP3,IL-18,IL-1βand Caspase-1 in liver tissue homogenates were investigated by western blot and real-time polymerase chain reaction（PCR）assays.Results:1.Compared to 6 weeks,at 8 weeks of modeling,the liver index in the model group increased obviously,HE staining showed that rats exhibited unclear nucleus boundaries,marked deformation of hepatocyte structures,liver inflammatory infiltration and necrosis,an obvious increase in balloon-like spots,and a significant increase in NAS（P<0.01）.AST,ALT,TC,TG and LDL-C increased significantly,and HDL-C decreased remarkably（P<0.01）.There was no observable change in most of the indicators in each dosing group after 1 week of treatment（P>0.05）.At 2weeks of administration,compared with the model group,there were no obviously results in the 25 mg/kg group,while ALT,AST,TC and TG were greatly reduced in the 50 mg/kg group（P<0.05）;Body weight,liver wet weight,liver index,ALT,AST,TC,TG and LDL-C were decreased（P<0.01）and HDL-C was increased obviously（P<0.05）in the 100 mg/kg group,and the 150 mg/kg group markedly improved biochemical indexes and pathology as well as decreased NAS（P<0.01）.2.It was found that the rats in the model group were obese,and had dull and yellow hair.The pathological results indicated that the nuclei of the rats were blurred,the hepatocyte structures were distorted,the boundaries of the hepatic lobules were unclear,and the hepatic cords were disorganized,the degrees of vacuolar changes and patchy necrosis were appeared in the livers,the surrounding inflammatory cells were infiltrated,the NAS was markedly increased（P<0.01）;and there were a lot of red lipid droplets in the liver tissue,which were dark and fused into slices.There were clear and intact hepatic cell nuclei in the administered group,neatly arranged liver lobules,reduced NAS and lipid droplets compared with the model group,which the improvement effect of high-dose group of baicalin magnesium was the most significant（P<0.01）.3.We observed that baicalin magnesium can alleviate liver injury,lipid deposition and oxidative stress in NASH rats.Compared with the control group,the levels of ALT,AST,LDL-C,TC,and TG were increased in the model group,while HDL-C levels were distinctly decreased（P<0.01）;compared with the model group,ALT,AST,LDL-C,TC,and TG were decreased in baicalin magnesium group,HDL-C was increased（P<0.05）.Compared to the control group,in the model group,SOD activity was significantly lower,MDA content and MPO activity was higher（P<0.01）.Compared with the model group,the activity of SOD was higher,MDA content and MPO activity was obviously lower in the administered group,which the effects of high-dose group of baicalin magnesium were the most remarkable（P<0.01）.4.Baicalin magnesium was found to ameliorate the inflammatory response in NASH rats.Compared to the model group,the levels of IL-6 and IL-1βdecreased in the administered group,while IL-10 was increased,reduced gene and protein expression of NLRP3,TNF-α,Caspase-1,IL-18and IL-1β（P<0.01）,which the improvement was the most obvious in high-dose group of baicalin magnesium.Conclusions:1.The modeling for 8 weeks was closer to the NASH model than 6weeks,and the therapeutic effect of 2 weeks of administration was more obvious in NASH rats.50 mg/kg and 150 mg/kg were used as the administration doses to further investigate the therapeutic effect and mechanism of baicalin magnesium on NASH rats.2.It was apparently superior to baicalin that baicalin magnesium exerted therapeutic effects on HFD-induced NASH rats by ameliorating lipid deposition,oxidative stress and inflammatory response,and the interaction mechanism may be related to the inhibition of the activation of NLRP3/Caspase-1/IL-1βsignaling pathway of inflammation.