The Effect Of Berberine Hydrochloride On Nonalcoholic Fatty Liver Disease Rat Model And Discusses The Mechanism

Nonalcoholic fatty liver disease (NAFLD) is a condition in which excess fataccumulates in the liver of a patient without a history of alcohol abuse. Nowthat obesity, type2diabetes, hyperlipidemia, metabolic syndrome, and so onmany factors alone or together formed the risk factor of NAFLD. In recentyears with the improvement of our living standard, the incidence ofnonalcoholic fatty liver disease is increasing, and the trend of age becomeslower. Today people pay more attention to fatty liver disease. At present it hasbecome a worldwide attention in the medical and social problems. And thestudy found that insulin resistance, lipid metabolism disorder, inflammatoryfactor and endotoxin in the pathogenesis of NAFLD played an important role.But there is no exact effective drug treatment. Berberine hydrochloride, as atreatment for intestinal infectious diseases drugs, in recent years have foundthat it can reduce blood sugar, improve insulin resistance,and have the role ofcorrecting lipid metabolism disorders. And berberine, has the resources isextensive, the price is low, and the advantages of with fewer side effects.Therefore NAFLD rat model was established by high fat diet, and applied toberberine hydrochloride intervention treatment, in order to explore thetreatment effect of berberine hydrochloride to rat nonalcoholic fatty liverdisease and find its possible mechanism. Even put forward useful suggestionin the future NAFLD study.Objective:To establish the model of nonalcoholic fatty liver disease（NAFLD）by highfat diet and use berberine hydrochloride intervention treatment, Observe theeffects of berberine hydrochloride on the level of body weight，liver serumtransaminase，lipid，insulin resistance index TNF-a and liver histology in ratswith NAFLD.To explore the possible mechanism of berberine hydrochloride in the treatment of NAFLD.Method:1：Thirty-six male Sprague Dawley rats were randomized into two groups afternormal feeding one a week: normal control group(n=10)were fed withcommon diet; High fat model group(n=26)were fed with high fatdiet(84%common food plus10%lard,5%yolk powder and1%cholesterol).Continuous feed12weeks, every week time weighing rats weight again. Allthe rat feed in the Second Hospital of Hebei Medical University digestinginternal medicine animal laboratory, the ambient temperature of25±2℃,light and shade every12hours.2：After high fat model group randomly divided into model group (n=8),berberine hydrochloride small dose group (n=8), berberine hydrochloride bigdose group (n=8). Berberine hydrochloride full suspension with distilledwater, large and small doses groups were given berberine hydrochloride300mg/kg d.) and150mg/kg d.) by intragastric administration and continuedhigh-fat diet. NC group and FC group was given the same distilled water byintragastric administration at daily9:00a.m. to time10:00. By the end ofsixteenth week, all rats were killed. And fasting for12hours before killed.3：Observe rat fur and behavior, Electronic weighing scales rat body weightand liver wet weight,and calculating liver index: liver wet weight/bodyweight%. All rats were killed by intraperitoneal injection of10%hydrationchlorine aldehyde. Aseptic conditions the inferior vena cava take5ml blood.3000r/min centrifugal15minutes, take serum used to:(1) Tachypleus amebocyte lysate method to determine the serum endotoxinlevel.(2) Radiation immune method to determine the fasting insulin serum (FINS)content, insulin resistance index (HOMA-IR) was also assayed. HOMA-IR=（(FPG×FINS）/22.5.(3) Using automatic biochemical analyzer detection aspertate aminotransferase(AST),alanine aminotransferase (ALT), triglycerides (TG), low densitylipoprotein (LDL), high-density lipoprotein (HDL), fasting plasma glucose (FPG) and so on each biochemical index.(3) Liver organization (size:1cm×1cm×0.5cm) from left lobe of liver wasfixed in4％paraform, then convention slices were made, slices were dyedby HE, and the extents of fatty degeneration and inflammatory cellinfiltration of slices of liver tissue were detected by light microscope. Take agram of liver tissue for the preparation of liver tissue homogenate, usingWestern Blot methods to determine the liver tissue TNF-α content.Results:1The condition of the body weight, liver weight and liver index of rats in eachgroup: The NAFLD rat model was successfully established with high fat dietfor12weeks. The mean body weight, liver weight and liver index of modelgroup(570.9±13.4,19.82±2.47and3.48±0.46%,respectively) were increasedsignificantly compared with normal group(507.4±11.7,12.91±0.78and2.55±0.16%,respectively)(p﹤0.01).Compared with modelgroup, the BBR high dose group(517.2±14.7,13.30±0.62and2.55±0.14%,respectively) the BBR small dosegroup(532.15±18.90,13.76±0.69and2.61±0.10%,respectively) were decreasedmarkedly(p﹤0.01, respectively).2Serum biochemical index:(1) Serum ALT level(U/L) of model group(67.88±8.68) were increasedsignificantly compared with normal group(41.63±6.48)(p﹤0.01). Comparedwith model group, ALT level of the high dose and small dose treatmentgroup(42.38±3.78and39.25±4.43,respectively) were decreased markedly (p﹤0.01). Serum AST level(U/L) of model group(215.88±12.99) were increasedsignificantly compared with normal group(157.80±17.72)(p﹤0.01).Compared with model group, AST level of each treatment group(159.63±8.05and163.88±7.14,respectively) were also decreased (p<0.01).(2) Serum TG、LDL and HDL level (mmol/L): Serum TG and LDL level ofmodel group(0.65±0.07,0.45±0.04,respectively) were increased significantlycompared with normal group(0.34±0.05,0.16±0.04,respectively),(P<0.01,respectively).The TG and LDL level of the high doses and small dose treatment group drop to(0.36±0.06,0.24±0.03)and(0.38±0.05,0.22±0.03), Compared with modelgroup reduced significantly (P<0.05);Serum HDL level of modelgroup(0.65±0.06) were significantly decreased compared with normalgroup(0.82±0.08)(p﹤0.01). Compared with model group, HDL level of thehigh dose and small dose treatment group(0.85±0.05,0.81±0.06) wereincreased markedly (p﹤0.01).3The changes in insulin resistance and fasting blood glucose: The modelgroup of fasting blood glucose, fasting plasma insulin and HOMA-IR(9.7±0.6,12.31±0.76,5.32±0.59) were increased markedly compared withnormal group (4.7±0.5,8.53±0.58,1.79±0.30)(p﹤0.01). Compared withmodel group, the high dose and small dose treatmentgroup(5.8±0.4,9.67±0.63,2.51±0.30) and（5.9±0.6,9.52±0.65,2.49±0.39）weresignificantly decreased, have statistical difference(p﹤0.01).4Serum endotoxin level:Serum endotoxin level of model group(0.24±0.02)were increased significantly compared with normal group(0.12±0.01)(p﹤0.01). Compared with model group, Serum endotoxin level of the high doseand small dose treatment group(0.14±0.01and0.17±0.01,respectively) weredecreased markedly (p﹤0.01).And high dose group reduce more apparent.5Liver tissue pathology: The naked eye: NC group rat liver structure has beennormal. The edge of the liver is sharp, smooth surface, and color is bright red.The fatty model rats’ liver of its appearance is diffuse enlargement, edge isblunt and thick, and the surface color is yellow. And with the surroundingtissue have adhesion.By light microscopy HE dyes display: Steatosis and inflammation were notseen in NC controlled group, while the liver cells cable is more inattentivearrangement, liver cell markedly swollen, and severe lipid degeneration werefound in model group, especially around central vein. Hepatic steatosis areareaches more than two-thirds. Part of the vision to see inflammatory cellsinfiltrating, and mild necrosis of liver cells in the model group. Steatosis andinflammation show significant improved in the each treatment group, liver cells are arranged in order, and appearance close to normal.6TNF-a content of liver tissue:Compared with normal group the model grouprat liver tissue TNF-a content is significantly higher, given drug berberineafter intervention liver tissue TNF-a content decreased obviously. And highdose group reduce more apparent.Conclusion:Rats model of NAFLD has been successfully established by fat-rich dietafter12weeks. BBR can improve insulin resistance, lipid metabolism,alleviate inflammation and restrain bacterial endotoxin, etc to presenting theprotective effect for NAFLD. But the treatment effect and the dose have nosignificant amount-activity relationship.