albiflorin granule significantly decreased the cholesterol gallstone formation by the regulation of insulin transduction signal

Asian Pacific Journal of Tropical Medicine 2016; 9(9): 877–881 H O S T E D BY 877 Contents lists available at ScienceDirect Asian Pacific Journal of Tropical Medicine journal homepage: http://ees.elsevier.com/apjtm Original research http://dx.doi.org/10.1016/j.apjtm.2016.07.010 Albiflorin Granule significantly decreased the cholesterol gallstone formation by the regulation of insulin transduction signal Bang-Jiang Fang1, Jun-Yi Shen1, Hua Zhang3, Shuang Zhou2*, Chuan-Zhu Lyu3, Yi-Qiang Xie4 Emergency Department, Long Hua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China Changhai Hospital of Traditional Chinese Medicine, Second Military University, Shanghai 200433, China Traumatology Department, Affiliated Hospital of Hainan Medical University, Haikou 571199, Hainan, China College of Traditional Chinese Medicine, Hainan Medical University, Haikou 571199, Hainan, China A R TI C L E I N F O ABSTRACT Article history: Received 17 May 2016 Received in revised form 16 Jun 2016 Accepted Jul 2016 Available online Aug 2016 Objective: To study the mechanism of insulin resistance in the cholesterol gallstone formation from insulin signal transduction pathway so as to reveal the possible mechanism and the effective role of Albiflorin Granule on preventing the cholesterol gallstones Methods: Serum triglycerides (TG), free fatty acid (FFA), and total cholesterol (TC) from different groups were measured and liver cells InsR, PKB, IKK-b protein expression levels were detected by western blotting Results: Albiflorin significantly decreased the cholesterol gallstone formation rate, increased glucose infusion rate in gallstone guinea pigs and improved insulin resistance Compared with the normal group, insulin receptor and PKB protein expression in GS group were significantly reduced IKK-b protein in the GS group increased significantly and Albiflorin could reduce IKK-b protein expression in guinea pig liver cells Conclusions: The model of insulin resistance in cholesterol gallstone guinea pig was successfully established, which plays an important role in the cholesterol gallstone formation All aspects of insulin signaling pathway are involved in gallstone formation Albiflorin can regulate various aspects of insulin signal transduction pathway to prevent the formation of gallbladder Keywords: Cholesterol gallstone Albiflorin Insulin resistance Insulin signal transduction Introduction Gallstone disease has become one of the most common digestive disorders in the world With the development of economic conditions in recent years, cholesterol gallstone (CGS) has become the main type of gallstones [1] Gallbladder stone may induce pancreatitis, severe biliary infection, and malignant biliary tumor The super saturation of biliary cholesterol is very important for the formation of cholesterol gallstones The main components of bile are the products of hepatic synthesis, intake, and secretion Thus, hepatic metabolism is related to the super saturation of biliary *Corresponding author: Shuang Zhou, Changhai Hospital of Traditional Chinese Medicine, Second Military University, Shanghai 200433, China Tel: +86 13918088023 E-mail: zhoushuang8008@163.com Peer review under responsibility of Hainan Medical College Foundation Project: This study was supported by the National Science Foundation of China (30672698) cholesterol Triglycerides (TG), free fatty acid (FFA), and total cholesterol (TC) are main hepatic metabolism indicators which associated with CGS formation Insulin resistance (IR) is a pathological condition in which cells fail to respond to the normal actions of the hormone insulin When insulin under conditions of insulin resistance, the cells in the body is resistant to the insulin and is unable to use it effectively, leading to high blood sugar Beta cells in the pancreas subsequently increase their production of insulin, further contributing to a high blood insulin level This often remains undetected and can contribute to a diagnosis of Type diabetes or latent autoimmune diabetes of adults [2] Rare type diabetes cases sometimes use high levels of exogenous insulin At the cellular level, much of the variance in insulin sensitivity between untrained, non-diabetic humans may be explained by two mechanisms: differences in phospholipid profiles of skeletal muscle cell membranes, and in intramyocellular lipid (ICML) stores within these cells [3] High levels of lipids in the bloodstream have the potential to result 1995-7645/Copyright © 2016 Hainan Medical College Production and hosting by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/) 878 Bang-Jiang Fang et al./Asian Pacific Journal of Tropical Medicine 2016; 9(9): 877–881 in accumulation of triglycerides and their derivatives within muscle cells, which activate proteins Kinase, ultimately reducing the glucose uptake at any given level of insulin [4,5] This mechanism is quite fast-acting and may induce insulin resistance within days or even hours in response to a large lipid influx [6] As short-term overdosing of insulin causes short-term insulin resistance, it has been hypothesized that chronic high dosing contributes to more permanent insulin resistance More researches show that IR is the important start factor for the formation of gallstones [7–9] Only insulin bind to the cell membrane insulin receptor (InsR) and it can play their physiological effect Therefore, the number of InsR anomalies and function defects may affect insulin signal transduction and lead to IR PKB is key signaling proteins in PI3K way on insulin regulation, so in the process of insulin resistance the PKB expression is abnormal PKB is the important factor to maintain blood sugar stable In the PKB knockout mouse models, the blood glucose reduction ability of insulin is significantly reduced and the mouse characterized with insulin resistance and diabetes symptoms Recent research shows that in the process of insulin signal transduction except for tyrosine phosphorylation a variety of signaling proteins regulates Ser/ Thr phosphorylation Ser/Thr protein kinase I kappa B predominate-beta kinase (IKb) plays an important role in the development of IR [10] In this study, the model guinea pig gallstones were established by feeding the high fat method The model was administered by Albiflorin Then, the serum TG, TC, FFA level and expression level of InsR, PKB, IKK-b were observed in guinea pig liver tissue Material and method 2.1 Materials The antibodies of InsR, PKB, IKKb were purchased from Abcam Ursodeoxycholic acid (UDCA) was purchased from Sanwei Pharmaceuticals (Shanghai, China) Albiflorin was purchased from Alfa Chemistry (Stony Brook, NY 11790, USA); Acrylamide was from sigma (St Louis, MO USA); Trisbase was from Boehringer; sodium dodecyl sulfate (SDS), glycine, ammonium persulfate and TEMED was from Sigma (St Louis, MO USA); Chemiluminescence chromogenic reagent kit and protein assay kit were from Pierce (Shanghai, China) kg/d) for weeks The gallbladder samples were taken for detection at the eighth weekend The experimental protocols were approved by the Committee of Animal Experimentation of the Shanghai University of Traditional Chinese Medicine 2.3 Gallstone identification by infrared spectrum The infrared spectroscopy method was used to identify the gallstones Mix mg gallstones powder and 200 mg dry potassium bromide powder, load to the molding, vacuum (1– 2) min, plus 10 T pressure (vacuum) at the same time, and then take out the sheeting for inspection by Magna FTIR-750 infrared spectrometer (Nicolet) 2.4 The detection of FFA FFA detection referred to Nanjing Jiancheng FFA detection kits Free fatty acid is a result of adipose decomposes; determine the content of free fatty acids that can be used to measure the fat decomposition FFA can conjugate with copper ions to form fatty acid and copper salt soluble in chloroform The content of copper salt is proportional to the content of free fatty acids To detect the copper ions in the by copper reagent can calculate, the content of FFA 2.5 The detection of TG TG test was referred to the Nanjing Jiancheng triglycerides detection kits Briefly, the mL serum was added into 300 mL reagent and incubated at 37  C for min, then detected optical density value on 546 nm by Thermo Scientific Microplate Reader (Multiskan MK3) The blank TG and the standard TG was implemented as the same procedure 2.6 The detection of TC TC test was referred to the Nanjing Jiancheng TC detection kits by COD-PAP colorimetric method Briefly, cholesterol was oxidized by COD to produce hydrogen peroxide, and then react with PAP to generate red quinone imine pigment The concentration of TC was detected by on 520 nm by Thermo Scientific Microplate Reader (Multiskan MK3) The blank TC and the standard TC was implemented as the same procedure 2.2 Animals and grouping 2.7 Western blot analysis White guinea pig with red eye, weighing (200–240) g, half male and half female, were obtained from the Experimental Animals Center of the Shanghai University of Traditional Chinese Medicine (Shanghai, China) Guinea pigs maintained under pathogen-free conditions at a room temperature of (23 ± 3)  C and air humidity of 55 ± 15% in a 12 h light/12 h dark cycle All guinea pigs were provided free access to water and divided into four groups: control group, gall-stone (GS) group, Albiflorin group and ursodeoxycholic acid (UDCA) group Guinea pigs in GC group were fed by lithogenous diet containing 1% cholesterol, 0.5% cholic acid and 15% butter fat for weeks For gallstone prevention studies, guinea pigs in Albiflorin group with a lithogenic diet supplemented with Albiflorin at 10 mg/kg every 12 h for weeks Those in UDCA group were administered with a lithogenic diet supplemented with UDCA (80 mg/ The liver proteins were homogenized in PBS with protease inhibitor cocktail The homogenates were centrifuged for 15 at 14 000 rpm in  C Supernatants of the tissues were collected and protein concentration was measured with a bicinchoninic acid assay kit An equal amount of protein from each sample (150 mg) was resolved in 10% Tris-glycine SDS polyacrylamide gel Protein bands were blotted to nitrocellulose membranes After incubation for h in blocking solution at room temperature, the membrane was incubated for 24 h with anti-InsR, PKB, IKKb antibodies at  C The secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin) was added and incubated at room temperature for h Peroxidase labeling was detected with the Western blotting detection system and analyzed by a densitometry system The relative protein levels of InsR and PKB were normalized to b-actin 879 Bang-Jiang Fang et al./Asian Pacific Journal of Tropical Medicine 2016; 9(9): 877–881 2.8 Statistical analysis Data are presented as means SE The significance of the difference in mean values within and among multiple groups was examined with an ANOVA for repeated measures followed by a Duncan's post hoc test Student's t-test was used to evaluate the significance of differences between two groups of experiments (Sigma Stat, SPSS) P < 0.05 was considered statistically significant Results 3.1 Detection gallstone by infrared spectrum Infrared and Raman spectroscopy are emerging biophotonic tools to recognize various diseases The detached gallstones present like sand or mud floc shaped The absorption peak can be detected by infrared spectrum analysis in 466, 377 and 050 nm−1 which can be identified as cholesterol stone [11] Strong correlation can be found between guinea pig gallbladder stone forming rate and the production of cholesterol crystals The gallbladder stone forming rate in pig gallbladder stone model is 83.25%, which is much higher than that of control group 5.26% Cholesterol crystal in model group is much higher than that of control group (Figure 1) (P < 0.01) The TC level in Albiflorin group is much lower than that of UDCA group (P < 0.01) 3.4 Albiflorin decreased the guinea pig serum FFA level From Table we can detect that the FFA levels in model and UDCA group are much higher than that of control group (P < 0.01) The FFA level in Albiflorin is lower than that of control group but there is no significant difference (P > 0.05) The FFA level in UDCA and Albiflorin groups are much lower than that of model group, but there is no difference (P > 0.05) between UDCA group and model group but the difference exists between Albiflorin and model group (P < 0.01) The FFA level in Albiflorin group is much lower than that of UDCA group (P < 0.01) High level FFA reduces the insulin receptors (InsR) [12] and inhibits liver glucose transportation, lastly Ins sensitivity was reduced Table Serum TG, TC, and FFA level of different groups (mmol/L) Group n TG Normal group GS group UDCA group Albiflorin group 19 5.58 ± 3.99 TC 0.43 ± 0.27 17 23.88 ± 11.45DD 0.88 ± 0.46DD DD DD 18 19.41 ± 10.58 0.82 ± 0.24 FFA 518.88 ± 205.42 971.39 ± 99.64DD 781.49 ± 167.88DD** 18 11.33 ± 7.85**## 0.51 ± 0.24**## 729.84 ± 178.07DD** DD P < 0.01, vs normal group; **P < 0.01, vs model group; ##P < 0.01, vs UDCA group 3.5 Albiflorin enhanced the protein expression level of InsR and PKB Figure Detection of gallstone by infrared spectrum 3.2 Albiflorin decreased the guinea pig serum TG We can find that the TG levels in model and UDCA group are much higher than that of control group (P < 0.01) The TG level in Albiflorin is higher than that of control group but there is no significant difference The TG level in UDCA and Albiflorin groups are much lower than that of model group, but there is no difference (P > 0.05) between UDCA group and model group but the difference exists between Albiflorin and model group (P < 0.01) The TG level in Albiflorin group is much lower than that of UDCA group (P < 0.01) 3.3 Albiflorin decreased the guinea pig serum TC The TC levels in model and UDCA group are much higher than that of control group (P < 0.01) The TG level in Albiflorin is higher than that of control group but there is no significant difference (P > 0.05) The TC level in UDCA and Albiflorin groups are much lower than that of model group, but there is no difference (P > 0.05) between UDCA group and model group but the difference exists between Albiflorin and model group Only insulin bind to the cell membrane insulin receptor (InsR) and it can play their physiological effect Therefore, the number of InsR anomalies and function defects may affect insulin signal transduction and lead to IR From the results, we can find that the InsR in model group is much lower than model group The low level of InsR may contribute to the reduced the binding ability between Ins and InsR and consequently result in the IR Relative protein expression level of InsR were as follows: Control group: (0.4923 ± 0.0485); GS group: (0.1287 ± 0.0178); UDCA group: (0.3490 ± 0.0293); Albiflorin group: (0.4468 ± 0.0296) We can find that the InsR expression level of Mode group decreased comparing with the control group After the treatment of Albiflorin, the InsR level increased comparing with UDCA treatment group Therefore, we can estimate that Albiflorin can prevent the formation of gallstones by the inhibition the process of IR The experiment results show that PKB protein expression of the guinea pig liver in model group was obviously lower than normal group, which is the same as the literature [13], PKB is key signaling proteins in PI3K way on insulin regulation, so in the process of insulin resistance the PKB expression is abnormal From the experiment, the PKB protein of Albiflorin treatment group is obviously enhanced compared with the model group (Figure 2), so as to improve the insulin resistance of gallstones in guinea pigs PKB is the important factors to maintain blood 880 Bang-Jiang Fang et al./Asian Pacific Journal of Tropical Medicine 2016; 9(9): 877–881 Figure InsR, PKB and IKKb protein level of different groups sugar stable In the PKB knockout mouse models, the blood glucose reduction ability of insulin is significantly reduced and the mouse characterized with insulin resistance and diabetes symptoms [13] The in vivo and in vitro [14,15] experiments confirmed that the insulin resistance and type diabetes, the PKB protein levels is significantly impaired (Table 2) (0.4468 ± 0.0296) than UDCA group (0.3490 ± 0.0293) Albiflorin enhanced the expression level of PKB and IKKb comparing with GS and UDCA group and the highest expression level is in control group Albiflorin regulates multiple pathways of insulin signal transduction to prevent the formation of gallstones Albiflorin is promising to become a clinic drug for the treatment of gallstones The majority of gallstone patients remain asymptomatic; however, interest toward the gallstone disease is continuing because of the high worldwide prevalence and management costs and the development of gallstone symptoms and complications For cholesterol gallstone disease, moreover, a strong link exists between this disease and highly prevalent metabolic disorders such as obesity, dyslipidemia, type diabetes, the metabolic syndrome Moreover, some risk factors for gallstone disease are modifiable and some preventive strategies have become necessary to reduce the onset and the severity of complications TG is molecules formed by long chain fatty acids and glycerol fat molecules Triglyceride is the most abundant lipid in the human body, most organizations are permitted to use energy triglyceride breakdown products, at the same time, the liver, fat and other organizations also can undertake the synthesis of triglycerides, stored in fat tissue Table Relative protein expression level of PKB and liver IKKb Group InsR Control group Mode group UDCA group Albiflorin group DD P < 0.01, vs normal group; 0.4923 0.1287 0.3490 0.4468 ** ± ± ± ± 0.0485 0.0178DD 0.0293DD** 0.0296DD**## P < 0.01, vs model group; IKK-b PKB ## 0.4551 0.1065 0.3155 0.4056 ± ± ± ± 0.0341 0.0071DD 0.0237DD** 0.0100DD**## 0.1675 1.4000 0.8288 0.7463 ± ± ± ± 0.0592 0.0859DD 0.0683DD** 0.0865DD**## P < 0.01, vs UDCA group; n = per group 3.6 Albiflorin enhanced IKK-b expression The IKKb expression level in control group is (0.1140 ± 0.0363), IKKb expression level in gallstone model is (0.5548 ± 0.1895), UDCA group was (0.4211 ± 0.0523), Albiflorin was (0.2444 ± 0.0542) IKK-b expression level in model group is higher than control group (P < 0.01) It has confirmed that IKK b protein can make IKB phosphorylation and make it separate from NF-kB, and then NF-kB enters into the nucleus; regulate a series of inflammatory reaction IKK-b is phosphorylation of serine kinase of the Insulin receptor and insulin receptor substrate (IRS) Discussion IR plays an important role in the gallbladder cholesterol calculus formation It has been confirmed that the IR was the important mechanism of the cause of lipid metabolism disorders, which is the important reason for the formation of cholesterol stone In this study, we successfully established guinea pig gallbladder cholesterol calculus model of insulin resistance and the formed gall-stone was identified by infrared spectrum (1 466 nm−1, 377 nm−1 and 050 nm−1) Albiflorin enhanced lower TC, TG and FFA level comparing with UDCA group and there is no significant difference (P > 0.05) than control group InsR protein expression is highest in control group (0.4923 ± 0.0485) and lowest in GS group (0.1287 ± 0.0178) Albiflorin enhanced higher InsR protein expression level The rise of serum total cholesterol increases the risk of gallstones [16] Biliary cholesterol may contribute to the formation of cholesterol gallstones, and regulation of these levels could be a useful therapeutic strategy for gallstones disease Albiflorin significantly low the TC levels, therefore it reduced cholesterol transport from the hepatocytes to the gallbladder Albiflorin bear the potential as drug for prevention cholesterol gallstones FFA is an important symbol of lipid toxicity, which gets more and more attention High blood FFA is the consequences of IR, which interfere with glucose, fatty acid cycle [17], inhibit glucose transportation to reduce glycogen synthesis [18] and stimulate the sugar dysplasia which induced glycogen output, and lastly reduce the liver's inactivate Ins ability [19] Cholesterol in bile is in the form of micelles and bubble Bubble is the main form of the cholesterol stone and cholesterol micelles phase is a buffer role Heaton and Van Erpecum research suggests that if there are enough bile salts in the bile, the bubbles not gather to form the nucleation; Only when cholesterol supersaturated, the nucleation formed [20] IKKb make the phosphorylation of serine of IRS307 which leads to weakened tyrosine phosphorylation The binding of insulin receptor and the insulin become weakened IKKb proteins may play important roles in insulin resistance in the activities [21] Kim et al [22] knockout IKKb in rats, and the experiment shows that excessive expression of IKKb can cause insulin resistance, and reduce the expression of IKKb or inhibit its activity can improve insulin sensitivity In guinea Bang-Jiang Fang et al./Asian Pacific Journal of Tropical Medicine 2016; 9(9): 877–881 pig gallstones model the liver tissue IKKb protein expression was enhanced and after the Albiflorin treatment the level was reduced Therefore, in the process of the formation of gallstones need further study of IKKb protein changes to understand in the pathogenesis of gallstones which caused by the high cholesterol Conflict of interest statement We declare that we have no conflict of interest Acknowledgment The research was supported by the National Science Foundation of China (30672698) References [1] Dikkers A, Tietge UJ Biliary cholesterol secretion: more than a simple ABC World J Gastroenterol 2010; 16(47): 5936-5945 [2] Cubbon RM, Kearney MT, Wheatcroft SB Endothelial IGF-1 receptor signalling in diabetes and insulin resistance Trends Endocrinol Metab 2015; 27(2): 96-104 [3] Haag M, Dippenaar NG Dietary fats, fatty acids and insulin resistance: short review of a multifaceted connection Med Sci Monit 2005; 11(12): Ra359-Ra367 [4] Schinner S, Scherbaum WA, Bornstein SR, Barthel A Molecular mechanisms of insulin resistance Diabet Med 2005; 22(6): 674682 [5] Morino K, Petersen KF, Shulman GI Molecular mechanisms of insulin resistance in humans and their potential links with mitochondrial dysfunction Diabetes 2006; 55(Suppl 2): S9-S15 [6] Bachmann OP, Dahl DB, Brechtel K, Machann J, Haap M, Maier T, et al Effects of intravenous and dietary lipid challenge on intramyocellular lipid content and the relation with insulin sensitivity in humans Diabetes 2001; 50(11): 2579-2584 [7] Aune D, Vatten LJ Diabetes mellitus and the risk of gallbladder disease: a systematic review and meta-analysis of prospective studies J Diabetes Complicat 2015; 30(2): 368-373 [8] Feakins RM Obesity and metabolic syndrome: pathological effects on the gastrointestinal tract Histopathology 2015; 68(5): 630-640 [9] Cazzo E, Gestic MA, Utrini MP, Machado RR, Jimenez LS, da Silva APC Influence of insulin resistance status on the development of gallstones following roux-en-y gastric bypass: a prospective cohort study Obes Surg 2015; http://dx.doi.org/10.1007/ s11695-015-1815-3 881 [10] Luo C, Yang H, Tang C, Yao G, Kong L, He H, et al Kaempferol alleviates insulin resistance via hepatic IKK/NF-kappaB signal in type diabetic rats Int Immunopharmacol 2015; 28(1): 744-750 [11] Yoo EH, Oh HJ, Lee SY Gallstone analysis using Fourier transform infrared spectroscopy (FT-IR) Clin Chem Lab Med 2008; 46(3): 376-381 [12] Chen CC, Lee TY, Kwok CF, Hsu YP, Shih KC, Lin YJ, et al Major urinary protein interacts with cannabinoid receptor type in fatty acid-induced hepatic insulin resistance in a mouse hepatocyte model Biochem Biophys Res Commun 2015; 460(4): 10631068 [13] Cho H, Mu J, Kim JK, Thorvaldsen JL, Chu Q, Crenshaw EB Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the protein kinase Akt2 (PKB beta) Science 2001; 292(5522): 1728-1731 [14] Oku A, Nawano M, Ueta K, Fujita T, Umebayashi I, Arakawa K, et al Inhibitory effect of hyperglycemia on insulin-induced Akt/ protein kinase B activation in skeletal muscle Am J Physiol Endocrinol Metab 2001; 280(5): E816-E824 [15] Kim YB, Peroni OD, Franke TF, Kahn BB Divergent regulation of Akt1 and Akt2 isoforms in insulin target tissues of obese Zucker rats Diabetes 2000; 49(5): 847-856 [16] Taheri H, Ghemian N, Taghavi Y, Shokry-Shirani J Biochemical profile of bile fluid in patients with malignant cholestasis in comparison with cholestasis due to gall stone Casp J Intern Med 2015; 6(3): 165-169 [17] Battilana P, Seematter G, Schneiter P, J´equier E, Tappy L Effects of free fatty acids on insulin sensitivity and hemodynamics during mental stress J Clin Endocrinol Metab 2001; 86(1): 124-128 [18] Roden M, Shulman GI Applications of NMR spectroscopy to study muscle glycogen metabolism in man Annu Rev Med 1999; 50: 277-290 [19] Bieger WP, Michela G, Barwicha D, Biehla K, Wirth A Diminished insulin receptors on monocytes and erythrocytes in hypertriglyceridemia Metabolism 1984; 33(11): 982-987 [20] van Erpecum KJ, van Henegouwen GPB, Stoelwinder B, Schmidt YMG, Willekens FLH Bile concentration is a key factor for nucleation of cholesterol crystals and cholesterol saturation index in gallbladder bile of gallstone patients Hepatology 1990; 11(1): 1-6 [21] Motta K, Barbosab AM, Bobinskic F, Boscherod AC, Rafacho A JNK and IKKbeta phosphorylation is reduced by glucocorticoids in adipose tissue from insulin-resistant rats J Steroid Biochem Mol Biol 2015; 145: 1-12 [22] Kim JK, Kim YJ, Fillmore JJ, Chen Y, Moore I, Lee J, et al Prevention of fat-induced insulin resistance by salicylate J Clin Invest 2001; 108(3): 437-446  ... expression level The rise of serum total cholesterol increases the risk of gallstones [16] Biliary cholesterol may contribute to the formation of cholesterol gallstones, and regulation of these levels... chloroform The content of copper salt is proportional to the content of free fatty acids To detect the copper ions in the by copper reagent can calculate, the content of FFA 2.5 The detection of. .. estimate that Albiflorin can prevent the formation of gallstones by the inhibition the process of IR The experiment results show that PKB protein expression of the guinea pig liver in model group