Albiflorin Granule significantly decreased the cholesterol gallstone formation by the regulation of insulin transduction signal

Bang-Jiang Fang， Jun-Yi Shen， Hua Zhang， Shuang Zhou， Chuan-Zhu Lyu， Yi-Qiang Xie

1Emergency Department， Long Hua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine， Shanghai 200032， China

2Changhai Hospital of Traditional Chinese Medicine， Second Military University， Shanghai 200032， China

3Traumatology Department， Affiliated Hospital of Hainan Medical University， Haikou 571199， Hainan， China

4College of Traditional Chinese Medicine， Hainan Medical University， Haikou 571199， Hainan， China

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

1Emergency Department， Long Hua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine， Shanghai 200032， China

2Changhai Hospital of Traditional Chinese Medicine， Second Military University， Shanghai 200032， China

3Traumatology Department， Affiliated Hospital of Hainan Medical University， Haikou 571199， Hainan， China

4College of Traditional Chinese Medicine， Hainan Medical University， Haikou 571199， Hainan， China

ARTICLE INFO

Article history:

Received 15 May 2016

Received in revised form 16 June 2016

Accepted 1 July 2016

Available online 20 September 2016

Cholesterol gallstone

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 ef f ective role of Albif l orin 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-β protein expression levels were detected by western blotting. Results: Albif l orin signif i cantly 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-β protein in the GS group increased significantly and Albif l orin could reduce IKK-β 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. Albif l orin can regulate various aspects of insulin signal transduction pathway to prevent the formation of gallbladder.

1. 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 inducepancreatitis， 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 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 ef f ectively， 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 2 diabetes or latent autoimmune diabetes of adults［2］. Rare type 2 diabetes casessometimes use high levels of exogenous insulin. At the cellular level，much of the variance in insulin sensitivity between untrained， nondiabetic humans may be explained by two mechanisms: dif f erences 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 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 fastacting and may induce insulin resistance within days or even hours in response to a large lipid inf l ux［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 af f ect 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 signif i cantly 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 predominatebeta kinase （IKβ） 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 Albif l orin. Then， the serum TG， TC， FFA level and expression level of InsR， PKB， IKK-β were observed in guinea pig liver tissue.

2. Material and method

2.1. Materials

The antibodies of InsR， PKB， IKKβ were purchased from Abcam. Ursodesoxycholic 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）； Tris-base 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）.

2.2. Animals and grouping

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） ℃ 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， Albif l orin group and ursodesoxycholic 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 8 weeks. For gallstone prevention studies， guinea pigs in Albiflorin group with a lithogenic diet supplemented with Albif l orin at 10 mg/kg every 12 h for 8 weeks. Those in UDCA group were administered with a lithogenic diet supplemented with UDCA （80 mg/kg/d） for 8 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 2 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 Mgna FTIR-750 infra-red 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. Brief l y， the 3 μL serum was added into 300 μL reagent and incubated at 37 ℃ for 5 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.

2.7. Western blot analysis

The liver proteins were homogenized in PBS with proteaseinhibitor cocktail. The homogenates were centrifuged for 15 min at 14 000 rpm in 4 ℃. 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 μg）was resolved in 10% Tris-glycine SDS polyacrylamide gel. Protein bands were blotted to nitrocellulose membranes. After incubation for 1 h in blocking solution at room temperature， the membrane was incubated for 24 h with anti-InsR， PKB， IKKβ antibodies at 4 ℃. The secondary antibody （horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin） was added and incubated at room temperature for 1 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 β-actin.

2.8. Statistical analysis

Data are presented as means±SE. The significance of the dif f erence 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 signif i cant.

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 signif i cant dif f erence. The TG level in UDCA and Albif l orin groups are much lower than that of model group， but there is no dif f erence（P＞0.05） between UDCA group and model group but the dif f erence exists between Albif l orin and model group （P＜0.01）. The TG level in Albif l orin 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 Albif l orin is higher than that of control group but there is no signif i cant dif f erence （P＞0.05）. The TC level in UDCA and Albif l orin groups are much lower than that of model group， but there is no dif f erence （P＞0.05） between UDCA group and model group but the dif f erence exists between Albiflorin and model group （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

3. 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 fl oc shaped. The absorption peak can be detected by infrared spectrum analysis in 1 466 nm-1， 1 377 nm-1and 1 050 nm-1which can be identif i ed 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）.

Figure 1. Detection of gallstone by infrared spectrum.

From Table 1 we can detected that the FFA levels in model and UDCA group are much higher than that of control group （P＜0.01）. The FFA level in Albif l orin is lower than that of control group but there is no signif i cant dif f erence （P＞0.05）. The FFA level in UDCA and Albif l orin groups are much lower than that of model group， but there is no dif f erence （P＞0.05） between UDCA group and model group but the dif f erence exists between Albif l orin 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 1Serum TG， TC， and FFA level of dif f erent groups （mmol/L）.

3.5. Albiflorin enhanced the protein expression level of InsR and PKB

Only insulin bind to the cell membrane insulin receptor （InsR） and it can play their physiological ef f ect. Therefore， the number of InsR anomalies and function defects may af f ect insulin signal transductionand lead to IR. From the results， we can fi nd 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: （0.492 3±0.048 5）； GS group: （0.128 7±0.017 8）； UDCA group: （0.349 0±0.029 3）； Albif l orin group: （0.446 8±0.029 6）.

We can fi nd that the InsR expression level of Mode group decreased comparing with the control group. After the treatment of Albif l orin，the InsR level increased comparing with UDCA treatment group. Therefore， we can estimate that Albif l orin 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 Albif l orin 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 sugar stable. In the PKB knockout mouse models， the blood glucose reduction ability of insulin is signif i cantly reduced and the mouse characterized with insulin resistance and diabetes symptoms［13］. The in vivo and in vitro［14，15］ experiments conf i rmed that the insulin resistance and type 2 diabetes， the PKB protein levels is signif i cantly impaired （Table 2）.

Table 2Relative protein expression level of PKB and liver IKKβ.

Figure 2. InsR， PKB and IKKβ protein level of dif f erent groups.

3.6. Albiflorin enhanced IKK-β expression

The IKKβ expression level in control group is （0.114 0±0.036 3），IKKβ expression level in gallstone model is （0.554 8±0.189 5），UDCA group was （0.421 1 ± 0.052 3 ）， Albiflorin was（0.244 4±0.054 2）. IKK-β expression level in model group is higher than control group （P＜0.01）. It has confirmed that IKK β protein can make IKB phosphorylation and make it separate from NF-κB， and then NF-κB enters into the nucleus； regulate a series of inf l ammatory reaction. IKK-β is phosphorylation of serine kinase of the Insulin receptor and insulin receptor substrate （IRS）.

4. 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 identif i ed by infrared spectrum （1 466 nm-1， 1 377 nm-1and 1 050 nm-1）. Albif l orin 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 （0.492 3±0.048 5） and lowest in GS group （0.128 7±0.017 8）. Albiflorin enhanced higher InsR protein expression level（0.446 8± 0.029 6） than UDCA group （0.349 0 ±0.029 3 ）. Albiflorin enhanced the expression level of PKB and IKKβ 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 2 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 3 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.

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. Albif l orin 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 buf f er role. Heaton and Van Erpecum research suggests that if there are enough bile salts in the bile， the bubbles do not gather to form the nucleation； Only when cholesterol supersaturated， the nucleation formed［20］.

IKKβ make the phosphorylation of serine of IRS307 which leads to weakened tyrosine phosphorylation. The binding of insulin receptor and the insulin become weakened. IKKβ proteins may play important roles in insulin resistance in the activities［21］. Kim et al［22］ knockout IKKβ in rats， and the experiment shows that excessive expression of IKKβ can cause insulin resistance， and reduce the expression of IKKβ or inhibit its activity can improve insulin sensitivity. In guinea pig gallstones model the liver tissue IKKβ 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 IKKβ 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 conf l ict of interest.

Acknowledgment

The research was supported by the National Science Foundation of China （30672698）.

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10.1016/j.apjtm.2016.07.010

Bang-Jiang Fang， Emergency Department， Long Hua Hospital Affi liated to Shanghai University of Traditional Chinese Medicine， Shanghai 200032，China.

E-mail: fangbji@163.com

Tel: 18917763257

?Corresponding author: Shuang Zhou， Changhai Hospital of Traditional Chinese Medicine， Second Military University， Shanghai 200433， China.

E-mail: zhoushuang8008@163.com

Tel: 13918088023

Foundation project: This study was supported by the National Science Foundation of China （30672698）.

Albif l orin

Insulin resistance

Insulin signal transduction