| Alcoholic fatty liver (AFL) identifies a liver in which lipids account for more than 5% of liver wet weight, or 1/3 above of liver cells have steatosis in liver biopsy, which result from excessive intake of alcohol. Steatosis is divided into mild, moderate and severe degrees based on the fat content, which is from one-third to half of the hepatocytes with steatosis; from half to two-third of the hepatocytes with steatosis; more than two-third of the hepatocytes with steatosis. Some show the diffuse small droplet fatty change characteristic. AFL is the earliest and mildest response of alcoholic liver disease (ALD), which includes progressive liver damage due to fatty infiltration, inflammation, fibrosis, and finally cirrhosis. Four pathological types is a progressive process, or different types exist at the same time.Due to Chinese wine culture and improvement of people’s living level, our country’s drinking population and alcohol consumption is increasing year by year. Public health survey report on drinking in 25 province of China in 2007 shows that our country’s drinking population exceeds 500 million, and men and women drinking rates were as high as 84.1%and 29.3%, while 65% of whom drinking excessive. Alcoholism has always been the primary factor to cause serious liver disease in Western countries. The incidence of alcoholic liver disease was up to 84% in the drinking crowd, and more than 80 percent of heavy drinkers suffer from AFL. Therefore, how to prevent the further development of alcoholic fatty liver is the focus of our current research.Risk factor is clear for AFL, which is long-term heavy drinking, but its pathogenesis is very complex. After drinking, ethanol in the gastrointestinal soon be absorbed and then mostly metabolized into acetaldehyde in the liver. It is currently considered the major cause of AFL is the direct damage of ethanol, the toxic effects of acetaldehyde, reactive oxygen species and lipid peroxidation injury, inhibition of fatty acid oxidation, endotoxemia and cytokine-related damage. LPS-induced Kupffer cells activation plays an important role in the pathogenesis of AFL, throughout the whole process of development of AFL. High alcohol concentrations can directly damage the intestinal epithelium, thereby impairing the ability of the epithelium to serve as a barrier preventing access of unwanted substances from the intestine to the bloodstream. Thus, gut-derived endotoxin can leak into bloodstream, which causes endotoxemia. Endotoxin in the blood circulation can activate Kupffer cells, and then activated Kupffer cells can greatly enhance NADPH oxidase and produce greater amounts of cytokines, particularly TNF-a and ROS, NO, superoxide anion, which result in excessive immune response and oxidative stress, eventually leading to injury of hepatocyte structure and function, and then fat metabolism disorders. A large number of experiments have presented that TNF-a antibody and TNF-a receptor-knockout mice or NADPH oxidase-deficient mice are resistant to liver damage caused by long-term exposure to ethanol and don’t increase the production of free radical. Therefore, preventing LPS-induced Kupffer cell activation or clear the inflammatory cytokines and reactive oxygen species released from activated Kupffer cells will be resistant to liver damage caused by long-term exposure to ethanol.The current method for treatment of AFL is abstinence and symptomatic drug treatment, such as nutritional support, antiatheroscloresis, unsaturated fatty acids and phospholipids, but it is ineffective for treatment of severe alcoholic fatty liver.Antioxidant compounds or anti-endotoxin therapeutic options is also used for the treatment of AFL, but we still can not obtain the desired effect, maybe most antioxidant compounds can’t target the inflammatory cytokines and reactive oxygen species released from activated Kupffer cells. The components of Chinese medicine are complex; the active ingredient is difficult to determine; preparation process is not stable enough; the finished product quality is difficult to control. Therefore, there is still no effective drug with fewer side effects for treatment of AFL, and it is very urgent to find more effective drugs for treatment of AFL.In 2001, Kawada et al. first discovered the expression of CYGB protein by proteomic analysis in rat stellate cells. The expression of CYGB protein was augmented in thioacetamide-induced fibrotic liver. It is initially named stellate cell-activated protein (STAP). Protein sequencing confirmed that it is hexacoordinate hemoglobin, representing a fourth member of the globin superfamily in mammals. Immunohistochemical analysis revealed that CYGB is ubiquitously expressed in several organs, such as kidney and pancreas, and localized in fibroblast-like cells. Afterwards it is named cytoglobin (CYGB). CYGB is a 21.4-kDa protein consisting of 190 amino acids and mapped at the 17q25.3 chromosomal segment.Since the discovery of CYGB a decade ago, its function is gradually being recognized. It is confirmed that CYGB is of peroxidase activities and nitric oxide dioxygenase and it can scavenge reactive oxygen species and make nitric oxide metabolized to nitrate, which is to protect cells against oxidative damage by free radical. So far, many experiments have demonstrated that exogenously overexpression of rhCYGB could protect hepatic stellate cells (HSC) from oxidative stress and suppress their differentiation to a myofibroblast-like phenotype both in vitro and in vivo. And animal experiment represented that rhCYGB could protect against liver fibrosis induced by CCl4 in rat. Similarly, oxidative stress is also a major cause in AFL, so we considered whether rhCYGB may suppress pathological development of AFL.This experiment was divided into two parts. In the first, we observed the efficacy of rhC YGB for treatment of alcoholic fatty liver in rats. In the second part, we observed whether rhC YGB inhibited LPS-induced activation of rat Kupffer cells to explore the mechanism of rhCYGB in treatment of alcoholic fatty liver.We used the constructed engineering bacteria E.coli PET28a-rhCygb-BL21 (DE3) to express the target recombinant protein. Protein renaturation and preliminary purification was accomplished by molecular sieve chromatography and then further purified by affinity chromatography. Protein concentration was detected by BCA assay. Purified rhCYGB was analyzed by SDS-PAGE (12% acrylamide). Analysis of grayscale was performed by using BandScan5.0 software and determined the purity of purified rhCYGB. Then we used the T-AOC assay to determine the antioxidant activity of rhCYGB after purification.The first part, forty male SD rats were randomly divided into 2 groups:the normal control group (A) with 9 rats and the model group (M) with 31 rats. Rats in normal control group drank water freely; rats in model group drank liquor freely. And the liquor was prepared by 55%(v/v) Red Star Erguotou and contained 10% sugar, the alcohol concentration of which increased gradually from 5,10,15,20,25, 30,35 to 40%(v/v). Each concentration was supplied for ten days and the concentration 40% for twenty weeks. During the experiment period, all rats were fed with ordinary forage. After 30W, two rats were randomly choosed from each group for pathological observation to confirm AFL model rats replicated successfully. Then the model group were randomly divided into 3 groups with 10 rats in each group:the blank model group (B), rhCYGB treatment group (C), reduced glutathione (Atomolan) treatment group (D). Normal control group received free access to water, and other groups continued to be free access to 40 percent of the liquor, and additionally, rhCYGB treatment group received 3 mg/Kg/d rhCYGB by subcutaneous injection; reduced glutathione treatment group received 0.12 g/Kg/d reduced glutathione by subcutaneous injection. The treatment continued for two months. All rats were fasted for 12 h, and then were killed after being narcotized with ether to leave over serum and hepatic tissue rapidly according to routine. Then we detected the transaminase levels and blood lipids and observe pathological changes.The results showed that the purity of the rhCYGB was 93.3%with antioxidant activity (95.58 ± 2.67) U/mg. So the prepared rhCYGB can be used as a drug in this experiment. After 30W, liver pathology in model group showed severe steatosis, while in the normal control lobule structure was visible, no steatosis, which confirmed AFL model rats replicated successfully. After two months of treatment, in the rhCYGB treatment group the transaminase levels and blood lipids decreased significantly, and the liver specimens is reddish-brown with soft texture and histological examination showed normal hepatocytes. Compared with the normal control, all indexes were not statistically significant (P>0.05). While in the reduced glutathione treatment group, although the transaminase levels and blood lipids decreased compared with the model group, histological examination still showed obvious steatosis and did not achieve the desired therapeutic effect.In the second part rat Kupffer cells isolated from livers of male Wistar rats were cultured in RPMI1640 medium supplemented with 10%fetal bovine serum at 37℃ and 5%CO2. For experiments, rat Kupffer cells were digested with 0.25% trypsin-EDTA and then seeded at 3×105 per milliliter in 96-well plates or 6-well plates. After cells adherence, cells were treated with 10 μg/mL LPS in the presence or absence of variant concentrations of rhCYGB (5,10 or 20 μg/mL) for 24 h. Four or two repeated wells were set for each group in every experiment, which was repeated three times. Simultaneously, culture medium incubated without cells was used as a blank control. Subsequently, the supernatants or cells were harvested for analysis. Cell proliferation rate was measured using the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The supernatants were collected for detecting TNF-α level with the rat TNF-α ELISA kit according to the manufacturer’s instructions. NADPH oxidase activity within cells was determined with quantitative colorimetric assay. Intracellular ROS, NO and superoxide anion was measured by fluorescent probe and then fluorescence intensity was detected by Ultra Multifunctional Microplate Reader and observed directly under the fluorescence microscope.The result showed that the purity of the Kupffer cell cultures was more than 95% as judged by the phagocytosis of India ink.10 μg/mL LPS could activate rat Kupffer cells, which was reflected that 10 μg/mL LPS increased cell proliferation rate by 81.6% ± 2.9% compared with the normal control, and enhanced NADPH oxidase activity in conjunction with a significant increase in the production of TNF-α, ROS, NO and superoxide anion. Supplementation with variant concentrations of rhCYGB attenuated these adverse changes in a dose-dependent manner induced by LPS administration. rhCYGB (20 μg/mL) alone had no impact on rat Kupffer cells untreated by LPS.These results suggest rhCYGB could reverse alcoholic fatty liver in rats and had a better therapeutic effect than reduced glutathione, which achieved the desired therapeutic effect. It may be the mechanism for rhCYGB in the treatment of alcoholic fatty liver that rhCYGB could inhibit rat Kupffer cell activation induced by LPS and reduce the activity of NADPH oxidase as well as decrease the levels of TNF-α, ROS, NO and superoxide anion, which avoided injury on hepatocyte caused by excessive inflammatory response and oxidative stress.CYGB, as a protective protein, has few side effects on the body and is up-regulated in various stress conditions including hypoxia, oxidative stress and fibrosis. Moreover, when exogenously overexpressed, CYGB is shown to protect against these adverse factors. Because of the limited ability, the organism can not produce enough CYGB to protect against these serious adverse factors, so we need to exogenous supply CYGB in order to achieve treatment goals. These experimental results show that rhCYGB could reverse alcoholic fatty liver in rats by inhibiting LPS-induced activation of Kupffer cells. Therefore rhCYGB as being a genetically engineered drug may be a promising candidate for development as a therapeutic agent for alcoholic fatty liver. |
