Study On The Protective Effect Of IGF-â…  For Liver Cirrhotic Rats From Intestinal Barrier Injury

Decompensated liver cirrhosis is the end stage of various chronic liver diseases. Hepatitis B is popular in China, chronic hepatitis B is still the main etiology of liver cirrhosis. Complications encompassing variceal bleeding, spontaneous bacterial peritonitis, hepatic encephalopathy, hepatorenal syndrome and hepatic pulmonary syndrome are common presentations of decompensated liver cirrhosis and often life threatening to liver cirrhotic patients. All the complications are the consequences of liver dysfunction and portal hypertension. So it is of great importance to elucidate the mechanisms of liver dysfunction and portal hypertension in liver cirrhosis. Elucidating the above mechanisms would faciliate novel therpay development, and improve patients’ survival ultimately.Intestinal barrier including intestinal barrier consists of microorganism barrier, mucus adhering to the apical surface of enterocytes, tight junction complexes sealing adjacent enterocytes, abundant intestinal endothelia, immune barrier(primary immune system and secondary immune system) and the enteric nervous system can prevent intra-intestinal toxins from entering blood circulation and protect hosts from toxin induced injuries. Previous studies have indicated that increased intestinal barrier permeability develops during liver cirrhosis, which increases intra-intestinal toxins entering into blood circulation and promotes bacterial translocation. Bacterial translocation and increased lipopolysaccharides(LPS) permeability through defective intestinal barrier could cause pro-inflammatory cytokines to be released from polymorphonuclear cells in the portal vein system. Pro-inflammatory cytokines induce nitric oxide synthase(NOS) overexpression and nitric oxide overproduction in the portal vein system, which could cause hyperdynamic circulatory state of the portal vein system, and portal hypertension will ensue subsequently. Additionally, LPS activates Kupffer cells secreting vasoconstrictors such as thromboxane A2 and cysteinyl leukotrienes, which increases intrahepatic resistance and promotes portal hypertension development. Moreover, Pro-inflammatory cytokines in portal vein system also attack intestinal epithelia and aggravate intestinal barrier dysfunction. So intestinal barrier interruption is not only the consequence of liver cirrhosis, but also an active participant in the development of liver damage and portal hypertension. Endotoxemia and intestinal barrier dysfunction(especially intestinal tight junction dysfunction) very likely form a vicious circle, breaking this vicious circle has great clinical significance to liver cirrhosis patients.Insulin-like growth factor Ⅰ(IGF-Ⅰ) is a polypeptide with an insulin like structure, and whose receptors are widely distributed in muscles, bones, intestines, testicles and livers. Apart from the pro-growth and trophic effects of IGF-Ⅰ on various tissues, IGF-Ⅰ could act on intestines to regulate intestinal barrier function. A previous study showed that IGF-Ⅰ reduced intestinal barrier permeability through increasing cyclooxygenase-2(COX-2) expression and decreasing tumor necrosis-alpha(TNF-alpha) expression of intestinal cells. The cytoprotective effect of prostaglandins produced by COX-2 might be one of the mechanisms of IGF-Ⅰ improving intestinal barrier function in liver cirrhosis. Some previous studies also indicated that IGF-I unregulated tight junction protein expressions in osteoblast- like MC3T3-E1 cells and reduced apoptosis in mesenchymal stem cells. However, whether IGF-I could regulate enterocytic tight junction proteins and apoptosis to improve intestinal barrier functions is not clear.The present study is aimed to investigate the dynamic changes of intestinal barrier, IGF-Ⅰ and endotoxins during the development of liver cirrhosis with carbon tetrachloride(CCL4) induced liver cirrhotic rats models. This study evaluates the effect of IGF-Ⅰ on endotoxin levels in portal vein, claudin-1 and occludin expressions in intestines, enterocytic apoptosis and portal pressure in liver cirrhotic rats. We also use Caco-2 cells to confirm the findings in cirrhotic rats. Transepithelial electrical resistance is employed to evaluate the effect of IGF-Ⅰ on cellular monolayer permeability, Claudin-1 and Occludin expressions are determined to reveal the mechanism of IGF-Ⅰ on cellular monolayer permeability. Furthermore, we evaluate the effect of IGF-Ⅰ on LPS induced apoptosis of enterocytes. Moreover, the association between serum IGF-Ⅰ and severity of liver cirrhosis in cirrhotic patients is also assessed.This research includes three parts.Part I: The mechnism of IGF-Ⅰ protecting liver cirrhotic rats from intestinal barrier injuryPart II: The mechanism of IGF-Ⅰ attenuating permeability of Caco-2 monocelluar layerPart III: The association between serum IGF-Ⅰ and severity of liver cirrhosis in cirrhotic patients Part I Study on the protective effect of IGF-Ⅰfor liver cirrhotic rats from intestinal barrier injuryObject: To evaluate the effect of external administration of IGF-Ⅰ on intestinal barrier in liver cirrhotic rats by detecting the expressions of intestinal tight junction proteins and intestinal apoptosis.Methods:1.30 healthy male adult Sprague-Dawley rats were divided into control group,liver cirrhotic group and liver cirrhotic treatment group randomly. The liver cirrhotic group was injected with CCL4(0.2ml/kg/d) intraperitoneally for 6 weeks. The liver cirrhotic treatment group was administered with CCL4(0.2ml/kg/d) intraperitoneally for 6 weeks, then was administered with recombinant human IGF-Ⅰ(0.2μg/kg/d) subcutaneously for 21 days afterwards.2. After treatments, portal pressure was measured in all rats. Blood in portal veins, liver tissues and terminal ileum tissues were collected.3. The serum ALT and AST levels were determined with the assay kit.4. The levels of serum IGF-Ⅰ were measured with the ELISA kits. Limulus Amebocyte Lysate test kit was employed to determine endotoxin concentration. HE staining and Masson trichrome staining were perform to evaluate liver histology, Image pro plus software was used to quantify the severity of liver cirrhosis.5. Western blot and Reverse transcription-polymerase chain reactions(RT-PCR) were used to evaluate claudin-1 and occludin expressions in terminal ileum.6. TUNEL assay was employed to evaluate terminal ileum apoptosis. Bax and Bcl-2 were also determined with RT-PCR to evaluate terminal ileum apoptosis.Results1. After 6 weeks of CCL4 administration, rats developed liver cirrhosis with ascites. Liver cirrhosis was confirmed with liver histology.2. Liver fibrosis in the liver cirrhotic treatment group was less severe than that in the liver cirrhosis group(P<0.001).3. Portal pressure was significantly lower in the liver cirrhotic treatment group than in the liver cirrhotic group(P<0.001).4. Serum ALT(P<0.001) and AST(P<0.001) levels were lower in liver cirrhotic treatment group than in the liver cirrhotic group.5. Serum IGF-Ⅰ was higher in liver cirrhotic treatment group than in the liver cirrhotic group(P<0.001). Plasma endotoxins were lower in the liver cirrhotic treatment group than in the liver cirrhotic group(P<0.001).6. The terminal ileum protein expression of Occludin(P<0.001) and Claudin-1(P<0.001) were higher in the liver cirrhotic treatment group than in the liver cirrhotic group. The terminal ileum mRNA level of Occludin(P<0.001) and Claudin-1(P<0.001) were higher in the liver cirrhotic treatment group than in the liver cirrhotic group.7. TUNEL indicated that terminal ileum apoptosis level was lower in the liver cirrhotic treatment group than in the liver cirrhotic group(P<0.001). Terminal ileum Bax mRNA level was lower in the liver cirrhotic treatment group than in the liver cirrhotic group(P<0.001). Terminal ileum Bcl-2 mRNA level was higher in the liver cirrhotic treatment group than in the liver cirrhotic group(P<0.001).Conclusions: External administration of IGF-Ⅰ restores serum IGF-Ⅰ levels to some extent in liver cirrhotic rats. External administration of IGF-Ⅰ improves intestinal barrier function in liver cirrhosis by increasing the expressions of tight junction proteins in intestines and decreasing intestinal apoptosis. Improved intestinal barrier function decreases endotoxin levels in portal veins, which improves liver function and attenuate portal hypertension. Additionally, IGF-Ⅰ can alleviate liver fibrosis.Part II The mechanism of the effect of IGF-Ⅰ on the permeability of Caco-2 cell monolayerObject: The aim is to evaluate the mechanism of the effect of external administration of IGF-Ⅰ on the permeability of Caco-2 cell monolayer by detecting the expressions of intestinal tight junction proteins and intestinal apoptosis.Methods:1. Caco-2 cells at the logarithmic phase were collected and incubated with serum-free DMEM for 24 hours and monolayer model was developed. After attachment to the wall, Caco-2 cells were administered with IGF-Ⅰ(100ng/ml), LPS(10μg/ml), IGF-Ⅰ(100ng/ml) plus LPS(10μg/ml) and no treatment for 24 hours respectively. Each group was replicated with 6 wells.2. Transepithelial Electrical Resistance(TEER) was used to determine the permeability of Caco-2 cell monolayer.3. Western blot and RT-PCR were used to evaluate claudin-1 and occludin expressions in Caco-2 cell monolayer.4. TUNEL assay was employed to evaluate Caco-2 cell monolayer apoptosis. Bax and Bcl-2 were also determined with RT-PCR to evaluate Caco-2 cell monolayer apoptosis.Results1. TEER was significantly higher in the IGF-Ⅰ group than that in the no treatment group(P<0.001). TEER was significantly higher in the IGF-Ⅰ plus LPS group than that in the LPS group(P<0.001).2. The protein expression of Occludin(P<0.001) and Claudin-1(P<0.001) were higher in the IGF-Ⅰ group than in the no treatment group. The m RNA level of Occludin(P<0.001) and Claudin-1(P<0.001) were higher in the IGF-Ⅰ group than in the no treatment group. The protein expression of Occludin(P<0.001) and Claudin-1(P<0.001) were higher in the IGF-Ⅰ plus LPS group than in the LPS group. The m RNA level of Occludin(P<0.001) and Claudin-1(P<0.001) were higher in the IGF-Ⅰ plus LPS group than in the LPS group.3. TUNEL indicated that apoptosis level was lower in the IGF-Ⅰ group than in the no treatment group(P<0.001). TUNEL indicated that apoptosis level was lower in the IGF-Ⅰ plus LPS group than in the LPS group(P<0.001).4. Bax m RNA level was lower in the IGF-Ⅰ group than in the no treatment group(P<0.001). Bcl-2 m RNA level was higher in the IGF-Ⅰ group than in the no treatment group(P<0.001). Bax m RNA level was lower in the IGF-Ⅰ plus LPS group than in the LPS group(P<0.001). Bcl-2 m RNA level was higher in the IGF-Ⅰ plus LPS group than in the LPS group(P<0.001).Conclusions: External administration of IGF-Ⅰ decreases the permeability of Caco-2 cell monolayer by increasing the expressions of tight junction proteins and decreasing apoptosis.Part III The association between IGF-Ⅰ levels and severity of liver cirrhosisObject: The aim is to evaluate the association between serum IGF-Ⅰ and severity of liver cirrhosis in cirrhotic patients.Methods:1. This descriptive-analytic cross sectional study was performed on patients with cirrhosis which were admitted to the first affiliated hospital of Dalian medical university during the years 2014.8-2015.4. The liver disease severity at admission was assessed by the Child-Push classification and by the MELD score. A group(Child A, 5-6 score), B group(Child B, 7-9 score), C group(Child C, 10-15 score), D group(MELD,≤11 score), E group(MELD, 11-18 score), F group(MELD, ≥18 score).2. Patients were evaluated within 24 h of admission by one of the researchers involved in the study.The following tests were performed for this study: Albumin, total bilirubin,creatinine,prothrombin time,international normalized radio and IGF-Ⅰ.Results:1. The common causes of liver cirrhosis in these patients were alcoholic liver diseases(14.85%),viral hepatitis(52.48%),autoimmune hepatitis(23.76%),others(8.91%).Upon admission, ascites in 58.42%, spontaneous bacterial perrtonitis in 11.88%,upper gastrointestinla bleeding was observed in 31.68%, hepatic encephalopathy in 48.51%.2. A negative correlation was observed between IGF-Ⅰand the total Child-Push score, MELD score, prothrombin time,total bilirubin and INR, A positive correlation was observed between IGF-Ⅰand albumin,no significant correlations was observed between IGF-Ⅰand creatinine.3. The higher Child-Push scores,the lower IGF-Ⅰlevels.Higher IGF-Ⅰlevels were observed in patients classified as Child-Push A as compared to patients classified asChild-Push B(P<0.001) or C(P<0.001),no differences were observed when comparing IGF-Ⅰlevels between Child-Push B and C(P=0.327).4. The higher MELD scores,the lower IGF-Ⅰlevels.Higher IGF-Ⅰlevels were observed in patients MELD(≤11) as compared to patients MELD(11-18)(P<0.001) or MELD(≥18)(P<0.001),no differences were observed when comparing IGF-Ⅰlevels between MELD(11-18) and MELD(≥18)(P=0.658).Conclusions: Our study evaluate the association between serum IGF-Ⅰand Child-Push classification,MELD scores.liver function indexs.so IGF-Ⅰcan be an index of severity of cirrhosis and also a marker of liver function.