Application of PCR-HRM in the study of relationship between leptin gene promoter polymorphisms and liver cirrhosis

AIM: To analyze the feasibility of PCR-high-resolution melting curve analysis (HRM) for detecting the site mutations of C2549 and G2548 in leptin gene promoter from the patients with liver cirrhosis, and to explore the relevance between mutant genotypes and physiological and biochemical indexes in liver cirrhosis patients. METHODS: Compared with the method of PCR-restriction fragment length polymorphism (RFLP), the present research used the method of PCR-HRM to analyze the site mutations of C2549 and G2548 in leptin gene promoter in control group (n=100) and liver cirrhosis group (n=100). The physiological and biochemical indexes of the patients were also detected and compared. RESULTS: Leptin gene promoter polymorphism was detected using PCR-HRM with effectiveness, high flux and accuracy. Preliminary results showed that the main mutation of the patients with liver cirrhosis was in C2549 site, but not found in G2548 site. Leptin, free leptin index (FLI), fasting insulin (FINS) and insulin resistance index estimated by homeostatic model assessment (HOMA-IR) in liver cirrhosis group were higher than those in control group. Insulin sensitivity index (ISI) and soluble leptin receptor (sOB-R) in liver cirrhosis group were lower than those in control group with significant difference except leptin level. Meanwhile, FLI showed positively correlated with FINS and HOMA-IR (r=0.45, r=0.53, P<0.05), and negatively with ISI (r=-0.34, P<0.05). In the patients with liver cirrhosis, C2549A heterozygous mutation was predominant. The indexes of HOMI-IR, leptin, sOB-R and FLI of C2549A homozygotes and heterozygotes were higher than those of the wildtypes, which showed significant difference except leptin and sOB-R levels (P<0.05). CONCLUSION: PCR-HRM can be more accurate for identifying leptin promoter polymorphism. The increase in the frequency of C2549A mutation may be closely related with liver cirrhosis. Existence of hyperinsulinemia and insulin resistance may be correlated with leptin level in the patients with liver cirrhosis.     

Effect of simple phototherapy or phototherapy combined with albumin therapy on severe jaundice in full-term neonates

AIM: To analyze the effect of non-exchange transfusion therapy, including simple phototherapy or phototherapy combined with albumin therapy, on severe jaundice in full-term neonates. METHODS: The full-term neonates (n=110) with serum total bilirubin (TBIL) level over 342 μmol/L recewed simple phototherapy or phototherapy combined with albumin therapy. The changes of serum bilirubin levels and neurological signs of these neonates were observed. RESULTS: Serum TBIL and indirect bilirubin (IBIL) levels in the 2 groups of hospitalized cases significantly reduced after the first day of treatment and at discharged (P<0.01). The reduced degrees of TBIL and IBIL levels in the neonates given phototherapy combined with albumin therapy were higher than those in the neonates given simple phototherapy. All these neonates did not have bilirubin encephalopathy on admission or at discharged. CONCLUSION: Both simple phototherapy and phototherapy combined with albumin therapy treat severe jaundice effectively and prevent acute bilirubin encephalopathy in full-term neonates.     

Association of polymorphism of Fc receptor γ chain gene at position-29 in promoter with the susceptibility to systemic lupus erythematosus in southern Chinese

AIM:To detect the association between the polymorphism of Fc receptor γ chain gene at position-29 in promoter and systemic lupus erythematosus(SLE).METHODS:The genotypes at position -29 in promoter of Fc receptor γ chain gene were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method in 180 patients with SLE and 140 ethnically matched controls in southern China.RESULTS:The frequencies of TT genotype(33.3%) and T allele (54.4%) at position -29 in patients with SLE were significantly higher than those in controls (17.2% and 42.9%, respectively), whereas, the frequencies of GG genotype (24.4%) and G allele (45.6%) in patients with SLE were remarkably lower than those in controls (31.4% and 57.1%, respectively) (P＜0.05). The TT genotype and T allele at position -29 were not associated with lupus nephritis in SLE patients (P＞0.05).CONCLUSION:Our results indicate that the T allele at position -29 in promoter of Fc receptor gene probably contributes to the susceptibility to SLE, but does not play a role in the occurrence of lupus nephritis.     

Effect of sorafenib on liver regeneration after partial hepatoectomy in liver cirrhotic rats

AIM: To study the effect of sorafenib on the liver regeneration after partial hepatectomy (PH) in cirrhotic rats. METHODS: Thirty Wistar rats with liver cirrhosis induced successfully with diethylnitrosamine (DEN) underwent 30% PH and then were randomly divided into 2 groups (n=15). The rats in experimental group were fed with sorafenib at dose of 30 mg·kg^-1·d^-1 from the 1st day to the 10th day after PH, while those in control group were fed with vehicle by gavage. The blood and liver tissues of the rats were collected after PH and at the end of the experiment. Liver regeneration rate (LRR) and proliferating cell nuclear antigen (PCNA) expression were assessed for determining the hepatocyte proliferation. The content of alanine transaminase (ALT), albumin (ALB), total bilirubin (TBIL), direct bilirubin (DBIL), angiogenesis related factors including vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 2 (VEGFR-2), platelet-derived growth factor receptor β (PDGFR-β) and micro-vessel density (MVD) were measured in both groups. RESULTS: LRRs on day 10 after PH were 45.43%±3.36% and 44.21%±2.77% in experimental group and control group, respectively (P>0.05), and the expression of PCNA in hepatic tissues of the rats was not found by the method of immunohistochemistry in both groups. Liver function index had no significant difference between the 2 groups (P>0.05). However, other than VEGF, sorafenib resulted in inhibition of VEGFR-2 and PDGFR-β expression and reduction of MVD in experiment group, and significant difference between the 2 groups was observed (P<0.01). CONCLUSION: Sorafenib does not influence live regeneration after PH in liver cirrhotic rats.     

Distribution characteristics of polymorphisms of interleukin-22 gene in Guangxi population and their association with serum lipid levels

AIM: To investigate the distribution characteristics of interleukin-22 (IL-22) gene rs2227485C/T and rs2227491A/G polymorphisms in Guangxi people and the distribution differences with other ethnic groups, and to explore the difference levels of common lipid indexes in different genotypes. METHODS: SNaPshot technique and DNA sequencing were used in 280 Guangxi persons to examine IL-22 genotypes and to analyzed the distribution frequencies of allele and genotype in these sites. The distribution frequencies in different sexes, and the differences between groups and diffe-rence levels of common lipid indexes in different genotypes were analyzed statistically. RESULTS: Three genotypes of CC, CT and TT were found in rs2227485C/T with the frequency distribution of 17.1%, 49.3% and 33.6%, respectively. No significant difference between different sexes of each genotype and allele frequency in the Guangxi population was observed (P>0.05). Compared with the distribution frequencies of genotype and allele in HapMap-TSI, HapMap-HCB, HapMap-JPT and HapMap-MEX, those in Guangxi population showed statistically significant differences (P<0.05). Three genotypes of AA, AG and GG were found in rs2227491A/G with the frequency distribution of 16.1%, 52.8% and 31.1%, respectively. There was no significant difference between different sexes of each genotype and allele frequency in the Guangxi population (P>0.05). The significant differences of genotype frequencies among Guangxi population, HapMap-TSI, HapMap-JPT and HapMap-MEX were detected (P<0.05). Compared with the other 4 populations, allele frequencies in Guangxi population had significant difference (P <0.05). There were significant differences in the levels of HDL-C and LDL-C among the 3 genotypes of rs2227491A/G. The level of HDL-C had difference between AG/AA genotype and GG genotype. In addition, the level of LDL-C had difference between AG/GG genotype and AA genotype (P<0.05). CONCLUSION: rs2227485C/T and rs2227491A/G polymorphisms of IL-22 gene have differences in different populations. The rs2227491A/G polymorphism may be associated with serum lipid levels.     

Effect of bitter melon on liver fibrosis induced by carbon tetrachloride

AIM: To investigate the effects of bitter melon (BM) on liver fibrosis induced by CCl₄ in Wistar rats. METHODS: Healthy male Wistar rats were randomly divided into 4 groups (with 8 each): olive oil control group (group C), olive oil CCl₄ model group (group M), CCl₄+BM at low concentration (BM 100 g/kg, group BM-L), CCl₄+ BM at high concentration (BM 200 g/kg, group BM-H). All rats except those in group C were subcutaneously injected with CCl₄ twice a week for 8 weeks to induce liver fibrosis. After injection of CCl₄ for 8 weeks, all rats were sacrificed and the samples of blood and livers were collected. The weight ratio of liver to body was measured. The serum level of MDA and the activity of SOD were tested. The contents of total protein and albumin, the activity of GSH-Px, the content of hydroxyproline and the activity of monoamine oxidase in the liver homogenate were determined. Hepatic inflammation and collagen deposition were observed under microscope with Masson staining. RESULTS: In the rats treated with BM, the weight ratio of liver to body, the serum level of MDA, the content of hydroxyproline and the activity of monoamine oxidase in the liver homogenate were lower than those in group M (P<0.01). The serum activity of SOD, the contents of total protein and albumin, and the activity of GSH-Px in the liver homogenate were enhanced (P<0.01). The livers of the model rats had remarkable inflammatory necrosis, collagen accumulation and fibrosis. The rats in BM-treated group showed slighter hepatic injury and collagen deposition, and the liver functions were much better than those in the model group. High dose of BM showed more obvious liver-protective effects. CONCLUSION: BM attenuates liver fibrosis by its antioxidant effect and the mechanisms of reducing hydroxyproline content and monoamine oxidase activity.     

Effects of rs35100176 polymorphism in IPO8 promoter on gene expression

AIM: To investigate the effect of rs35100176 CCT insertion/deletion polymorphism in the promoter region of importin 8 (IPO8) gene on its mRNA expression. METHODS: A 342-bp fragment of IPO8 gene promoter containing the rs35100176 polymorphism was amplified from 49 DNA samples and sequenced. The IPO8 promoter fragments containing CCT 3-nucleotide insertion or deletion were amplified using the corresponding homozygote DNA samples. The PCR products were sequenced and inserted into the luciferase reporter vector pGL3-Basic. Recombinant vectors were transfected into the cells by Fugene 6.0 and the expression of the reporter gene was detected by a dual-luciferase reporter assay system. The mRNA expression level of IPO8 was detected by real-time PCR in 3-nucleotide insertion or deletion homozygote cells. RESULTS：The sequencing results showed that there were 3 kinds of genotypes in the rs35100176 polymorphism, CCT/CCT,CCT/- and -/-, and the gene frequencies were 1837%, 5510% and 2653%, respectively. The recombinant expression vectors pGL3-3N Insertion and pGL3-3N Deletion were successfully constructed. The luciferase assay showed that pGL3-3N Insertion produced significantly lower luciferase activity than that by pGL3-3N Deletion. Real-time PCR showed that HEK293 cells with 3-nucleotide insertion homozygote expressed relative lower IPO8 mRNA than Saos-2 cells with 3-nucleotide deletion homozygote. CONCLUSION：The CCT 3-nucleotide insertion variant decreases the promoter activity of IPO8, thus affecting the gene expression.     

Role of injury and phenotype shift of liver sinusoidal endothelial cells in the development of portal hypertension of cirrhosis in rats

AIM: To study the role of injury and phenotype shift of liver sinusoidal endothelial cells in the development of portal hypertension of liver cirrhosis in rats. METHODS: The rat liver cirrhosis model was established by peritoneal injection of dimethylnitrosamine (DMN) (at a dose of 10 mg·kg^-1, 3 times a week, for 4 weeks). The dynamic changes of liver cirrhosis were observed at different time points (1 day, 2 days, 3 days, 1 week, 2 weeks, 4 weeks, 6 weeks and 8 weeks). The pressure of portal vein (Ppv), the expression of CD44, von Willebrand factor (vWF), endothelin-1 (ET-1) mRNA and endothelial nitric oxide synthase (eNOS) mRNA, the serum hyaluronic acid (HA) content and liver ET-1 content were measured. RESULTS: Compared with the normal control rats, CD44 positive staining was weak in the 1 day model rats, and the numbers of fenestrae of sinusoidal endothelial cells (SECs) rapidly decreased, but serum HA content rapidly increased (P<0.05). vWF positive staining in the 2-day model rats was stronger than that in normal control rats (P<0.05). There was a positive correlation between the Ppv and the vWF expression, serum HA content in the DMN-induced liver cirrhosis rats (P<0.05). Compared with the normal control rats, ET-1 mRNA expression increased in the 2-day and 3-day model rats, and ET-1 content lightly increased. eNOS mRNA expression was stronger in the 1-day, 2-day and 3-day model rats than that in normal control rats, meanwhile eNOS always expressed at a low level. CONCLUSION: The injury and phenotype shift of SECs is a pathological basis in the development of portal hypertension of DMN-induced liver cirrhosis in rats. Imbalance of ET-1 and NO production increases intrahepatic resistance, which plays an important role in the development of portal hypertension.     

Study on the association of the polymorphism at the position -418A/G and -384C/T in the Apo(a) promoter

AIM: To investigate two single nucleotide polymorphisms (SNP) in the apolipoprotein(a) promoter at positions -418 and -384 and to compare distributing difference of genotype frequencies of single nucleotide among different races and to explore the influencies of them on serum lipid level and their association with coronary heart disease (CHD). METHODS: Using PCR-RFLP (BsgI,BfaI) method, we determined genotypes of these two SNPs in 156 unrelated healthy controls of HanZu Chinese and 56 unrelated CHD patients of HanZu Chinese and 56 unrelated African Blacks, then cloned polymerase chain reaction (PCR) products into T-vector and sequenced it by M13 currency primer, correspondingly. RESULTS: (1) There was no polymorphism at position -418A/A and -384C/C in control group. Only one CHD patient's genotype determined was -418G/G, other were -418A/A and -384C/C in CHD patients. (2) Only two African Blacks' genotype determined was -418G/G, other were -418A/A and -384C/C in African Blacks. (3) However, the Apo(a) promoter sequence was in coincident with the sequence publicized in GenBank and the base at positions -418 was adenine (A) and -384 was cytosine (C). CONCLUSION: The mutation frequencies at position -418 and -384 were low in the Chinese Han Population of Hubei and perhaps no single nucleotide polymorphisms was at two positions. No association with serum lipid levels and CHD was observed. There were great variabilities to the SNPs in the Apo(a) promoter among different races.     

Effect of urantide on liver function and histomorphology in atherosclerotic rats

AIM:To investigate the effect of urantide on the liver function and histomorphology in the rats with atherosclerosis (AS).METHODS:The AS Wistar rat model was induced by intraperitoneal injection of vitamin D₃ (VD₃) and feeding with high-fat diet. The rats were randomly divided into normal control group, AS model group, positive medicine group and urantide group. The liver function indexes of the rats were measured by biochemical test, and the pathological changes of the aorta and liver of the rats were observed by hematoxylin-eosin (HE) staining. The mRNA expression of urotensin Ⅱ (UⅡ) and GPR14 at mRNA and protein levels in rat livers was determined by RT-qPCR and Western blot. RESULTS:The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyltransferase (γ-GT), lactate dehydrogenase (LDH), total bilirubin (TBIL), indirect bilirubin (IBIL) and alkaline phosphatase (ALP) in AS model group were significantly increased compared with normal control group (P<0.05). The above indexes in urantide group were remarkably decreased compared with AS model group (P<0.05). No change of the levels of direct bilirubin (DBIL), total protein (TP), globulin (GLB) and albumin (ALB) in each group was observed. Urantide postponed hepatocyte fatty degeneration and repaired hepatocyte injury in the AS rats. Compared with normal control group, the mRNA and protein levels of UⅡ and GPR14 in the liver were significantly increased in AS model group (P<0.05). With the prolongation of dosing time, the mRNA and protein levels of UⅡ and GPR14 in the liver were significantly decreased in urantide group compared with AS model group (P<0.05). CONCLUSION:Urantide significantly attenuates the liver damage caused by liver fatty degeneration in AS rats.     

Application of ESC-derived hepatic stem cells in therapeutic liver repo pulation

AIM: To study the proliferation, differentiation and the capacity of forming teratomas of ESC-derived hepatic stem cells in mouse pre-treated with retrorsine and 70% partial hepatotomy. METHODS: The ESC-derived hepatic stem cells, labelled with CFDA SE, were transplanted into BALB/c mouse liver. The distribution, incorperation and proliferation of transplanted cells were observed under fluorescent microscopy. Hepatic function was assayed by detecting albumin level in serum. The situation of forming teratomas in vivo was also evaluated.RESULTS: 1 week post-transplantation, some scattered region was green under fluorescent microscopy. The aera of green region increased apparently in 2 weeks, and cord-like structure was observed. Immunofluorescent staining of albumin demonstrated some positve cells, but there was no significant difference for albumin level in serum (P>0.05). No teratoma was formed in the experimental group, while a large teratoma was observed in control group in 6 weeks post-transplantation.CONCLUSION: The ESC-derived hepatic stem cells are normally incorporated into mouse liver parenchymal structure, proliferate and differentiate further in vivo and possess some hepatic functions without forming teratomas.     

Effects of diammonium glycyrrhizinate on the lipid peroxidation and interstitial collagenase activity in fibrotic liver in rats

AIM: To investigate the effect of diammonium gycyrrhizinate (Ganlixin) against liver fibrosis through preventing lipid peroxidation and regulating interstitial collagenase activity. METHODS: The liver fibrotic model was induced through subcutaneous injection of CCl4 and the feeding with high fat and low protein in rats. Diammonium gycyrrhizinate was administered (70 mg/kg rat BW). Hepatic inflammation and collagen were observed with H-E and Sirius red staining. The liver function including serum ALT, AST activity, Alb and total bilirubin levels were determined. The hepatic lipid peroxidation including SOD and GSH-Px activities, MDA and GSH content were also measured. Hepatic hydroxyproline (Hyp) content was detected with Jamall's method. The activity of interstitial collagenase in liver was assayed by the reaction with ［3H］ labeled type I collagen, and the gene expression of α1(Ⅰ) pro-collagen was analyzed by RT-PCR. RESULTS: The model rats had remarkable inflammatory necrosis, collagen accumulation and fibrosis at liver, while the diammonium gycyrrhizinate treated group showed slighter hepatic injury and collagen deposition, and the much better liver function than the model. The diammonium gycyrrhizinate-treated group had lower levels of hepatic MDA, Hyp and α1 (Ⅰ) pro-collagen mRNA expression than those in the model group, but had higher levels of interstitial collagenase activity, GSH content, SOD and GSH-Px activity than those in the model group. CONCLUSION: Diammonium gycyrrhizinate has a good effect against liver fibrosis, which may be related to the prevention from lipid peroxidation and improvement of interstitial collagenase activity in fibrotic livers.     

Changes of aquaporin 4 expression after hepatic ischemia-reperfusion injury in rats

AIM: To study the relationship between the changes of aquaporin 4 (AQP4) expression and the liver functions in the process of hepatic ischemia-reperfusion (I/R) injury in rats. METHODS: Forty-eight Wistar rats were used to establish the animal model of hepatic I/R injury. The rats were subject to ischemia for 30 min and were randomly divided into 4 groups according to the time of reperfusion: 2 h group, 1 day group, 3 days group and 7 days group. The corresponding control animals were also set up. The serum was collected for detecting direct bilirubin (DB), indirect bilirubin (IB) and alanine transaminase (ALT). The pathological changes of the liver tissues were observed under microscope with HE staining. The protein expression of AQP4 was measured by the method of immunohistochemistry and the mRNA expression of AQP4 was detected by RT-PCR. RESULTS: Under microscope, degeneration and necrosis of the hepatic cells were observed in the liver tissues in I/R injury groups. Compared with sham operation group, the concentrations of DB, IB and ALT activity in I/R injury groups increased obviously, peaking on the first day after operation, then declining continuously and restoring to the normal levels on the 7th day after operation. The expression of AQP4 were significantly decreased in I/R injury animals in 2 h group, 1 day group and 3 days group, and reached the minimum level on the first day. The mRNA expression levels of AQP4 were also deceased in hepatic I/R injury rats in 2 h group, 1 day group and 3 days group, and reached the minimum level on the first day after operation, then increased slowly and restored to the normal levels on the 7th day after operation. CONCLUSION: Hepatic ischemia-reperfusion induces a decrease in AQP4 expression and impairs the liver functions, indicating an important role of AQP4 in the pathogenesis of liver ischemia-reperfusion injury.     

Effects of traditional Chinese medicine Dan-shao-hua-xian capsule on expression of miR-200s in rat fibrotic livers

AIM: To investigate the effect of Dan-shao-hua-xian (DSHX) capsule on the expression of the family of microRNA-200 (miR-200s) in rat fibrotic livers. METHODS: Forty male Wistar rats weighing 180 g to 220 g were divided into 5 groups (control group, two model groups and two interference groups). The rats in model groups and interference groups were induced by hypodermic injection of CCl4 for 4 weeks and 8 weeks. The rats in interference groups were also treated with DSHX capsule (0.5 g/kg) once daily for 4 weeks and 8 weeks at the same time. The liver index and serum activity of ALT and AST were analyzed. The liver fibrosis was observed under microscope. Additionally, the expression of miR-200a, -200b, -200c, -141 and -429 was determined by quantitative real-time PCR. RESULTS: The liver index, and serum activity of ALT and AST in model groups and 4-week interference group were obviously higher than those in normal control group. The apparent liver fibrosis was observed in 8-week model group. The expression of miR-200a,-200b, -200c, -141 and -429 in the liver of 8-week model groups was obviously higher than that in control group. CONCLUSION: In the process of liver fibrosis induced by CCl4, the obvious changes of miR-200s may play an important role in the development of liver fibrosis. The miR-200s might be the potential target that DSHX capsule inhibits the process of liver fibrosis.     

Sulforaphane inhibits diethylnitrosamine-induced hepatocellular carcinoma tumorigenesis in rats

AIM: To investigate the inhibitory effect of sulforaphane (SFN) on the hepatocellular carcinoma (HCC) induced by diethylnitrosamine (DEN) and its possible mechanism. METHODS: DEN was repeatedly injected into the SD rats to induce HCC model, and different doses (0.19 mg/kg, 0.38 mg/kg and 0.57 mg/kg) of SFN were given at the initial symptoms of fibrosis or cirrhosis. The morphological changes of liver specimens and the number of cancerous nodules were observed, and the degree of hepatocyte injury and hepatocellular carcinogenesis were evaluated by HE and Masson staining. The levels of alkaline phosphatase (ALP), aspartate aminotransferase (AST), total bilirubin (TBIL), alanine aminotransferase (ALT), interleukin (IL)-1α, IL-6, IL-10, IL-1β, tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β) in liver tissues were measured by ELISA. The activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), and content of mdlondialdehyde (MDA) in liver tissues were detected by spectrophotometry method. RESULTS: Macroscopic observation showed that the number of cancerous nodules in SFN intervention groups was lower than that in DEN group, and the dosage of SFN was negatively correlated with the degree of liver canceration. HE staining and Masson staining showed that SFN inhibited the liver canceration and inflammatory cell infiltration induced by DEN, and the degree of alleviation was positively correlated with the dosage of SFN. The data of ELISA showed that SFN attenuated the hepatocyte injury induced by DEN, and the higher the concentration of SFN was used, the lower the levels of AST, ALT, TBIL and ALP in liver tissues were detected. The levels of inflammatory factors IL-1α, IL-6, IL-1β and TNF-α in liver tissues were decreased after administration of SFN, and the degree of reduction was positively correlated with the concentration of administration, while the levels of inflammatory factors IL-10 and TGF-β were positively correlated with the concentrations of SFN. The activity of SOD, CAT and GPx was decreased with the increase in SFN concentration. CONCLUSION: SFN has a certain inhibitory effect on the liver cancer development induced by DEN, which may be related to the anti-inflammatory, antioxidant and liver injury-reducing effects of SFN.     

Morphology of endoplasmic reticulum and expression of GRP78 in rat fibrotic liver

AIM: To observe the changes of endoplasmic reticulum and the biomarker of endoplasmic retidum stress (ERS), glucose-regulated protein 78(GRP78),in the process of liver fibrosis induced by carbon tetrachloride (CCl₄). METHODS: Male Wistar rats weighing 180 g to 220 g were divided into control group and liver fibrosis group. The rats in liver fibrosis group were induced by hypodermic injection of CCl₄ for 4 weeks or 8 weeks. The liver index and the serumactivity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed. The liver fibrosis and the morphological changes of endoplasmic reticulum were observed under light and electronic microscopes, respectively. Additionally, the expression of GRP78 at mRNA and protein levels was determined by real-time PCR and the method of immunohistochemistry, respectively. RESULTS: The liver index, serum ALT and AST activity in liver fibrosis group were obviously higher than those in control group. Swelling and reduced number of endoplasmic reticulum were observed in the hepatocytes of fibrotic rats compare to the controls. The levels of GRP78 protein and GRP78 mRNA in the liver of hepatic fibrotic rats were obviously higher than those in the control rats. CONCLUSION: In the process of liver fibrosis induced by CCl₄, the obvious morphological changes of endoplasmic reticulum and increased expression of ERS protein indicate that ERS plays an important role in the liver fibrosis.     

Establishment and evaluation of a hyperbilirubinemia and kernicterus model in neonatal rats

AIM：To establish and evaluate a hyperbilirubinemia and kernicterus model in neonatal SD rats. METHODS：Three-day-old SD rats were randomly divided to 7 experimental groups by litter and body weight, and were intraperitoneally injected with physiological saline (control group), and 6.25 μg/g (T1), 12.5 μg/g (T2), 25 μg/g (T3), 50 μg/g (T4), 100 μg/g (T5) and 200 μg/g (T6) bilirubin, respectively, twice every day for 3 d. All rats were photographed, weighed and killed 12 h after the last injection. The contents of the stomach were drawn and weighed, and the index was calculated. The liver/body weight ratio was determined, the total and unconjugated bilirubin in the serum and total bilirubin in the brain were calculated, and the contents of ATP and water in the brain were measured. HE and Nissl staining were used to observe the pathological changes. RESULTS：Along with the increase in bilirubin, gradual exacerbation of the general performance of the rats, and yellowish discoloration of the skin and mucous membranes were observed.The degree of the activity gradually reduced, and the weight gain was suppressed. The weight of T6 group showed negative growth, and the 72 h mortality rate was close to 100%. The mortality rate in T4 and T5 groups continued to rise 1 week after injection. Compared with control group, the weight of stomach contents and stomach content index in T3~T5 groups significantly decreased (P<0.01). The liver/body weight ratio in T5 group was significantly higher (P<0.05). The concentrations of serum total and unconjugated bilirubin and brain bilirubin levels in T1~T5 groups were gradually increased, while the brain water content had no difference among groups. The brain ATP content in T1~T5 groups increased at the beginning and reached its peak in T3 group, but compared with control group, that in T4 group and T5 group significantly reduced (P<0.05). HE results showed that, with the increase in bilirubin concentration, the number of the neurons in the cerebral cortex of the rats decreased. In T4 group and T5 group, the neuronal structural disorder, cell swelling, nuclear pyknosis, fragmentation and dissolution, increase in non-homogeneous structure of the material dyed red, and disappearance of nuclear staining were observed. Nissl staining showed that, compared with control group, in T1 group and T2 group, the cortical neurons became smaller, Nissl bodies decreased, and cytoplasmic staining changed little. The cortical neuronal tigroid body color became light gradually, neuron cells become small, and Nissl bodies decreased obviously in T3, T4 and T5 groups. The T4 and T5 rat ce-rebral cortical neurons dissolved or even disappeared. CONCLUSION：Newborn 3-day-old SD rats receiving intraperitoneal injection of bilirubin at doses of 12.5, 25, 50 and 100 μg/g, 2 times a day, can induce hyperbilirubinemia, and 50 and 100 μg/g can cause bilirubin encephalopathy.     

Role of caspase-3 dependent hepatocyte apoptosis in liver ischemia-reperfusion injury in cirrhotic rats

AIM:To investigate whether hepatocyte apoptosis is contributed to liver ischemia-reperfusion (I/R) injury and the relationship between liver caspase-3 activity and hepatocyte apoptosis in cirrhotic rats. METHODS:Liver ischemia-reperfusion is induced by Pringle maneuver. The cirrhotic rats were randomized into two groups: Group A: simple hepatic blood inflow occlusion (HBIO); Group B: HBIO + inhibitor, before HBIO, ZVAD-fmk 15 mg/kg was injected via dorsal penis vein; Group C: healthy rat, simple HBIO. The ischemia time was 30 min in these groups. Serum aspartate aminotransferase(AST), liver caspase-3 activity, and apoptotic hepatocytes were examined in the three groups. RESULTS: After 6 h of reperfusion, the liver caspase-3 activity was markedly elevated and reached its peak, which was statistically higher than that of before I/R . The same change occurred in hepatocyte apoptosis between 6 h of reperfusion and before I/R (20.9%±4.9% vs 0.5%±0.3%, P＜0.01). As the reperfusion prolonged, the caspase-3 activity and apoptotic hepatocyte decreased gradually. The 7th-day survival rate was 62.5% in group A. The serum AST, liver caspase-3 activity and apoptotic hepatocytes were significantly higher in group A than those in group B and C, representing the most severe liver injury among the three groups. CONCLUSION:Hepatocyte apoptosis is the major form of cell death in liver ischemia-reperfusion injury in cirrhotic rats. Hepatoctye apoptosis induced by I/R is caspase-3 dependent, and inhibiting caspase-3 can alleviate liver injury. The caspase-3 dependent hepatocyte apoptosis is highly contributed to the pathological phenomenon that the ischemic sensitivity of cirrhotic liver is higher than normal liver.     

Genetic polymorphisms of RTN4 gene in Chinese Guangxi population

AIM: To investigate the distribution characteristics of rs2920891A/C and rs17046647A/G polymorphisms of RTN4 gene in Guangxi population, and to compare the differences among different populations. METHODS: The genotypes of RTN4 gene at rs2920891A/C and rs17046647A/G in 323 healthy persons of Guangxi were performed by the technique of SNaPshot and DNA sequencing. The results were compared with the alleles and genotypes of other populations (HapMap-CEU, HapMap-HCB, HapMap-JPT and HapMap-YRI in HapMap). RESULTS: In Guangxi population, 3 genotypes, AA, AC and CC, and 2 alleles, A and C, were found in rs2920891A/C. The allele frequencies between male and female showed significant differences (P<0.05). The genotype and allele frequencies compared with HapMap-JPT, HapMap-CEU and HapMap-YRI had differences with statistical significance (P<0.05). Three genotypes, AA, AG and GG, and 2 alleles, A and G, were found in rs17046647A/G. The genotype and allele frequencies between male and female showed no significant differences (P>0.05), but there were significant differences of the genotype and allele frequencies as compared with HapMap-JPT, HapMap-CEU and HapMap-YRI (P<0.01).CONCLUSION: The rs2920891A/C and rs17046647A/G polymorphisms of RTN4 gene in Chinese Guangxi population are different from those in other races.