Effects Of Prenatal Hypoxia On The Susceptibility Of Fibrosis In The Adulthood Offspring Rats

Objectives: 1. To investigate PH(prenatal hypoxia) on the liver growth and development of fetal and offspring rats; 2. To evaluate the effects of PH on the susceptibility of liver fibrosis in fatal and adult offspring rats. Methods: The pregnant rats from gestation day 4 to day 20(GD4-20) were fed under the hypoxic environment(10±0.5% O2) to establish PH model. The rats were divided into two groups: normoxia group and PH group. Body weight, liver weight, and liver weight/body weight ratio of fetal and offspring rats were calculated; Total protein, albumin, total bilirubin and direct bilirubin, alanine aminotransferase, and aspartate aminotransferase in the serum of 20 days, 1-month, 3-month-old offspring rats were detected by automatic biochemical analyzer. HE staining of liver tissue to evaluate liver pathology. 5-month-old offspring male Sprague-Dawley(SD) rats were randomly divided into normoxia offspring group,PH offspring group, normoxia+CCl4-treated group, PH+CCl4-treated group. Normoxia and PH group rats were injected with saline, normoxia+CCl4-treated group and PH+CCl4-treated group were injected with 40% CCl4 olive oil suspension. Body weight and liver weight of rats in four groups were determined at the end of 2, 4, and 6 weeks;HE and Masson staining to detect the liver fibrosis process, stages of hepatic fibrosis were evaluated according to semi quantitative histological scoring system(SSS system).Results: 1. Body weight, liver weight, and liver weight/body weight ratio of the fetal rats in PH group were significantly lower than the normoxia group(P<0.05); 2. HE staining: Fetal hepatocytes were irregular and the liver without lobular architecture in normoxia. More hepatic sinusoids appeared in hypoxia group. 3. Compared to the normoxia+CCl4-treated group, liver weight/body weight ratio of the rats in PH+CCl4-treated group decreased at the first two weeks while increased significantly at the fourth and sixth week(P<0.05). 4. HE staining: Normoxia+CCl4-treated and PH+CCl4-treated groups appeared liver tissue damage. Administrated for two weeks,rats in PH+CCl4-treated group showed a small amount of hepatic steatosis; liver pseudolobules formed at the fourth week in PH+CCl4-treated group. 5. Masson trichrome staining: The liver tissue showed numerous collagen fibers and pseudolobules in PH+CCl4-treated group after 4 weeks. While the phenomena appeared in normoxia+CCl4-treated group for 6 weeks. Conclusion: PH affects the growth of liver and may increase the susceptibility to adult liver fibrosis.Objectives: 1. To observe the the influence of hypoxia on cell growth, cell cycle,proliferation and apoptosis of HSC-T6. 2. To observe the link between hypoxia and liver fibrosis via detecting expression of profibrogenic proteins in HSC-T6. Methods:HSC-T6 was cultured under hypoxia(1% O2) and normoxic(21% O2) state. Cells were detected with cell counter and cell cycle and proliferation were analyzed by flow cytometry. HSC-T6 was stained with TUNEL kit to determine the apoptosis cells.Expressions of HIF-1α, TGF-β1 and PDGFR-β were detected by Western blot. Results:Cultured for 48 and 72 h and compared with normoxia group, cell viability significantly enhanced and cell proliferation index significantly increased in hypoxia group. Hypoxia increased the expression of HIF-1α significantly; TGF-β1 of hypoxia group was significantly higher than the normoxia; Expression of PDGFR-β in hypoxic cells was significantly higher than normoxia group. Conclusions: Hypoxia changes cell cycle of HSC-T6 and enhanced cell viability and proliferation. Hypoxia increases the expression of HIF-1α, which up-regulates TGF-β1 and then increases the expression of PDGFR-βlater, thus activating HSC-T6.Objective: To determine the effects of hypoxia on PI3K/Akt signaling pathway in HSC-T6. Methods: HSC-T6 was exposed to PI3K/Akt signaling pathway inhibitor LY294002 after passage and divided into four groups: N、NL、H、HL. Cultured for 3, 6,12, 24, 48, and 72 hours under hypoxia(1% O2) and normoxia(21% O2). Expressions of p110β, Akt-1, p-Akt of HSC-T6 were detected by Western blot. Results: Expression of p110β increased significantly at the time points of 3-72 hours except for 24 hours.Expression of p-Akt increased significantly under hypoxia at the time points of 3-72 hours except for 12 hours. p-Akt and p110β decreased significantly in hypoxia+LY294002 and normoxia+LY294002 groups for 3-72 hours. Conclusion:Hypoxia up-regulates the expression of PDGFR-β, which contributing to the expression of p110β and p-Akt. Incubated with LY294002, expression of p110β and p-Akt decreased, which indicates that HSC-T6 activation under hypoxia was associated with the stimulation of PI3K/Akt signaling pathway.