| BackgroundChronic liver disease is the main problem affecting human health.The common progress includes chronic hepatitis,liver fibrosis and cirrhosis.For chronic liver disease,excessive chronic stress can lead to the aging of liver cells and further aggravate the progression of liver disease.Therefore,to explore the injury induction and the aggravation of hepatocyte senescence on the basis of chronic liver disease,It has been a key point and difficult point to find the effective prevention and cure scheme.The occurrence and development of liver fibrosis process mainly continued stimulus effects on liver cell,start the liver inflammation cells and secrete a variety of inflammatory cytokines,which activate the effector cells(such as hepatic stellate cells(HSC,hepatic sinus endothelial cells,Kupffer cells),extracellular matrix formation and deposition,so that the fibrosis formation.Among them,hepatocytes are the target cells of virus,alcohol,fat,autoimmunity and other stimulating factors.Hepatocyte senescence is a kind of transformation after continuous injury of hepatocytes,and is the root of chronic progression of liver fibrosis.objectivesIn this paper,CCl4 was used to induce rats to establish a liver fibrosis model.After successful modeling,chronic stress was sustained in rats.By observing p21,SABG,IGF-1 and GH-R in liver tissues,the continuous chronic stress and the senescence of liver cells were evaluated on the basis of hepatic fibrosis.In order to find a way to reverse hepatocyte senescence in the future.Materials and methods1.Liver fibrosis rat model was established with CCL4.Normal SD male rats(n=80)were selected to weigh 200g~250g and were divided into normal control group(n=20)and liver fibrosis model group(n=60).The hepatic fibrosis model group was given 40% CCl4 subcutaneous injection,3ml/kg,2 times/week,and the first dose was doubled,8 weeks.The liver model control group was injected with olive oil,dose and time same as above.In addition,both groups were given the same feeding care.2.Chronic stress in rats with liver fibrosis.Using the chronic and unpredictable stress method,10 random stimuli were randomly designed to stimulate rats.(1)Fasting for 24 h.(2)Ban water for 24 h.(3)Thermal stimulation: use a small cup of 100 ° C hot water,tighten the cap and put into the cage,natural cooling.(4)Cold stimulation: use plastic bottles to hold water in the water and put them in a squirrel cage until they are naturally defrosted.(5)Tilt breeding: raise above 35 °,squirrel cage side to maintain 24 h.(6)Strange environment: move rat from rat cage to big glass steel 24 h.(7)Strange smell: 5ml of oil or glacial acetic acid in a glass is placed next to the cage at 24 h.(8)Behavior binding 2h: bind the rats’ lower limbs 2h with string.(9)Electric shock: current 0.1ma,4times/min,continuous stimulation for 10 min.(10)Day and night alternate: the day use the opaque cloth to cover the squirrel cage to simulate the night,after dark with the light to illuminate the rat cage simulation day.One of the 10 stimulus methods was randomly selected each day,and the stimulation was replaced at 8 o ’clock every morning for 4 weeks.The rats that had been successfully constructed were randomly divided into 2 groups.Hepatic fibrosis + chronic stress intervention group(n=30): after the establishment of hepatic fibrosis model,chronic stress intervention was given for 4 weeks.Hepatic fibrosis + chronic stress control group(n=20): after the establishment of hepatic fibrosis model,no chronic stimulation was given for 4 weeks.Meanwhile,in the experimental group of liver fibrosis induced by CCl4,the remaining rats were taken as the blank control group(n=15)after the normal control group was executed.Stop injecting olive oil solution,the diet feeding method is unified.Observe and record the daily diet,sleep,urine and mental status of rats in each group.3.Specimen collection and testing.After 8 weeks of CCl4 induction,rats were killed,and the liver was excised,HE staining and Masson staining were performed on liver pathological tissues to observe whether the model was successfully constructed.At the beginning of chronic stress 0 weeks,2 weekends,4 weekends,some rats were put to death in batches,and the liver right lobe tissue was used to detect p21,SABG,IGF-1,GH-R to assess the aging of liver cells.HE staining and Masson staining were performed on liver pathological tissues to observe whether the liver fibrosis was aggravated after chronic stress intervention.results1.Rats were given 40% CCl4 solution under subcutaneous injection of 3ml/Kg,2times/w,the first dose doubled,and the liver fibrosis model was successfully established in 8 weeks.2.Chronic stress intervention after 4 weeks,chronic stress intervention group rats weight(391.16±12.36)g and intervention control rats weight(443.22±12.0)g,and blank control group rats weight(569.94±9.20)g.intervention was significantly lower than the control group and the blank control group,comparing the two groups statistically significant difference(P<0.05).3.After 4 weeks of chronic stress intervention,the pathological manifestations of liver tissue in the chronic stress intervention group showed further aggravation of liver fibrosis compared with the control group.Said with semi-quantitative scoring system of liver fibrosis,and chronic stress intervention group semi-quantitative score results shown(14.25±1.49)intervention is significantly higher than control group(7.60±1.14),statistically significant difference(P < 0.05).4.After 4 weeks of chronic stress intervention,The level of p21 in liver tissue was higher in the stress intervention group(23.57±3.24)% than in the intervention control group(14.98±1.64)% and the blank control group(7.78±0.73)%.In liver tissues,SABG level stress intervention group(24.60±1.33)% was significantly higher than the intervention control group(15.21±0.96)% and the blank control group(7.42±0.89)%,and the difference between the two groups was statistically significant(P<0.05).Immunofluorescence results of IGF-1 stress intervention group in liver tissues were shown as follows: +,the fluorescence intensity was significantly weaker than that of the intervention control group: ++,and the blank control group +++.The immunofluorescence results of the GH-R intervention group in liver tissues were shown as follows: +,and the fluorescence intensity was significantly weaker than that of the intervention control group: ++,and the blank control group +++.conclusions1.Chronic stress intervention in rats with liver fibrosis further aggravates malnutrition.2.Chronic stress intervention can aggravate the degree of fibrosis in rat liver.3.With chronic stress intervention,the levels of senescence markers p21 and SABG in rat liver tissues increased significantly,suggesting that chronic stress intervention can promote the senescence of rat liver cells.4.Chronic stress intervention can reduce the content of IGF-1 and GH-R in liver cells,suggesting that chronic stress intervention can aggravate the injury of liver cells and aggravate the degree of liver fibrosis. |
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