| Objective:Liver fibrosis is the pathological basis of kinds of chronic liver disease.It is considered now that the main mechanism of liver fibrosis is the imbalance of liver extracellular matrix (ECM) synthesis and degradation,who leading to excessive deposition of ECM in the liver.Hepatic stellate cells (HSC)is a mesenchymal cell,located in Disse space.Under normal circumstances, HSC holding in a resting state,and when HSC is activated it expresses higher levels ofα-SMA,and synthesizes ECM,including collagen. Therefore the activation and proliferation of HSC has become the central link of liver fibrosis.Connective tissue growth factor (CTGF) is a cell growth regulatory factors, and is closely related to the cell proliferation,differentiation organization and the fibrosis of tissues and organs,it plays an important role in the occurrence and development in liver fibrosis.Inhibit or even reverse the development of liver fibrosis on clinical is extremely important,but now is still a lack of reliable and effective drug. Curcumin is the major active pharmacological ingredient, isolated from Curcuma Chinese,has anti-inflammatory,anti-lipid peroxidation,anti-tumor, the protection of liver and many other pharmacological effects.Large numbers of studies has confirmed that curcumin may play a role of anti-hepatic fibrosis through a variety of ways.This experiment,by detecting the expression of CTGF in mice liver tissue of CCL4-induced liver fibrosis,and the effect of curcumin on liver cell line HSC-T6 proliferation,explore the role of CTGF in the development of liver fibrosis and the possible mechanisms of the prevention and treatment of curcumin on liver fibrosis,and provide a new target for the treatment of liver fibrosis.Methods: 6 ~ 8-week-old female C57BL/6J mice were randomly divided into 4 groups,normal control group,model control group,curcumin preventive group and curcumin treatment group.In addition to the normal control group,all groups of mice were given carbon tetrachloride 5ul/g body weight (with a 1:9 mixture of olive oil) by intraperitoneal injection,2 times a week,a total of 8 weeks. Curcumin preventive group and curcumin treatment group of mice were given a gavage treatment 50 mg/kg body weight separately with the test the first week and the seventh week,3 times a week,the normal control group were given distilled water with equivalent dose,3 times a week.All mice were killed 8 weeks later.Detect the level of alanine aminotransferase (ALT) and hyaluronic acid (HA) of decapitated mice blood serum;Do liver tissues hematoxylin - eosin (HE) staining and Masson staining of collagen to observe liver inflammation and fibrosis in pathological histology;Detect the content of connective tissue growth factor (CTGF)andα-smooth muscle contractile proteins (α-SMA)with immunohistochemical method,use Beijing University of Aeronautics and Astronautics true color pathological image analysis system,each sample three slices,each slice take five vision image to analysis,take positive areal density (areal density = positive-positive cell area / total area of vision×100%) on average; Reverse transcription polymerase chain reaction (reverse transcriptase polymerase chain reaction, RT-PCR) detect the content of CTGFmRNA of liver tissue; Flow cytometry detect the effect on cell cycle and the apoptosis rate of curcumin on cultured rat hepatic stellate cells (HSC-T6).Results:1 General situations of mice: In normal control group, mice act freely, have a good mental state, shiny and tidy hair, good appetite, normal weight gain per week. With the extension of time, model group rats of carbon tetrachloride injected become bowel, offensive individually, dull hair, disorders, low spirit, loss of appetite, weight gain slow or no gain. Curcumin treatment group has a gradual improvement in symptoms with administration increase in the number of days. After 8 weeks, compared with the normal group, model group,preventive group and treatment group decreased the body weight,the difference has statistical significance (P value <0.05). Compared with model group, preventive group and treatment group increased in body weight, the difference has statistical significance (P value <0.05). Preventive group compared to treatment group in body weight, there were no significant difference (P> 0.05).2 Serological indicators: experimental animal serum ALT levels,the model control group(168.62±13.75 U/L)> curcumin treatment group(104.86±11.68 U/L)> curcumin preventive group(82.98±9.57U/L)>normal control group (47.91±8.08 U/L) , the ALT levels of between two two comparison are statistically significant, P <0.05; experimental animal serum HA levels, model control group (1609.95±125.91ng/ml)> curcumin treatment group (1290.26±127.59ng/ml)> curcumin preventive group (953.39±98.48ng/ml)> normal control group (759.10±77.31ng/ml), the HA levels of between two two comparison are statistically significant, P <0.05.3 Liver Pathology detection3.1 Pattern of liver tissue: the normal control group,the liver of mice is reddish-brown,bright and shiny;model control group,the liver of mice is darker then the normal control group in colourl,surface is less smooth and volume decreased slightly than the normal control group, the edge of liver become blunt and texture is solid,it can be seen yellow fat particle scattered at the surface of liver;The liver of curcumin preventive group and curcumin treatment group mice are reddish-brown, shiny,no significant narrowing volume.3.2 Liver pathology HE staining In normal control group of mice,the liver structure is organizational,liver cell size uniformity,no degeneration and necrosis,hepatic lobule exists and liver cells within the cord arranged neatly; In the model control group, hepatic lobule is damaged, with Hepatic cord disorders,there is infiltration of inflammatory cells,liver cells edema, part of the formation notholobule can be seen; In curcumin preventive group,a small amount of fibrous tissue hyperplasia can be seen in Health District,no formation of noth- olobule,a small amount of inflammatory cell infiltration,hepatocyte edema degeneration reduced significantly;The situation of curcumin treatment group is between the model control group and curcumin prevention group.3.3 Liver pathology Masson staining We focuse on the distribution of blue collagen fibers under a microscope.In the normal control group,only a small amount of blue perivascular fibers,thin and short, the structure of liver unit is normal, no fibrosis was seen;In the model control group,liver fibers can be seen increased significantly,widely distributed, and interconnected to form larger coarse fiber spacing,most of which located in the Meeting areas and perivascular tube; Smaller blue fibers can be seen running around in the blood vessels in the curcumin preventive group,the number of fibers is slightly more than the normal control group;In the curcumin treatment group,it was seen some coarse fiber,most of which running around in the blood vessels.The fibers of curcumin treatment group is more than curcumin preventive group,but less than the model group.4 Immunohistochemical staining 4.1α-SMA immunohistochemical staining: Compared with the blank negative control group,it is regard as positive expression that cells and tissues stained brown.The normal control group has no positive staining.In the other groups,α-SMA was mainly expressed at the Health District in the vessel wall,the junction liver fibrous tissue with the liver parenchyma,a small amount expressed in the liver parenchyma of the sinus perirenal space,occasionally was seen in the central vein wall.α-SMA expression of four groups: model control group(8.21±1.48)> curcumin treatment group(6.25±1.69)> curcumin preventive group(4.78±1.32)> normal control group(1.51±0.95), two two compared among the groups,P <0.05, differences with statistical significance.4.2 CTGF immunohistochemistry: Compared with the blank negative control group,it is regard as positive expression that cells and tissues stained yellow or brown. Normal control group has no expression of CTGF(0.31±0.19)%,and in the model control group we can see the most expression of CTGF(5.49±0.29)%,distributed at District Meeting,the portal vein and fiber spacing and hepatic stellate cells.In curcumin preventive group(0.95±0.21)%,the positive expression is less than in the curcumin treatment group(1.82±0.32)%, two two compared, P <0.05, differences with statistical significance.5 The expression of CTGFmRNA in liver tissue: Compared with normal control group(0.15±0.02), CTGFmRNA expression of model control group(0.42±0.02),curcumin preventive group(0.27±0.01) and curcumin treatment group(0.34±0.01) have a marked increase.The CTGFmRNA expression of four groups are: model control group> curcumin treatment group > curcumin preventive group> normal control group, two two compared among the groups, P <0.05, differences with statistical significance.6 Flow cytometry detects the effect of curcumin on cultured rat hepatic stellate cells in vitro, HSC-T6 cell cycle and apoptosis rate.6.1 Effect of curcumin on cell morphology Curcumin 40μmol/L in vitro cultured rat HSC, 24 h, observe cell morphological change with inverted phase contrast microscope, results showed: after curcumin treatment, HSC cell shrinkage, apoptosis phenomenon, apoptotic cell volume is smaller than normal cells, nuclear chromatin condense along nuclear membrane, the cell concentration can also happen, and apoptotic bodies also can be seen.6.2 Effect of curcumin on HSC cell cycle and apoptosis6.2.1 Curcumin with 10μmol/L, 20μmol/L, 30μmol/L, 40μmol/L, 50μmol/L respectively affect HSC-T6 cells 24h, deterct cell cycle and apoptosis with flow cytometry (Beckman), and detect cell cycle and apoptosis rate with Single histogram ststistic Determination analysis software. The results showed that: The sub-diploid apoptotic peak appers significantly with the concentration of curcumin at 10,20,30,40,50μmol/L, the apoptosis index in each group (%) is 6.8±0.6,11.8±1.5,21.3± 2.1,29.4±3.7,48,6±4.3, compared with the control group (1.9±0.6), differences with statistical significance. (P <0. 01).6.2.2 Withen 10 ~ 50μmol/L concentration range, with the increase in the concentration of curcumin, G0/G1 phase cells increased slightly, while the proportion of S phase cells decreased gradually, G2/M phase was significantly increased, Which indicates that curcumin can make HSC stagnate at the phase of G2/M, thereby inducing cell apoptosis.Conclusion:1 Liver fibrosis mouse model,in line with the serumal biochemical and pathological features,can be successfully set up after 8 weeks'intraperitoneal injection with 10% CC14 soluted with olive oil.2 Connective tissue growth factor is closely related to the occurrence of liver fibrosis,as a promoting factor to liver fibrosis,its over-expression promotes the activation and proliferation of HSC,and promotes the formation of extracellular matrix,thus involved in the occurrence of liver fibrosis and development.3 Curcumin can prevent carbon tetrachloride-induced liver fibrosis in mice,and has a certain therapeutic effect on mice has been induced to liver fibrosis by carbon tetrachloride.The mechanism may be related to the inhibition of CTGF expression, or is related to the inhibition of the activation and proliferation of HSC,and promoting HSC apoptosis. |
PDF Full Text Request