| Liver fibrosis is that diffuse liver extracellular matrix (especially collagen) excessively depose. It is not an independent disease, but it is the common pathological process of many chronic liver disease, including alcohol, viral infections, genetic metabolic, self-Immunity, and so can lead to liver fibrosis. Currently, most anti-hepatic fibrosis drugs had no significant clinical effect, or drug toxicity is too large, the actual application is limited. JAK / STAT signaling pathway is one of a cell signaling pathway factor,which involvs in cell proliferation, differentiation, apoptosis and immune regulation, and many other important biological processes. Research has shown that JAK / STAT signal transduction pathway very close ties with liver fibrosis, but the specific molecular mechanisms remain is unclear. Therefore, this experimental study investigated the role of the JAK / STAT signal transduction pathway in the pathogenesis of hepatic fibrosis and the expression of JAK / STAT signal transduction pathway in hepatic fibrosis process by Chinese medicine Fuzhenghuayu. Experiment was divided into two parts,in the first part we use liver fibrosis model ,witch carbon tetrachloride induced, study JAK / STAT signal transduction pathway dynamics in the process of liver fibrosis model and spontaneous reversal, in order to clarify the mechanisms of JAK / STAT Signal transduction pathway in the pathogenesis of hepatic fibrosis. In the second part we use the model witch is induced liver fibrosis by carbon tetrachloride,they were given the intervention of Fuzhenghuayu to clarify the effetion that Fuzhenghuayu JAK / STAT signal transduction pathway made of fibrosis, provide experimental basis for traditional Chinese medicine Fuzhenghuayu, witch can be continued researched and seek for a broader way for the development of the clinical treatment of liver fibrosis . Part I The dynamic changes of liver fibrosis of JAK / STAT signal transduc- tion pathway in the spontaneous reversalObjective: Liver fibrosis refers to the role of different causes, the cause of chronic liver cell injury and activation of certain non-parenchymal cells, such as the fiber factor induced by cytokines and activation, causing decomposition of fiber and fiber proliferation imbalance, resulting in fibrous deposition to form excessive tissue fibrosis, the consequences of its development is the formation of irreversible liver cirrhosis stage. Large number of experimental studies confirmed that hepatic stellate cells (the hepatic stellate cell) is the major cause of liver fibrosis cells, the activation is the cellular basis of hepatic fibrosis and the leading cause of liver fibrosis in different common central link and the main source of ECM synthesis. Therefore, HSC activation, has extremely close ties with the development of liver fibrosis, activation of HSC has been the treatment of liver fibrosis as the main target. Currently, studies have shown that JAK / STAT signaling pathway is closely related with liver fibrosis,but its molecular mechanisms remain unclear.Therefore, we have established the model of hepatic fibrosis induced by CCl4 .Reversal the dynamic changes of jak1 and stat3 in the jak / stat signaling pathway. Clarify the molecular mechanism of the JAK / STAT signal transduction pathway in hepatic fibrosis and spontaneous reversal and provide a theoretical basis for the development of antifibrotic drugs.Methods: We selected healthy male Wistar 64 rats of the age of 8 weeks, weighing 180 200g, They were purchased from Experimental Animal Center of Hebei Medical University. After a week of the feeding adaptation thy were randomly divided into two groups: 18 in control group,48 in the spontaneous reversal of liver fibrosis model group (model group). Control group:rats were infused normal saline by intraperitoneal injection (0.2ml/100g), 3 times per week for 8 weeks; model group : rats were infused 30% CCL4 olive oil by intraperitoneal injection (0.2ml/100g), 2 times a week; to 8 weeks liver fibrosis is formed, CCL4 stop deliverying.8 rats were killed after the first start modeling 4,6,8,10,12 weeks, At 4,6,8,10,12 weekend, we randomly selected 3rats to kill in the control group,at week 14, all rats in control and model group were killed, specimens were taken. serum specimens from liver function ALT, AST; place sacrificing part of the liver tissue frozen tube in the refrigerator at -80℃cryopreservation for the detection of liver hydroxyproline and total extracted RNA, reverse transcription - Polymerase chain reaction (RT-PCR) detection jak1, stat3mRNA expression; sacrificing part of the liver in 10% neutral formalin-fixed, paraffin-embedded, hematoxylin - eosin (HE) staining and Masson pathological changes in liver tissue staining; immunohis- tochemical staining of liver tissue jak1, stat3 expression.Results:1 The health of rats: the activities of normal rats is ability, appetite, hair glossy and neat, the spirit and nutritional status is good, weight gain per week is normal. Model rats with intraperitoneal injection of CCL4 time, began to decrease appetite, and listlessness, poor nutritional status, loss of hair color, weight gain is slow, individual is irritability. 8 weeks after intraperitoneal injection of CCL4 disabled, the rats increased appetite, mental and nutritional status were better than before.2 The changes of serum ALT and AST level in experimental group : In the model 4,6,8,10,12,14 serum ALT levels in serum were 72.38±4.25 U/L,143.67±8.97U/L,264.61±14.63U/L,182.41±10.57U/L,112.64±14.15 U/L,50.27±2.05U/L were higher (16.69±2.04U/L), and the difference between the groups were significantly (P <0.05); AST were: 182.35±4.12U/L,323.53±11.50U/L,501.04±24.15U/L,401.22±21.35U/L,140.20±10.33U/L,94.00±11.21 U/L,Were higher (17.21±2.34U/L), and the difference between the groups were significantly (P <0.05). (Table1)3 The changes of hydroxyproline (HYP) content in Liver tissue in experimental group: The first rat model of liver tissue were 4,6,8,10,12,14 weekend HYP levels:195.97±17.90,346.35±15.25,478.80±8.16,414.62±10.04,361.42±14.06,296.58±16.51,were higher (104.57±14.72), and the difference between the groups were significantly (P <0.05). In model group, the hydroxyproline (HYP) levels of reversal of liver tissue were significantly lower than the modeling period.4 Pathological changes in the live in experimental group: liver tissue was reddish-brown soft, shiny, smooth edges in control by the naked eye,. Model group, liver tissue and tissue surrounding were adhesion, dark red, compared with normal liver tissue volume is small, blunt edge, the surface is a sense of vector.5 The immunohistochemistry of JAK1 and STAT3 in experimental group: The positive cells of immunohistochemical of JAK1 is the expression of brown particles in cytoplasm; the expression of positive cell of immunohistochemistry of STAT3 is brown particles in nuclei. The control group, only a small amount of liver tissue has distribution of positive cells. With the modeling time,in the first part of the model group positive cells of JAK1 and STAT3 of the liver tissue gradually increased;After 8 weeks we disable carbon tetrachloride, liver cells reduced inflammation and fibrosis, The positive cells ofJAK1 and STAT3 gradually Reduced. In model group JAK1, STAT3-positive optical density values of 4,6,8,10,12,14 weekend (0.55±0.02,1.23±0.15,2.73±0.17,2.12±0.11,1.69±0.18,1.20±0.12),(0.82±0.03,1.53±0.11,2.89±0.13,2.20±0.14,1.73±0.13,1.24±0.03)were significantly higher(0.21±0.04),(0.24±0.06)with statistical significance(P <0.05).6 Jak1 and stat3 mRNA expression in Liver tissue in experimental group: In the model group 4,6,8,10,12,14 weekend, liver expression of JAK1 and STAT3 mRNA were(0.76±0.04,1.54±0.06,2.48±0.03,1.99±0.10,1.51±0.11,1.12±0.10),(0.81±0.09,1.65±0.07,2.66±0.10,2.02±0.04,1.61±0.11,1.20±0.07)were significantly higher (0.52±0.03) (0.56±0.02), P <0.05; and each time point were significantly different between , with statistical significance (P <0.05). In model group, the JAK1 and STAT3 mRNA expression of reversal of liver tissue was significantly lower than the modeling period.7 The correlation of JAK1, STAT3 mRNA expression levels of experimental rat liver tissue and liver HYP: The levels of experimental rat liver tissue and liver HYP expression levels of JAK1, STAT3 mRNA in liver tissue are linear regression, P <0.05, statistically significant. levels of JAK1 in Experimental rat liver tissue and liver HYP, The level of STAT3 mRNA expression has the correlation between HYP in liver tissue .(Fig30,Fig31)Conclusion:1 Carbon tetrachloride in rats by intraperitoneal injection for 8 weeks induced liver fibrosis with typical characteristics of an animal model, fibrosis induced by carbon tetrachloride to eliminate factors, animal models can be partially reversed liver fibrosis.2 JAK1 and STAT3 protein and mRNA expression is increased in the dynamic progression of liver fibrosis by JAK / STAT signal transduction pathway.they took part in liver fibrosis. The level of JAK1 and STAT3 mRNA expression has the correlation between HYP in liver tissue.3 JAK1 and STAT3 protein and mRNA expression was significantly decreased in the process of spontaneous reversal of hepatic fibrosis by JAK / STAT signal transduction pathway.it took part in hepatic fibrosis reversal of rats, suggesting that JAK / STAT signal transduction pathway is important in the pathogenesis of hepatic fibrosis.Part II The effection of JAK-STAT Pathway in rat liver fibrosis by fuzhenghuayuObjective: Due to various factors,the liver cell is damaged by chronic and inflammation, leading to hepatic stellate cells (hepatic stellate cell, HSC) are activated, causing a large number of ECM synthesis, resulting in excessive deposition of fibrous connective tissue to form fibrosis. Therefore, HSC is a major source of ECM synthesis, but also the different pathogenic factors lead to liver fibrosis in a common center link, HSC activation has been seen as the main target of treatment.of liver fibrosis.Currently, HSC activation of signal transduction pathways have more access of NF-κB, TGF-β/Smad pathway, MAPK pathway, and JAK / STAT signaling pathway has been confirmed that many of HSC activation during liver fibrogenesis has an important role. Recently, the Chinese role in liver fibrosis was significantly, increasing attention has been paid, Chinese medicine has a multi-target, multi-channel integrated pharmacological effects. Fuzhenghuayu composed of 6 herbs.They are Salvia, Cordyceps sinensis, peach kernel, Schisandra and of extraction of clinical anti-hepatic fibrosis drugs, traditional Chinese medicine theory is based on its "Yin Deficiency, Blood Stagnation network"in Pathogenesis of liver fibrosis. This study the effect and mechanism of Fuzhenghuayu Recipe on hepatic fibrosis JAK / STAT signaling pathway. Provide a new theoretical basis and clarify the mechanism for antifibrotic drugs.Methods: 8 week old Wistar rats were 57 healthy male, weighing 180 200g, were purchased from Experimental Animal Center of Hebei Medical University. After a week of adaptation to feed, were randomly divided into three groups: 9 in the control group, model group 24, the intervention group Fuzhenghuayu capsule 24. Control group received normal saline intraperitoneally (0.2ml/100g), 2 times a week for 8 weeks; model group were given intraperitoneal injection of olive oil 30% CCL4 (0.2ml/100g), 2 times a week for 8 weeks; righting Huayu Capsule modeling intervention group from the beginning by 0.2ml/100g weight (crude drug capsule containing Fuzhenghuayu 0.046g/ml) given Fuzhenghuayu liquid capsule once a day 1 for 8 weeks. 4,6,8 in the first week in control group rats were killed three randomly selected, and the remaining two were taken the first week the rats were sacrificed. 4,6,8.8specimens were taken. serum specimens from liver function ALT, AST; place sacrificing part of the liver tissue frozen tube in the refrigerator at -80℃cryopreservation for the detection of liver hydroxyproline and total extracted RNA, reverse transcription - Polymerase chain reaction (RT-PCR) detection jak1, stat3mRNA expression; sacrificing part of the liver in 10% neutral formalin-fixed, paraffin-embedded, hematoxylin-eosin (HE) staining and Masson pathological changes in liver tissue staining; immunohistochemical staining of liver tissue jak1, stat3 expression.Results:1 Normally rats: control group spirit, good appetite, hair neat and shiny, for the live active, normal weight gain per week. Model group with the modeling time, listlessness, loss of appetite, slow weight gain, coat color to yellow and dull, individual become irritable, and even individual of peritoneal effusion. Fuzhenghuayu party intervention group spirit, diet, shiny coat color compared with model group, mild symptoms.2 Serum ALT, AST levels in rat: Fuzhenghuayu intervention group were the first 4,6,8 weekend ALT: 58.02±9.02 U / L, 104±13.12 U / L, 147.00±15.73U / L, were higher than (17.37±5.04U/L), and each time point were significantly different between (P <0.05); AST were: 118±13.23 U/L,228.49±13.01U/L,302.17±17.19U/L Always make control group (19.20±4.93 U/L), and also between the various time points were significantly different (P <0.05). The same time point, compared with model group, prevention group ALT, AST were lower (P <0.05). Compared with model group, the ALT, AST levels of intervention group Fuzhenghuayu side were lower.(Table1)3 The changes of hydroxyproline (HYP) content in Liver tissue in fuzhenghuayu group: The intervention group on 4,6,8 Fuzhenghuayu weekend HYP (132.67±11.24,246.36±13.16,444.67±13.24ug/g) were higher (114.26±18.72ug/g), P <0.05; And the same time point in each group and were significantly different between the time difference was statistically significant (P <0.05). the HYP levels of Fuzhenghuayu intervention group was significantly lower than the model group.4 Pathological changes in the live in experimental group: Naked eye: In the control group the liver color is red, shiny, smooth surface, sharp edges, no adhesion with surrounding tissue, texture soft and crisp, touch fragile. In model group, at4 and 6 weekend, no significant changes in size, some slightly larger, pale yellow, fatty tissue attached to the surface, easy to peel, blunt liver edge, texture and tough, not brittle, cut greasy; volume of more than 8 weeks has no significant change, some slightly reduced, the color is dark red, nodular surface has sediment samples, and more adhere with omental adhesions, easily separated, the liver edge was dull, touch slightly harder, not brittle. Fuzhenghuayu party intervention group is better than the model group the liver. HE and Masson staining of collagen: In control group, structur of liver lobule e is normal, hepatic cord arranged in neat rows, no inflammatory necrosis and fibrosis (Fig1, Fig8). Model group, hepatic lobules at 4 weeks are disorders, liver cells showed fatty degeneration and changes in water samples, showing inflammatory cell infiltration and mild fibrous tissue hyperplasia, portal area expanded; intervention group showed mild liver cell degeneration and fatty changes of water, Portal area a small amount of inflammatory cell infiltration, mild fibrosis (Fig2, Fig3, Fig9, Fig10). At 6 weeks, the liver cells showed diffuse fatty degeneration, accompanied by mononuclear cells consisting mainly of mixed inflammatory cells, some of lobular architecture is destroyed, fibrous tissue hyperplasia; intervention group, part of the liver steatosis, accompanied by point, focal necrosis , mild proliferation of fibrous tissue, portal area shows a lot of mixed inflammatory cell infiltration (Fig4, Fig5, Fig11, Fig12). 8 weeks, most of the damage model group, hepatic lobules, extensive infiltration of inflammatory cells, extensive proliferation of fibrous tissue, a false lobule; intervention group, part of the destruction of hepatic lobules, fibrous tissue hyperplasia, inflammatory cell infiltration, a pseudo lobule, the Department of District expanded, showing that small bile duct proliferation and inflammatory cell exudate (Fig6, Fig7, Fig13, Fig14).5 Fuzhenghuayu intervention group, at the same time point compared with the model, The expression of JAK1 and STAT3 are few. In model group JAK1, STAT3-positive optical density values of 4,6,8 weekend (0.55±0.02,1.23±0.15,2.73±0.17),(0.82±0.03,1.53±0.11,2.89±0.13) and intervention group on 4,6,8 in Fuzhenghuayu weekend JAK1, STAT3-positive optical density values were (0.48±0.06,0.86±0.16,1.65±0.10), (0.57±0.04,1.12±0.12,1.90±0.08) were significantly higher (0.21±0.04),(0.24±0.06), P <0.05; and the same time points in each group were significantly Differences (P <0.05), model group and the group Fuzhenghuayu intervention group also significantly different between time points, with statistical significance (P <0.05). JAK1 and STAT3 protein in liver tissue in Fuzhenghuayu party intervention group was significantly lower than the untreated group. 6 The immunohistochemistry of JAK1 and STAT3 in fuzhenghuayu group :The intervention group on 4,6,8 Fuzhenghuayu weekend JAK1 and STAT3 mRNA liver expression, respectively (0.52±0.06,0.96±0.12,1.52±0.14),(0.62±0.02,1.05±0.10,1.64±0.11)were higher the control group(0.33±0.08) (0.42±0.02), P<0.05; and the same in each group were significant differences between time points (P<0.05), model group and Fuzhenghuayu intervention group at different time points between groups also Significantly different, with statistical significance (P<0.05). JAK1 and STAT3 mRNA in liver tissue in Fuzhenghuayu intervention group was significantly lower than the untreated group.Conclusion:1 Fuzhenghuayu can improve liver function, degree of inflammation and necrosis of liver tissue fibers, with the prevention and treatment of liver fibrosis.2 Fuzhenghuayu can inhibit jak1/stat3 mRNA and protein expression in liver, reduce inflammation and necrosis of liver tissue, reduce the degree of liver fibrosis and prevent the progress of liver fibrosis, suggest that its presence in the treatment of liver fibrosis in the great prospects. |
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