Study On The Anti-hepatic Fibrosis Mechanism Of Hujin Granule And Its Medicated Rat Serum

The incidence of liver disease is very high in China, and about 10 percent of them are hepatitis virus carriers. Furthermore, he people who infected hepatitis virus is about 50 percent. Hepatic cirrhosis (especially posthepatitic cirrhosis) is very common, along with poor effective prevention and cure, and seriously harms patients' physical and mental health. It is reported that hepatic cirrhosis comes from various kinds of chronic liver diseases, and hepatic fibrosis is the common pathologic basis of various kinds of chronic liver diseases. Hepatic fibrosis, which is necessary vital phase in chronic liver disease development, is pathologically repair reaction followed by liver injury which displays periapical fibroma of collagen fibers, degeneration and necrosis of hepatocyte, destruction of structure and function of hepatic tissue, consequently induce liver dysfunction and poor prognosis. On the other hand, hepatic fibrosis can be protected and cured. Interrupting or reversing the occurrence and development of hepatic fibrosis before hepatic cirrhosis will make a mighty advance on chronic liver diseases.In the later stage of disease, hepatic cirrhosis patients are usually complicated with gastrointestinal bleeding, abdominal dropsy and hepatocerebral disease. However in the process stage, hepatic cirrhosis is reversible, and appropriate and prompt treating measures can block the transition of hepatic fibrosis to hepatic cirrhosis.At present, drugs for treating hepatic fibrosis still remain in the phase of study. Traditional drugs such as Colchicines and Malotilate are limited used because of their not very significant effects and relatively severe side effects. Another drug, interferon , is more effective in treating hepatic fibrosis revealed by clinical and experimental tests, it is a pity that it is very expensive. So scholars pay more and more attentions on traditionalChinese herbs to develop some effective drugs on hepatic fibrosis. Clinic and basic researches indicate that some Chinese herbs have obvious effect on hepatic fibrosis with small side effects. Meanwhile, the price is very low. So the Chinese herbs would have widely exploited in the future.According to TCM theory, hepatic fibrosis belongs to hypochondrium pain or abdominal mass. Pathogenesis of hepatic fibrosis includes stagnation of qi, blood stasis and dysfunctions of liver and spleen, in which blood stasis is the basis. Therefore, the therapeutical principle of hepatic fibrosis includes activating blood circulation to dissipate blood stasis, benefiting vital energy to promoting blood flow, soothing liver and strengthening spleen, softening hard mass and removing food retention and so on.Hujin Granule (HJG) is a clinical empirical prescription, in which the principal durgs Rhizoma Polygonicuspidati and Radix Curcumae are used to promote blood circulation and dissipate blood stasis, promote qi circulation to relieve pain;Fructus Crataegi and Radix Notoginserg activate blood circulation to dissipate blood stasis, Rhizoma Alismatis removes dampness and promotes diuresis, these three drugs are used as ministerial drug;Ganoderma replenishes qi and reinforces deficiency as adjuvant drug and messenger drug. All of them together have the effects of activating blood circulation to dissipate blood stasis and promoting qi circulation to remove stagnant. This study investigates the therapeutical effect of HJG on hepatic fibrosis rats induced by dimethylnitrosamine(DMN), discusses the antihepatic fibrogenic mechanisms of HJG, and especially the effect of HJG on the expression of Smad protein in the TGF-01 signal transduction pathway. In vitro, by using the method of Chinese herb serum pharmacology, we studied the effect of the medicated serum on collagen I, TGF-P1 and its receptor II mRNA expression in hepatic stellate cells (HSC) so as to explore the antihepatic fibrogenic cytomolecular mechanism of HJG.Experiment in vivo:The hepatic fibrosis rat model was induced by DNM. The details of method are as follows: Animals were intraperitoneally injected with 0. 5% DMN (2ml/kg) at 2/3 of total dosage for successive three days at first week, and then at total dosage for successive three days every week. All the modeling time is 4 weeks. In addition, the control group' s animals were simultaneously injected saline water at the same administrating route and dosage. 4 weekslater, the experimental rats injected with DMN were randomly divided into five groups: model control group, Dahuangzhechongwan(DHZCW) group and the low(0.6g/kg), mean(1.2g/kg) and high(2. 4g/kg) groups of HJG. All the rats were administered once per day with corresponding drugs and physiological saline for successive 4 weeks. At the end of this experimentation, we valued the liver function by measuring the indexes of ALT, AST, TP, ALB in serum, chose HA, LN, PCIII as the hepatic fibrosis indexes and measured the indexes of MDA, SOD, Hyp in liver tissue. The liver tissue was fixed by 10% formalin, then we observed the liver tissue under the light microscope via the HE and VG dyeing method. Furthermore we measured the indexes of TGF-PK Smad3, Smad7, and a -SMA in liver tissue by immunohistochemical method, and discussed the effect of HJG on the expression of these indexes mentioned above in the liver tissue of the hepatic fibrosis mode rats. Measurement data was analyzed by statistic software SPSS11.0 for Windows, numeration data was analyzed by software epi6. 0.Experiment in vitro:SPF grade female SD rats were used. Give HJG to the experimental rats by intragastric administrating at the dose of 2ml/100g(10 times as the 60kg man' s dosage) twice per day for successive three days. At the last time, the rats were fasted diet for 12 hours at night. At the fourth day the rats were administered the whole dosage of HJG, 1 hour later, blood specimen was taken from abdominal aorta, at room temperature, 2 hours later centrifuged for 20 minutes at 4°C and 3000r/min, then serum was separated and inactivated at 56° C for 30min, then frozen respectively at below zero 80° C after filtration sterilization.HSC-T6 culture:HSC was cultured after revival and inoculated at the content of lOVml, culture solution was changed each two or three days till the second cell monolayer appeared.HSC-T6 disposal by the medicated serum and its collectionHSC-T6 was inoculated in 25 milliliter culture flask with DMEM culture solution contained 10 percent calf serum, 4ml each flask. HSC-T6 was exerted on disposal factor after 48 hours, then put in 4'C refrigerator synchronization.￡ for one hour, and then divided into following groups: control group(DMEM4. 0ml);* TGF-131 (lOng/ml) stimulus group: (DMEM3.8ml plus TGF- P 1 200 u 1);10% normal \serum group (DMEM3. 4ml plus TGF-01 200 u 1 and plus normal serum 400 ul);5% medicated serum group (DMEM3. 6ml plus TGF-01 200 ul and plus medicated serum 200u 1);10% HJG serum group (DMEM3.4ml plus TGF-01 200u1 and plus medicated serum 400 ul);20% medicated serum group (DMEM3. 0ml plus TGF-Pl 200 u 1 and plus medicated serum 800 pi) . DMEM mentioned above groups contained 2% calf serum. Cultivanted HSC-T6 for 24 hours, discarded the supernatant, added Trizol (lml per flask), flushed out again and again, collected suspension, then put the suspension in -80°C refrigerator. Each flask repeated three times and at the third time extracted RNA all together.Index and measurement :Extracted total RNA by one step method according to Trizol kit instruction, measured the content and purity of RNA by DNA/RNA radiometer, and then stored the total RNA prepared for use in below 80°C refrigerator. By using the method of RT-PCR, we measured the expression of the collagen I, TGF-P 1 and T￡RII mRNA of HSOT6. We took 6ul reaction product of PCR for electrophoresis with 1. 8% agarose gel for 30min, and got Electropherogram by image analysis machine. Semiquantitative analysis was used to evaluate the relative level of gene fragment with standard of GAPDH gray scale. Results :1 General condition of model rats: The incidence of model rats induced by DMN was 25%, and the other survival rats mostly bring about ascitic fluid.2 Successfully created the hepatic fibrosis model. The ALT and AST levels in serum of model group were obviously higher than those of normal control group. Compared with model group, the ALT and AST levels of each HJG group decreased. The degree of decrease presented dose-dependent. These results indicated that HJG had a good effect of protecting liver and lowering enzyme action. The ALB level of model group decreased, but after the intervention of HJG, the level increased, and the ratio of A/G was corrected. This indicated that HJG could recovery the level of ALB and had the effect of protecting liver. The levels of HA, PCIII, LN in serum of model group were markedly higher than those of normal control group, but those indexes levels of each HJG group, especially of mean dose group, significantly decreased. These results indicated that HJG had therapeutic effect on the experimental hepatic fibrosis model rats, could lessen liver cell injured, ameliorated liver function, and decreased the levels of HA, LN and PCIII in serum, therefore lightened thedegree of hepatic fibrosis.3 The levels of Hyp and MDA of model rats' liver tissue obviously increased, simultaneously the content of SOD decreased. After HJG treatment, the levels of Hyp and MDA decreased to some extent, and the level of SOD increased. These results indicated that HJG could obviously reduced the anormal increase of MDA in hepatic fibrosis model rats and raised the level of SOD. That to say, the antihepatic fibrogenic mechanism maybe is concerned with anti-lipid peroxidation.4 Pathological results indicated that each dose of HJG could lessen hepatic cell necrosis, obviously inhibit hyperplasia of f ibrolast and collagen fiber, so HJG could improve the constitution of hepatic tissue, recovery hepatic lobules constitution, and reduce fibrous tissue.5 Immunohistochemical results indicated that the TGF-P1 expression of hepatic fibrosis model rats was stronger than that of normal control group. The Smad3 expression was mainly seen in the fiber compartment. Simultaneouly the Smad7 expression of normal rats liver cell was higher than that of model rats, which indicted that this protein participated in keeping normal physiological function of hepatocyte. When hepatic fibrosis happened, the Smad7 expression was inhibited and the feedback regulation of TGF-P1 was decreased. After HJG treatment, the constitution of hepatic tissue improved obviously, the Smad7 expression in liver increased, meanwhile, the expression of TGF-01 and Smad3 presented down regulation. These results demonstrated that HJG could efficiently inhibit the activity of TGF-0 1 which can promote the process of hepatic fibrosis, and has some intervention in signal transduction of TGF-P1.6 The results of study in vitro indicated that medicated serum could obviously down regulate the TGF-fll and TPRII mRNA expressions of HSC-T6, which indicated that HJG could inhibit the synthesis of cytokine TGF-01. Simultaneously the binding between TGF-0 1 and its receptor was blocked, the signal transmission of TGF-01 was first inhibited at the receptor level, therefore, TGF-0 1 could not educe the function of anti-hepatic fibrosis through the pathway. According to the results of HJG serum on the expression of Collagen I mRNA, HJG could inhibited the expression of Collagen I mRNA,/ this indicted that HJG could interfere in the collagen synthesis and had effect of anti-hepatic fibrosis. The inhibitory action of HJG serum on expressionsof Collagen I, TGF-p 1 and TPRII Mrna was obvious when used 10% ~ 20% HJG serum.To sum up, TGF- P1 plays an important role in the occurrence and development of hepatic fibrosis, and HSC could express TGF-PL TGF-PI expression could up-regulate via autocrine and paracrine action, consequently accelerated the proceeding of hepatic fibrosis. Therefore, preventing and curing hepatic fibrosis can further be considered from inhibiting the expression of TGF- P 1 and its signal protein. HJG could inhibit the expression of TGF-p 1 and signal protein in hepatic fibrosis model rats, and down-regulate the expression of Collagen I mRNA, TGF-p 1 and TPRII mRNA in HSC, consequently block TGF-PI and its signal transduction at receptor level. Therefore HJG has good effect on treating hepatic fibrosis.