Effects Of YiGanKang Decoction On Ca～(2+) And MAPK Signaling Pathway In Hepatic Stellate Cells

Liver fibrosis is a common pathological basis and an inevitable stage of chronic liver diseases developing into hepatic cirrhosis, and therefore the process is an important link to influence the prognosis of chronic liver diseases. At present,it is thought that liver fibrosis is reversible while hepatic cirrhosis is not. There is a high incidence rate of hepatic diseases in our country, and such as chronic hepatitis B and C are commonly encountered and seriously influence the people's life quality. It has become a quite important countermeasure to stop or reverse the liver fibrosis progress due to no effective antiviral measure to cure chronic liver diseases. It is well known that the activation and proliferation of hepatic stellate cells (HSCs) are key links of liver fibrosis and HSCs are major collagen-synthesizing cells, so suppressing the activation and proliferation of HSCs is an important measure for anti-fibrosis. The activation and proliferation of HSCs is a process associated with numerous cytokines, such as platelet-derived growth factor (PDGF) and transforming growth factor-β1(TGF-β1). These cytokines interact with their receptors and make signal transduction.Sequentially, HSCs were activated by intracellular signaling pathway systems. in turn, the activated HSCs can activate more cytokines, forming a continuous amplification circle. Therefore, it has become a new target and a new hot point in the treatment of liver fibrosis to inhibit cell signaling pathway systems. Both Ca2+ and mitogen-activated protein kinase (MAPK) signaling pathway play the most crucial roles among the cell signaling pathway systems. With the gradual clarification of pathologic mechanism of liver fibrosis, it is possible to prevent, treat and reverse the liver fibrosis. However, up to now, there is no effective therapeutic method against liver fibrosis in modern medicine; while the good preventing and treating efficacies against liver fibrosis and low toxicity of traditional Chinese medicine have brought to the extensive attention of the scholars at home and abroad at present. In recent years, a lot of experiments and clinical researches have confirmed that blood-activating and stasis-eliminating traditional Chinese medicine has a better therapeutic efficacy in the treatment of liver fibrosis. YiGanKang decoction is an excellent recipe used to treat chronic liver diseases which is meticulously researched and developed on the basis of the basic theory of traditional Chinese medicine and the clinical experience for several decades. Its good therapeutic efficacy and low side effect were received by patients. Although the studies on treating liver fibrosis by the use of traditional Chinese medicine have made an encouraging progress, the study on active mechanism and ingredients has been a hot point and a difficult point in the new drug research and development of traditional Chinese medicine. When compound preparations are added to a reaction system in vitro, their non-specific physicochemical factors such as pH, osmotic pressure, tannin, inorganic salts and so on seriously interfere the experiments and lead to false positive or false negative results. In 1985, the seropharmacological method was put forward in Japan, which is used to accurately reproduce the active ingredients of compound preparations of traditional Chinese medicine playing roles in vivo. At present, drug-containing serums are extracted from normal animal or human in many studies, however, the pharmacological action is a result of interaction of drugs and bodies. When bodies have different physiological and pathological states, their metabolic pathways of drugs are different; consequently, the drug concentrations, quality and quantity of metabolites and induced active ingredients are also different. Therefore, to use the drug-containing serums from normal animal or human to perform experiments in vitro is difficult to reflect the real process of biological transformation of drugs in diseased bodies to the maximal degree. Our studies adopted a "comparative seropharmacological method " based on this idea. According to this method, different medicated serum whichextracted respectively from normal rats or CCl4-induced liver fibrosis rats was used to incubate HSCs. So, two kind of YiGanKang decoction drug-containing serums were acted on cells, and Radix Salviae Miltiorrhizae(RSM) ---main ingredients of two kind drug-containing serums were used as controls in order to observe the effects of the whole preparations exactly. Three important target spots in signaling pathway systems of proliferation's course of HSCs [i.e. 1.intracellular free calcium; 2. transcription of c-fos and c-jun gene; 3. activation of extracellular signal regulated kinase(ERK) and c-Jun-N-terminal kinase(JNK)]were researched in this article. For the first target spot, i.e. intracellular free calcium, besides drug-containing serums extracted from rats, drug-containing serums extracted from patients with type B hepatitis were used to research it. The article aimed at further researching the effect and mechanism of YiGanKang decoction on activation and proliferation of HSCs and seeking new target spots of drug action, which will lay an experimental foundation for developing new drugs of traditional Chinese medicine in the treatment of liver fibrosis. Part I: Effects of drug-containing serums extracted from rats with Liver fibrosis on intracellular free calcium in HSCs Objective: To investigate the effects of drug-containing serums of YiGanKang decoction and Radix Salviae Miltiorrhizae(RSM) extracted from normal rats and CCl4-induced liver fibrosis rats respectively on intracellular free calcium in HSCs. Methods: HSCs strain was cultured in vitro. After the model of hepatic fibrosis was established in SD rats, YiGanKang or Radix Salviae Militiorrhizae decoction was given via gastrogavage to the rats of treatment groups to prepare the medicated serum, and the dosage was 10 times of dose per kg per day for adults in 2 divided doses for 6 consecutive days, while the same volume of 0.9% Nacl was given to the rats of non-treatment groups. on the 7th day, the routine dose was orally given again; the blood sample wasdrawn from the vena cava after 2 hour; the serum was isolated and inactivated with water bath at 56℃; finally, the serum was filtered to eliminate bacteria. Just before using the serum, RPMI-1640 culture medium was added to prepare culture media of 10% drug-containing serum, which was incubated with the subcultured HSCs. The experiment was divided into the following groups: A: serum of extracted from normal rats; B: medicated serum of RSM extracted from normal rats; C: medicated serum of YiGanKang extracted from normal rats group; D: serum from CCl4-induced liver fibrosis rats; E: medicated serum of RSM extracted from CCl4-induced liver fibrosis rats; F: medicated serum of YiGanKang extracted from CCl4-induced liver fibrosis rats. After 24h incubation with above every group serum which were added blindly to HSCs , HSCs were loaded with Fluo-3/AM, a Ca2+ marker, and were observed with laser scanning confocal microscopy(LSCM). The experimental data were expressed as mean±S( x ±s). One-way ANOVA and q test were performed by using SPSS11.5 statistical software; P < 0.05 was considered statistically significant. Results: â‘ Compared with HSCs treated by serum from CCl4-induced liver fibrosis rats, calcium Fluorecence Intensity of HSCs treated by both drug-containing serums of YiGanKang decoction and of Radix Salviae Militiorrhizae significantly decreased (groupD:61.32±12.62; groupB:39.23±18.77;groupC: 30.65 ±9.17;groupE: 32.52 ±6.59;groupF: 28.44 ±7.71, AllP<0.05). Compared with HSCs treated by two kind drug-containing serums of Radix Salviae Militiorrhizae(30.65±9.17 vs 39.23±18.77and 28.44±7.71 vs 32.52±6.59), calcium Fluorecence Intensity of HSCs treated by two kind drug-containing serums of YiGanKang decoction had no obvious decrease correspondingly(P>0.05). â‘¡Compared with HSCs treated by serum from CCl4-induced liver fibrosis rats, Changing Ratio of [Ca²⁺]i Fluorecence Intensity of HSCs treated by both drug-containing serums of YiGanKang decoction and of Radix Salviae Militiorrhizae significantly decreased (groupD: 1.21 ±0.49; groupB: 0.11 ±0.05;groupC: 0.10 ±0.06;groupE: 0.086 ±0.026;groupF: 0.075±0.033, AllP<0.05). Compared with HSCs treated bytwo kind drug-containing serums of Radix Salviae Militiorrhizae(0.10±0.06 vs 0.11±0.05and0.075±0.033 vs 0.086±0.026), Changing Ratio of calcium Fluorecence Intensity of HSCs treated by two kind drug-containing serums of YiGanKang decoction had no obvious decrease correspondingly, (P>0.05). Conclusions: Both YiGanKang and Radix Salviae Militiorrhizae decoction could decrease calcium Fluorecence Intensity and Changing Ratio of calcium Fluorecence Intensity in HSCs; At the point of decreasing intracellular free calcium in HSCs, Radix Salviae Militiorrhizae maybe the principal ingredient in the whole recipe of YiGanKang decoction; Both YiGanKang and Radix Salviae Militiorrhizae decoction maybe inhibit the HSCs activation and proliferation by decreasing intracellular free calcium in HSCs; The actions of decreasing biological transformation of drugs and increasing cytokines in CCl4-induced liver fibrosis rats collectively caused the results of two kind seropharmacological methods no significant difference. Part II: Study of the Seropharmacological effects of YiGanKang and Radix Salviae Militiorrhizae decoction on c-fos and c-jun gene expression in HSCs Objective: To investigate the effects of YiGanKang and Radix Salviae Militiorrhizae decoction medicated serum extracted from normal rats and CCl4-induced liver fibrosis rats respectively on c-fos and c-jun gene expression in HSCs. Methods: The preparations of the medicated serum of YiGanKang and Radix Salviae Militiorrhizae decoction were same as foresaid. The experiment was divided into the following groups: A: serum of extracted from normal rats; B: medicated serum of RSM extracted from normal rats; C: medicated serum of YiGanKang extracted from normal rats group; D: serum from CCl4-induced liver fibrosis rats; E: medicated serum of RSM extracted from CCl4-induced liver fibrosis rats; F: medicated serum of RSM extracted from CCl4-induced liver fibrosis rats. After 24h incubation with above every group serum which were added blindly to HSCs, c-fos mRNA and c-jun mRNA expression in HSCs were detected by reverse transcription polymerase chainreaction(RT-PCR). P < 0.05 was considered statistically significant. Results: â‘ YiGanKang and Radix Salviae Militiorrhizae decoction medicated serum extracted from normal rats and CCl4-induced liver fibrosis rats could inhibit c-fos mRNA expression in HSCs. Compared with HSCs treated by serum from CCl4-induced liver fibrosis rats, c-fos mRNA of HSCs treated by both drug-containing serums of YiGanKang decoction and of Radix Salviae Militiorrhizae significantly decreased (0.577±0.038,0.560±0.049;0.631±0.044,0.593±0.081 vs 0.941±0.070) (AllP<0.05). Compared with HSCs treated by serum from normal rat(s0.898±0.075), c-fos mRNA of HSCs treated by both drug-containing serums of YiGanKang decoction and of Radix Salviae Militiorrhizae significantly decreased too(AllP<0.05). Compared with HSCs treated by two kind drug-containing serums of Radix Salviae Militiorrhizae, c-fos mRNA expression of HSCs treated by two kind drug-containing serums of YiGanKang decoction had no obvious decrease correspondingly,(0.577±0.038 vs 0.631±0.044 and 0.560±0.049 vs 0.593±0.081) (AllP>0.05). â‘¡YiGanKang and Radix Salviae Militiorrhizae decoction medicated serum extracted from normal rats and CCl4-induced liver fibrosis rats could inhibit c-jun mRNA expression in HSCs. Compared with HSCs treated by serum from CCl4-induced liver fibrosis rats, c-jun mRNA of HSCs treated by both drug-containing serums of YiGanKang decoction and of Radix Salviae Militiorrhizae significantly decreased ( 0.538±0.078 ,0.504±0.025 ; 0.575±0.009 , 0.531±0.044 vs 0.924±0.068 , AllP<0.05). Compared with HSCs treated by serum from normal rats(0.910±0.093), c-jun mRNA of HSCs treated by both drug-containing serums of YiGanKang decoction and of Radix Salviae Militiorrhizae significantly decreased too(AllP<0.05). Compared with HSCs treated by two kind drug-containing serums of Radix Salviae Militiorrhizae, c-jun mRNA expression of HSCs treated by two kind drug-containing serums of YiGanKang decoction had no obvious decrease correspondingly, (0.538±0.078 vs 0.575±0.009 and 0.504±0.025 vs 0.531±0.044, AllP>0.05). Conclusions: Activated HSCs expressed more c-fos and c-jun mRNA.Itindicated c-fos gene and c-jun gene actively participated in the course of the HSCs activation and proliferation; Both YiGanKang and Radix Salviae Militiorrhizae decoction medicated serum extracted from normal rats and CCl4-induced liver fibrosis rats could inhibit c-fos and c-jun mRNA expression in the course of liver fibrosis forming ; At the point of decreasing c-fos and c-jun mRNA expression in HSCs, Radix Salviae Militiorrhizae maybe the main ingredient in the whole recipe of YiGanKang decoction; Both YiGanKang and Radix Salviae Militiorrhizae decoction maybe inhibit the HSCs activation and proliferation by decreasing c-fos and c-jun mRNA expression in HSCs;The actions of decreasing biological transformation of drugs and increasing cytokines in CCl4-induced liver fibrosis rats collectively caused the results of two kind seropharmacological methods no significant difference. Part III: Study of the effects of medicated serum of YiGanKang decoction on ERK and JNK activation in HSCs Objective: To observe two main subtypes of MAPK family—ERK and JNK activation in HSCs and investigate the effects of medicated serum of YiGanKang and Radix Salviae Militiorrhizae decoction on the two proteins activation in HSCs. Methods: The preparations of the medicated serum of YiGanKang and Radix Salviae Militiorrhizae decoction were same as foresaid. The preparations of the medicated serum of YiGanKang and Radix Salviae Militiorrhizae decoction were same as foresaid. The experiment was divided into the following groups: A: serum of extracted from normal rats; B: medicated serum of RSM extracted from normal rats; C: medicated serum of YiGanKang extracted from normal rats group; D: serum from CCl4-induced liver fibrosis rats; E: medicated serum of RSM extracted from CCl4-induced liver fibrosis rats; F: medicated serum of YiGanKang extracted from CCl4-induced liver fibrosis rats. After 24h incubation with above every group serum which were added blindly to HSCs, phosphorylated extracellular signal-regulated kinase (P-ERK) protein expression were detected by Westernblot and phosphorylated c-Jun-N-terminal kinase (P-JNK) protein expression were measured respectively. The functional activity of JNK and ERK kinases was studied using phospho-specific antibodies. P<0.05 was considered statistically significant. Results: â‘ The results of Western blot analysis revealed that, there were two subtypes of ERK protein phosphorylation in rat HSCs.which were ERK1(MT 44000) and ERK2(MT 42000). At the same time, there were two subtypes of JNK protein phosphorylation in rat HSCs.which were JNK1(MT 46000) and JNK2(MT 54000). â‘¡The results Western blot crossing showed that, compared with HSCs treated by serum from CCl4-induced liver fibrosis rats, ERK1/2 protein phosphorylation level of HSCs treated by both drug-containing serums of YiGanKang decoction and of Radix Salviae Militiorrhizae significantly decreased (28.16±5.37 vs 6.47±2.95,18.03±3.99, AllP<0.01). Compared with HSCs treated by two kind drug-containing serums of Radix Salviae Militiorrhizae(6.47±2.95 vs 18.03±3.99;8.35±2.31vs 18.17±4.02), ERK1/2 protein phosphorylation level of HSCs treated by two kind drug-containing serums of YiGanKang decoction had obvious decrease correspondingly(AllP<0.01). â‘¢Compared with HSCs treated by serum from CCl4-induced liver fibrosis rats, JNK1/2 protein phosphorylation level of HSCs treated by both drug-containing serums of YiGanKang decoction and of Radix Salviae Militiorrhizae significantly decreased (4.31±1.54,17.70±2.83 vs 32.66±7.08,AllP<0.01). Compared with HSCs treated by two kind drug-containing serums of Radix Salviae Militiorrhizae(4.31±1.54 vs 17.70;7.29±3.20vs 19.53±2.48), JNK1/2 protein phosphorylation level of HSCs treated by two kind drug-containing serums of YiGanKang decoction had obvious decrease correspondingly (AllP<0.01). Conclusions: ERK protein phosphorylation and JNK protein phosphorylation were detected in activated HSCs which indicated MAPK signal transductive pathway actively participated the course of HSCs activation and proliferation; Medicated serum of YiGanKang and Radix Salviae Militiorrhizae decoction extracted from normal and liver fibrosis ratscould inhibit ERK and JNK at protein phosphorylation level. With respect to the above-mentioned effects, YiGanKang decoction has more evidently inhibitory action that embodies the advantage of the composition of the whole recipe, and the medicated serum from liver fibrosis rats was not superior to that from normal rats due to decreasing biological transformation of drugs and increasing cytokines in CCl4-induced liver fibrosis rats. Part IV: Study of the effects of drug-containing serums extracted from patients with type B viral hepatitis on intracellular free calcium in HSCs Objective: To investigate the effects of drug-containing serums of YiGanKang decoction extracted from patients with type B viral hepatitis on intracellular free calcium in HSCs. Methods: HSCs strain was cultured in vitro. 15 patients with chronic B type viral hepatitis were phlebotomized and the blood sample as A group(before taking the drug);then all patients took YiGanKang decoction to prepare the medicated serum(B group:after taking the drug ), and the dosage was normal dose for adults in 2 divided doses for 6 consecutive days. On the 7th day, the routine dose was orally given again; the blood sample was drawn after 2 hour; the serum was isolated and inactivated with water bath at 56℃; finally, the serum was filtered to eliminate bacteria. Just before using the serum, RPMI-1640 culture medium was added to prepare culture media of 10% drug-containing serum, which was incubated with the subcultured HSCs. The rest experiment were same as as Part 1 and t test were performed by using SPSS11.5 statistical software; P < 0.05 was considered statistically significant. Results: â‘ Compared with HSCs treated by serum from group A, calcium Fluorecence Intensity of HSCs treated by drug-containing serums of YiGanKang decoction significantly decreased (25.25±6.61 vs 53.01±14.96, P<0.01). â‘¡Compared with HSCs treated by serum from group A, Changing Ratio of [Ca²⁺]i Fluorecence Intensity of HSCs treated by drug-containing serums of YiGanKang decoction significantly decreased (0.095±0.028 vs 1.00±0.37, P<0.001). Conclusions: drug-containing serums of YiGanKang decoction could...