The Study Of The Effects Of Melatonin, Agonist And Antagonist Of MAChR On The Rat Hepatic Stellate Cells

OBJECTIVE To clarify the effects of melatonin (MT) on liver pathological examination,laboratory examination and serum marks of hepatic fibrosis (HF) in HF model rats induced by carbon tetrachloride (CCl4) .Because hepatic stellate cells (HSC) play a core role in the process of HF, in vitro , the influences of MT, pilocarpine nitrate (PN) and atropine sulfate (AS) on the biological behaviours of HSC such as morphology, synthesis or secretion of extracellular matrix (ECM) and apoptosis, were investigated,for the further research on exploring the mechanisms of MT,PN or AS on HF, and supplying experimental basis to find out therapeutic drugs for HF in the future.METHODS The rat HF model was prepared by back subcutaneous injection of CCL4. In vivo,rats in MT groups were intragastric administrated (ig) MT (0.125, 0.5, 2.0 mg/(kg?d)) for 8 wk,the levers of biochemical indicators were detected by automatic-biochemical analyzing equipment (A-BAE) ,the levers of serum hyaluronic acid (HA) and procollagen typeⅢ(PCⅢ) were assayed by RIA,the pathological changes were determined by HE stain. In vitro,HSC isolated from model group rats were exposed to MT (10, 0.1μmol/L and 1 nmol/L), PN (1 mmol/L, 10, 0.1μmol/L),AS (1 mmol/L, 10, 0.1μmol/L), MT (10μmol/L)+PN (1 mmol/L, 10, 0.1μmol/L),MT (10μmol/L)+AS (1 mmol/L, 10, 0.1μmol/L), while HSC from normal group rats were exposed to NS.After 48 h of HSC co-cultured with drugs,the morphrage changes of HSC creeped on the microscope slides were observed with light microscope,the apoptosis rates of HSC were determined by FCM,and the levers of HA and PCⅢin cultured HSC were determinded by RIA. HSC isolated from model group rats were exposed to PN(10μmol/L),MT(10μmol/L)+PN(10μmol/L) , MT(0.1μmol/L)+PN(10μmol/L) ,and negative control group were treated with isodose DMEM without serum, after 48 h of HSC co-cultured with drugs,the expression of collagen typeⅠmRNA (ColⅠmRNA) were detected by reverse transcriptase polymerase chain reaction (RT-PCR).RESULTS1 Effects of MT on laboratory examination, serum marks of HF and liver pathological examination in HF rats induced by CCl4.1.1 Effects of MT on laboratory examination in HF rats induced by CCl4.After 12 wk of the HF model prepared with CCL4., compared to the normal rats group, in HF model rats, the levers of TP ,ALB decreased and A/G declined, while ALT,AST and ALP increased significantly. Compared to the HF model rats group, in CLC group, the levers of ALB and A/G increased,and ALT and ALP decreased significantly,but AST had no obviously change, TP significantly increased in MT (0.5 mg/(kg·d), 8 wk) group rats, and ALB and A/G increased significantly, with ALT,AST and ALP decreased significantly in all MT groups. Compared to the CLC group, the levers of TP and ALB increased in MT (0.5 mg/(kg·d), 8 wk) group rats, while ALT increased significantly in MT (2.0 mg/(kg·d), 8 wk) group1.2 Effects of MT on serum marks in HF rats induced by CCl4.After 12 wk of the HF model prepared with CCL4., compared to the normal rats group, in HF model rats, the levers of HA and PCⅢincreased. Compared to the HF model rats group, the levers of HA and PCⅢsignificantly diminished in CLC and all MT groups. Compared to the CLC group, the lever of HA was higher in MT (2.0 mg/(kg·d), 8 wk) group, but lower in MT (0.5 mg/(kg·d), 8 wk) group.1.3 Effects of MT on liver pathological examination in HF rats induced by CCl4.After 12 wk of the HF model prepared with CCL4., compared to the normal rats group, in HF model rats, the rats liver pathological examination showed shrinkage, swelling, necrosis, collapse, destruction and fatty degeneratsion of hepatocyte, fibrous tissue hyperplasyed and infiltrated between hepatocytes, with inflammatory cells infiltration and pannus formation. CLC and MT diminished the pathological changes differently.2. Effects of MT, PN and AS on the morphrage of HSC.After 48 h of HSC co-cultured with drugs in vitro, compared to HSC from normal group rats, the HSC from HF model group rats nearly creeped over the microscope slides,and grew better. Compared to HSC in the model control group, HSC co-cultured with MT,or AS,or MT combinated AS grew rarefaction, with some HSC shrinked,while HSC co-cultured with PN creeped close over the glass slides and grew better, HSC co-cultured with MT combinated PN creeped on much area of the glass slides,grew worse,with a little HSC shrinked.3. Effects of MT, PN and AS on the apoptosis of HSC.After 48 h of HSC co-cultured with drugs in vitro, compared to HSC from normal group rats, the apoptosis rates of HSC from HF model group significantly increased. Compared to model control group, the apoptosis rates of HSC co-cultured with MT (10μmol/L), PN, AS, or MT(10μmol/L) combinated AS(10, 0.1μmol/L) increased significantly,while the apoptosis rates of HSC co-cultured with MT(10μmol/L) combinated PN(10μmol/L) decreased significant.4. Effects of MT, PN and AS on the synthesis or secretion of HA and PCⅢin HSC.After 48 h of HSC co-cultured with drugs in vitro, compared to HSC from normal group rats, the levers of HA and PCⅢin HSC from HF model group significantly increased.Compared to model control group, the levers of HA and PCⅢin HSC co-cultured with MT, AS, or MT combinated AS decreased significantly,while in HSC co-cultured with PN or MT combinated PN ,the levers of HA increased but PCⅢhadn't significantly changed.5. Effects of MT, PN on the expression of ColⅠmRNA in HSC.PN promoted the expression of ColⅠmRNA,and the effect was inhibited by MT. CONCLUSIONS1. MT can protect rats liver effectively and relieved the rat HF induced by CCl4.In this research experiments,we had successfully established a CCl4-induced rat chemical damage model, proved that,in different degree, MT in various doses(0.125, 0.5, 2.0 mg/(kg·d),8 wk) could raise the levers of proteins (including TP,ALB) indicating the liver synthesis functions, decrease the levers of liver functional enzymes (such as AST,ALT and ALP ) reflecting the liver metabolism functions, decline the serum concentrations of HA and PCⅢindicating degrees of HF, and effectively relieve the liver injuries. All these results demonstrated that MT can relieve or even reverse hepatic damages when lesions occurred in liver functions and mophrage. Additionally, the pathological results implied MT in medium dose (0.5 mg/(kg·d),8 wk) could protect liver better.2. MT probably play an anti-fibrogenesis role via mechanisms of inducing HSC into apoptosis,inhibiting the synthesis or secretion of HA and PCⅢ.In the research experiments, we considered two conditions both in vivo & in vitro to explore the role of MT on changes of HA, PCⅢand ColⅠmRNA which mirror the condition of ECM in HF, and demonstrated that MT could effectively diminish the levers of HA and PCⅢboth in vivo & in vitro, additionally inhibit the expression of ColⅠmRNA promoted by PN in vitro, implying that MT could play an anti-fibrogenesis role in the synthesis or secretion stage of ECM in HSC. Meanwhile,we investigated the affects of MT on HSC apoptosis, and showed MT (10μmol/L) could promoted the apoptosis of HSC,which should be a major mechanism. Forthermore, experiment results suggested that the inhibition effect of the synthesis or secretion of ECM in HSC which occurred in lower concentration, should not completely depend on effect of inducing HSC apoptosis which occurred in lower concentration. 3. The agonist - PN, and antagonist - AS of muscarinic acetylcholine receptor (mAChR) probably participate in the mechanisms of modulating the apoptosis,synthesis or secretion of ECM in HSC.In the experiments in vitro,we investigated the influences of the agonist - PN, and antagonist - AS of muscarinic acetylcholine receptor (mAChR) on HSC,and concluded that PN in all series concentrations (1 mmol/L, 10, 0.1μmol/L) could promote the synthesis or secretion of HA and PCⅢin HSC, moreover,it could enhance the mRNA expression of ColⅠ, essential contents in ECM, in HSC, hence strong supported that PN could promote the proliferation of HSC in sequence with more activity apoptosis. AS in different concentrations (1 mmol/L, 10, 0.1μmol/L) could diminish the levers of HA and PCⅢeffectively, and induce the HSC to apoptosis. Considered both roles of agonist - PN, and antagonist - AS of mAChR, we concluded that the both really participated in the mechanisms of modulating the apoptosis,synthesis or secretion of ECM in HSC, implying that influence on the mAChR can modulate biological behaviors as above of HSC.4.MT can interfere in the effects of PN or AS on modulating the apoptosis, synthesis or secretion of ECM in HSC.Further research showed that, MT in 10μmol/L variously influenced the pharmacology effects of PN or AS in series concentrations. MT affected much effects of PN (such as regulating HSC to proliferate , apoptosis or express ColⅠmRNA ), and influenced the roles of AS on inducing the apoptosis or inhibiting the produce of ECM in HSC, while the concrete combined effects related to both drugs'concentrations.