| Liver cirrhosis is a common and recurrent disease seriously endangering humanphysical and mental health. It is the necessary pathway for chronic hepatic diseases toprogress into liver cirrhosis and the common pathological basis of various chronichepatic diseases. As a recurrent pathological repair reaction induced by variousliver-injury factors (inflammation, poisoning, chronic cholestasis, dysmetabolism, bloodcirculation disorder and malnutrition), i.e. liver cell degeneration necrosis-inflammation reaction-fibrous connective tissue proliferation, its pathologicalcharacteristic is the increase of fibrous connective tissue in portal area to form tiny cordand thin septum and extend toward liver lobule but not form pseudolobule andregenerated nodule. Liver fibrosis is reversible.If disease cause exists continuously, liver fibrosis aggravates gradually, then liver lobuleand blood vessel are reconstructed, pseudolobule and nodule form, and thus livercirrhosis occurs to cause irreversible lesions and many adverse effects on liver. Forexample, blood vessels in fiber septum directly shunt blood of hepatic artery and portalvein out of liver to decrease the contact with liver parenchyma; collagen fibersedimentation capillarized liver sinus to hinder the contact of blood flow withhepatocytes; the communicating branches opening of hepatic artery and portal vein andthe fibrosis in lobule center hinders blood flow from entering hepatic vein to aggravateportal hypertension. Its clinical manifestations include malnutrition, portal hypertension,splenomegaly and ascites.Thus the key to treat chronic hepatic diseases and prevent liver cirrhosis and livercancer is to terminate the lesion at liver fibrosis stage and even reverse it to normal levelthrough proper measures.Amydae Carapax is well-known as common traditional Chinese medicine forprevention & treatment of liver fibrosis and its drug form coexisted historically (powderor decoction). The present study tested the effect of Amydae Carapax extract on liverstellate cell multiplication and activation regulating mechanism and screened its pharmacological activity. It aimed to specify its material base for prevention &treatment of liver fibrosis, provide basis for research on drug form relation, and furtherexplore the effect and mechanism of its prevention & treatment of liver fibrosis in rats.1. In vitro test1.1 Effect & drug form relation of Amydae Carapax on liver stellate cell multiplication,1.2 Effect of Amydae Carapax extract on activation regulating mechanism of liverstellate cells.Objective To explore whether Amydae Carapax extract inhibited the expression ofvarious proteins in LX-1 cells (such as C-1 and C-â…¢) and whether the expressionregulation of various cell factors (such asα-SMA) could inhibit HSC activation andthus inhibit liver fibrosis progress. Methods Amydae Carapax extract was preparedthrough method in Test 1.1, drugs administered, Western-blot analysis made throughroutine method, Typeâ… collagen, Typeâ…¢collagen andα-SMA protein expression inLX-1 cells detected with internal reference of actin, blank control, TGF-β1 stimulationcontrol and IFN-γcontrol. Results Compared with those in each control group, theexpression ofα-SMA, Typeâ… collagen and Typeâ…¢collagen in LX-1 cells decreasedsignificantly at dose-efficacy dependence in treatment group of Amydae Carapax(molecular weight:＜6000). Conclusion Amydae Carapax (molecular weight:＜6000)could significantly inhibit the expression of proteins in LX-1 cells (such as C-1, C-â…¢andα-SMA), the activation of HSC and thus the occurrence progress of liver fibrosis.2. In vivo test: Action mechanism of Amydae Carapax on prevention & treatment ofliver fibrosis in ratsObjective To explore the action mechanism of relevant drug forms of Amydae Carapaxon prevention & treatment of liver fibrosis in rats.Methods 36 female SD rats were randomly divided into 6 groups: normal group, liverfibrosis model group, prevention & treatment group and treatment group of AmydaeCarapax decoction and prevention & treatment group and treatment group of AmydaeCarapax micro-powder. For groups other than normal group, 0.12ml/100g 40ï¼…CCl4(body weight) was injected subcutaneously at the back qd alt. continuously for 12weeks; modeling was made for 6 weeks to form moderate fibrosis. In drug prevention & treatment group, Amydae Carapax decoction and Amydae Carapax micro-powder werelavaged orally in 1.35g crude drug/100g (body weight) respectively qd at the same timeof modeling; and in drug treatment group, above drugs were lavaged orally from Week7 of modeling. At the end of Week 12 of test, serum and liver tissue of rats weresampled and then sent for examination. Test data was expressed in x±s for t test, andP＜0.05 meant significant difference.Results2.1 Routine pathological stainingIn CCl4 liver fibrosis model group, under HE and sirius red staining of rats, numerousliver cells had fatty degeneration, normal liver lobule structure was damaged, and therewere numerous thick proliferous collagen fiber between portal area and lobule; andunder tissue section, there were numerous pseudolobuli. In Prevention & treatmentgroup and treatment group of Amydae Carapax decoction, compared with those inmodel control group, fatty degeneration degree of rat liver cell was weaker, portal areaexpanded and had fiber septum but mostly unconnected, SSS score decreasedsignificantly (P＜0.01). All above observations in Prevention & treatment group andtreatment group of Amydae Carapax micro-powder were similar to those in modelgroup.2.2 Immunohistochemical results2.2.1α-SMA Normal group: It was expressed at lower level only in portal area andliver sinus of normal rat liver. Model group: It was expressed widely diffusively mainlyin fiber septum, liver sinus wall, vascular endothelial cells, and bile duct epithelial cellsbut very few in liver cytoplasm. Prevention & treatment group and treatment group ofAmydae Carapax decoction: Its expression characteristic was same to those in modelgroup but at significantly less and weaker area and intensity of expression (P＜0.05). Itwas not significantly different among prevention & treatment group and treatmentgroup ofAmydae Carapax micro-powder and model group.2.2.2 Collagen Typeâ… Normal group: It was expressed at lower level only in portalarea and liver sinus of normal rat liver. Model group: It expressed widely diffusivelymainly in fiber septum, liver sinus wall, vascular endothelial cells, and bile duct epithelial cells but very few in liver cytoplasm. Prevention. & treatment group andtreatment group of Amydae Carapax decoction: Its expression characteristic was sameto those in model group but at significantly less and weaker area and intensity ofexpression (P＜0.01). It was not significantly different among prevention & treatmentgroup and treatment group ofAmydae Carapax micro-powder and model group.2.2.3 Collagen Typeâ…¢Normal group: It was expressed at lower level only in portalarea and liver sinus of normal rat liver. Model group: It expressed widely diffusivelymainly in fiber septum, liver sinus wall, vascular endothelial cells, and bile ductepithelial cells but very few in liver cytoplasm. Prevention & treatment group andtreatment group of Amydae Carapax decoction: Its expression characteristic was sameto those in model group but at significantly less and weaker area and intensity ofexpression (P＜0.01). It was not significantly different among prevention & treatmentgroup and treatment group ofAmydae Carapax micro-powder and model group.2.2.4 Collagen Typeâ…£Normal group: It was expressed at a lower level only in portalarea and liver sinus of normal rat liver. Model group: It was expressed widelydiffusively mainly in fiber septum, liver sinus wall, vascular endothelial cells, and bileduct epithelial cells but very few in liver cytoplasm. Prevention & treatment group andtreatment group of Amydae Carapax decoction: Its expression characteristic was similarto those in model group but at significantly less and weaker area and intensity ofexpression (P＜0.01). It was not significantly different among revention & treatmentgroup and treatment group ofAmydae Carapax micro-powder and model group.2.2.5 Laminin Normal group: It expressed at lower level only in portal area and liversinus of normal rat liver. Model group: It expressed widely diffusively mainly in fiberseptum, liver sinus wall, vascular endothelial cells, and bile duct epithelial cells but veryfew in liver cytoplasm. Prevention & treatment group and treatment group of AmydaeCarapax decoction: Its expression characteristic was same to those in model group butat significantly less and weaker area and intensity of expression (P＜0.05). It was notsignificantly different among prevention & treatment group and treatment group ofAmydae Carapax micro-powder and model group.2.2.6 TGF-β1 Normal group: It was expressed at a lower level only in liver cytoplasm of normal rat liver. Model group: It was mainly expressed in degenerated hepaticcytoplasm. Prevention & treatment group and treatment group of Amydae Carapaxdecoction: Its expression characteristic was same to those in model group but atsignificantly less and weaker area and intensity of expression (P＜0.01). It was notsignificantly different among prevention & treatment group and treatment group ofAmydae Carapax micro-powder and model group.2.2.7 NFκB p65 Normal group: It was expressed at lower level only in portal area andliver sinus of normal rat liver.Model group: It was expressed widely diffusively mainly in liver cytoplasm and also inliver perisinusoidal cells, vascular endothelial cells, bile duct epithelial cells and musclefibroblast. Prevention & treatment group and treatment group of Amydae Carapaxdecoction: Its expression characteristic was same to those in model group but atsignificantly less and weaker area and intensity of expression (P＜0.01). It was notsignificantly different among prevention & treatment group and treatment group ofAmydae Carapax micro-powder and model group.2.2.8 PDGF-BB Normal group: It expressed at lower level only in liver sinus of normalrat liver. Model group: It expressed widely diffusively mainly in liver cytoplasm andalso in liver perisinusoidal cells and muscle fibroblast. Prevention & treatment groupand treatment group of Amydae Carapax decoction: Its expression characteristic wassame to those in model group but at significantly less and weaker area and intensity ofexpression (P＜0.01).It was not significantly different among prevention & treatmentgroup and treatment group ofAmydae Carapax micro-powder and model group.2.2.9 TIMP-1 Normal group: It expressed at lower level only in liver sinus of normal ratliver. Model group: It expressed widely diffusively mainly in liver cytoplasm and also inliver perisinusoidal cells and muscle fibroblast. Prevention & treatment group andtreatment group of Amydae Carapax decoction: Its expression characteristic was sameto those in model group but at significantly less and weaker area and intensity ofexpression (P＜0.01). It was not significantly different among prevention & treatmentgroup and treatment group ofAmydae Carapax micro-powder and model group.2.3 Serum biochemical index and liver collagen content 2.3.1 HA level (radio-immunoassay): HA level was significantly higher in model groupthan that in normal group (P＜0.01), was significantly lower in prevention & treatmentgroup and treatment group of Amydae Carapax decoction than those in model group andprevention & treatment group and treatment group of Amydae Carapax micro-powder(P＜0.01), and was not significantly different among prevention & treatment group andtreatment group ofAmydae Carapax micro-powder and model group.2.3.2 Collagen content in liver: By referring to detection method reported by Jimenez etal, collagen content was calculated through the following formula: Collagenμg/section=(A540nm-7.78ï¼…A630nm)×1000/37. It was significantly higher in model group thanthat in normal group (P＜0.01);2.3.3 Liver function (automatic biochemical analyzer): Compared with those in normalgroup, ALT, AST, AKP and r-GT were significantly higher (P＜0.01), but ALB wassignificantly lower (P＜0.05) in model group. Compared with those in model group andprevention & treatment group and treatment group of Amydae Carapax micro-powder,ALT, AST, AKP and r-GT were significantly lower (P＜0.01), but ALB was significantlyhigher (P＜0.05) in prevention & treatment group and treatment group of AmydaeCarapax decoction. Liver function index was not significantly different amongprevention & treatment group and treatment group of Amydae Carapax micro-powderand model group.2.3.4 Liver tissue oxidation index (biochemical method): Compared with those innormal group, activities of SOD and GSH-PX were significantly lower (P＜0.05～0.01),but MDA was significantly higher (P＜0.05) in model group. Compared with those inmodel group and prevention & treatment group and treatment group of AmydaeCarapax micro-powder, activities of SOD and GSH-PX were significantly higher(P＜0.05), but MDA was significantly lower (P＜0.01) in prevention & treatment groupand treatment group of Amydae Carapax decoction. Liver tissue oxidation index wasnot significantly different among prevention & treatment group and treatment group ofAmydae Carapax micro-powder and model group.Conclusion: Treatment with Amydae Carapax decoction could resist lipid perioxidation,improve pathological function of liver tissue, improve liver function and regulate cell factor level. Thus it could inhibit HSC activated multiplication and ECMsynthesis/secretion, promote ECM degradation and significantly prevent/treat liverfibrosis in rats. But Amydae Carapax micro-powder did not significantly prevent/treatliver fibrosis.
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