Amygdalin Ameliorates Liver Fibrosis through Inhibiting Activation of TGF-β/Smad Signaling / 中国结合医学杂志

OBJECTIVE@#To observe the effect of amygdalin on liver fibrosis in a liver fibrosis mouse model, and the underlying mechanisms were partly dissected in vivo and in vitro.@*METHODS@#Thirty-two male mice were randomly divided into 4 groups, including control, model, low- and high-dose amygdalin-treated groups, 8 mice in each group. Except the control group, mice in the other groups were injected intraperitoneally with 10% carbon tetrachloride (CCl4)-olive oil solution 3 times a week for 6 weeks to induce liver fibrosis. At the first 3 weeks, amygdalin (1.35 and 2.7 mg/kg body weight) were administered by gavage once a day. Mice in the control group received equal quantities of subcutaneous olive oil and intragastric water from the fourth week. At the end of 6 weeks, liver tissue samples were harvested to detect the content of hydroxyproline (Hyp). Hematoxylin and eosin and Sirius red staining were used to observe the inflammation and fibrosis of liver tissue. The expressions of collagen I (Col-I), alpha-smooth muscle actin (α-SMA), CD31 and transforming growth factor β (TGF-β)/Smad signaling pathway were observed by immunohistochemistry, quantitative real-time polymerase chain reaction and Western blot, respectively. The activation models of hepatic stellate cells, JS-1 and LX-2 cells induced by TGF-β1 were used in vitro with or without different concentrations of amygdalin (0.1, 1, 10 µmol/L). LSECs. The effect of different concentrations of amygdalin on the expressions of liver sinusoidal endothelial cells (LSECs) dedifferentiation markers CD31 and CD44 were observed.@*RESULTS@#High-dose of amygdalin significantly reduced the Hyp content and percentage of collagen positive area, and decreased the mRNA and protein expressions of Col-I, α-SMA, CD31 and p-Smad2/3 in liver tissues of mice compared to the model group (P<0.01). Amygdalin down-regulated the expressions of Col-I and α-SMA in JS-1 and LX-2 cells, and TGFβ R1, TGFβ R2 and p-Smad2/3 in LX-2 cells compared to the model group (P<0.05 or P<0.01). Moreover, 1 and 10 µmol/L amygdalin inhibited the mRNA and protein expressions of CD31 in LSECs and increased CD44 expression compared to the model group (P<0.05 or P<0.01).@*CONCLUSIONS@#Amygdalin can dramatically alleviate liver fibrosis induced by CCl4 in mice and inhibit TGF-β/Smad signaling pathway, consequently suppressing HSCs activation and LSECs dedifferentiation to improve angiogenesis.

Effect of bone marrow mesenchymal stem cell transplantation on CCl₄-induced liver injury in rats and intervention effects of Yiguanjian / 中国中药杂志

The aim of this paper was to observe the function of bone marrow mesenchymal stem cell (BMSC) transplantation in process of liver injury induced by carbon tetrachloride (CCl₄) and the intervention effect of Yiguanjian (YGJ), a compound of Chinese herbal medicine. Wistar rats were randomly divided into five groups: normal group, model group, cell transplantation (CT) group, YGJ group and cell transplantation plus Yiguanjian (CTY) group. Liver injury was induced through subcutaneous injection with CCl₄ at a dose of 3 mL·kg⁻¹ body weight for 4 weeks, twice a week. They were injected for a total of 9 times. After the first injection with CCl₄, rats in the CT group and CTY group were injected with the third-generation BMSCs at dose 1×10⁶ (suspended in 1 mL saline solution) via tail vein. Rats in the YGJ and CTY groups were also intragastrically administered with Yiguanjian once a day. Rat serum ALT and AST activities were increased significantly on the second day after injection with CCl₄, while BMSC transplantation and Yiguanjian decreased their activities. After 4 weeks of injection with CCl₄, serum ALT, AST and -GT activities, and serum TNF- and IL-6 expressions were increased, while TBIL were decreased in model rats compared with normal rats. Meanwhile, liver cells edema, plasmatic loose, and numerous lipid droplets were observed in rats of the model group. BMSC transplantation aggravated liver injury compared with model rats, which was manifested by decreasing SOD activity, increased MDA, TG, TNF- and IL-6 levels, and aggravated necrosis level of hepatocytes, fusion of lipid droplets, and collagen deposition in liver tissue. Yiguanjian decreased liver injury induced by CCl₄ alone and CCl₄ plus BMSC transplantation. SRY gene hybridization method was used to detect the positive SRY expressions in heart, liver, spleen, lung and kidney, especially in liver, while Yiguangjian decreased liver SRY expression. Wnt and -catenin showed high expressions in rats of normal group, which were decreased significantly in rats of models group, while Yiguanjian increased their expressions. In conclusion, BMSC transplantation could exacerbate liver injury, while Yiguanjian could protect liver injury induced by CCl₄ and BMSC transplantation, which was related to decreasing the homing of BMSCs to liver and up-regulating Wnt/-catenin signaling pathway.

Observations on the Efficacy of Acupuncture plus Medicine in Treating Senile Hepatic Cirrhosis / 上海针灸杂志

Objective To investigate the clinical efficacy of acupuncture plus medicine in treating senile hepatic cirrhosis.Methods Eighty patients with senile hepatic cirrhosis were randomized to treatment and control groups, 40 cases each. Both group received conventional antiviral and anti-inflammatory treatments. In addition, the control group took domperidone tablets and spironolactone tablets orally and the treatment group received acupuncture. Serum HBV-DNA content was measured, hepatic fibrosis indicators (typeⅢ procollagen, serum hyaluronic acid and laminin) were examined, and clinical symptoms (ascites, hydrothorax, lower limb edema and abdominal varices) and gastrointestinal symptoms (reflux vomiting disappearance time, borborygmus recovery time and defecation frequency) were observed in the two groups before and after treatment. The clinical therapeutic effects were compared between the two groups.Results The total efficacy rate was 95.5％ in the treatment group and 77.5％ in the control group; there was a statistically significant difference between the two groups (P<0.05). There were statistically significant pre-/post-treatment differences in serum HBV-DNA content and hepatic fibrosis indicators in the two groups (P<0.05). There were statistically significant post-treatment differences in serum HBV-DNA content and hepatic fibrosis indicators between the treatment and control groups (P<0.01). There was a statistically significant post-treatment difference in the number of cases of ascites, hydrothorax or lower limb edema between the treatment and control groups (P<0.05). There were statistically significant post-treatment differences in the gastrointestinal symptoms between the treatment and control groups (P<0.01).Conclusion Acupuncture plus medicine is an effective way to treat senile hepatic cirrhosis.

Antifibrotic effect of total flavonoids of Astmgali Radix on dimethylnitrosamine-induced liver cirrhosis in rats / 中国结合医学杂志

<p><b>OBJECTIVE</b>To study the effect of total flavonoids of Astmgali Radix (TFA) on liver cirrhosis induced with dimethylnitrosamine (DMN) in rats, and the effect on peroxisome proliferator-activated receptor γ (PPARγ), uncoupling protein 2 (UCP2) and farnesoid X receptor (FXR).</p><p><b>METHODS</b>Fifty-three Sprague-Dawley rats were randomly divided into a control group (10 rats) and a DMN group (43 rats). Rats in the DMN group were given DMN for 4 weeks and divided randomly into a model group (14 rats), a low-dosage TFA group (14 rats) and a high-dosage TFA group (15 rats) in the 3rd week. Rats were given TFA for 4 weeks at the dosage of 15 and 30 mg/kg in the low- and high-TFA groups, respectively. At the end of the experiment blood and liver samples were collected. Serum liver function and liver tissue hydroxyproline content were determined. hematoxylin-eosin (HE), Sirus red and immunohistochemical stainings of collagen I, smooth muscle actin (α-SMA) was conducted in paraffinembedded liver tissue slices. Real time polymerase chain reaction (PCR) was adopted to determine PPARγ, UCP2 and FXR mRNA levels. Western blot was adopted to determine protein levels of collagen I, α-SMA, PPARγ, UCP2 and FXR.</p><p><b>RESULTS</b>Compared with the model group, TFA increased the ratio of liver/body weight (low-TFA group P<0.05, high-TFA group P<0.01), improved liver biochemical indices (P<0.01 for ALT, AST, GGT in both groups, P<0.05 for albumin and TBil in the high-TFA group) and reduced liver tissue hydroxproline content (P<0.01 in both groups) in treatment groups significantly. HE staining showed that TFA alleviated liver pathological changes markedly and Sirus red staining showed that TFA reduced collagen deposition, alleviated formation and extent of liver pseudolobule. Collagen I and α-SMA immunohistochemical staining showed that staining area and extent markedly decreased in TFA groups compared with the model group. TFA could increase PPARγ, it regulated target UCP2, and FXR levels significantly compared with the model group (in the low-TFA group all P<0.05, in the high group all P<0.01).</p><p><b>CONCLUSION</b>TFA could improve liver function, alleviate liver pathological changes, and reduce collagen deposition and formation of liver pseudolobule in rats with liver cirrhosis. The antifibrotic effect of TFA was through regulating PPARγ signal pathway and the interaction with FXR.</p>

Study on inhibitory effect of calycosin on hepatic stellate cell activation in rats by up-regulating peroxisome proliferator-activated receptor γ / 中国中药杂志

To observe the effect of calycosin on the proliferation and activation of primary hepatic stellate cells (HSCs) in rats, and prove calycosin shows the effects through peroxisome proliferator-activated receptor γ(PPARγ) and farnesoid X receptor (FXR). The results indicated that calycosin could inhibit HSC proliferation and expressions of activation marker smooth muscle actin-α and type I collagen. With the increase in HSC activation time, FXR expression reduced, but with no notable impact from calycosin. Calycosin could up-regulate PPARγ expression and its nuclear transition in a concentration-dependent manner. Its prohibitory effect on HSC activation could be blocked by PPARγ antagonist. In conclusion, calycosin could inhibit HSC activation and proliferation, which may be related with the up-regulation of PPARγ signal pathway.

Xiayuxue Decoction (symbols; see text) attenuates hepatic stellate cell activation and sinusoidal endothelium defenestration in CCl4-induced fibrotic liver of mice / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the effects of ancient Chinese medical formula Xiayuxue Decoction ([symbols; see text], XYXD) on activation of hepatic stellate cells (HSCs) and defenestration of sinusoidal endothelial cells (SECs) in CCl4-induced fibrotic liver of mice.</p><p><b>METHODS</b>High performance liquid chromatography was used to identify the main components of XYXD and control the quality of extraction. C57BL/6 mice were induced liver fibrosis by CCl4 exposure and administered with XYXD for 6 weeks simultaneously. Liver tissue was investigated by hematoxylin-eosin and Sirius-red staining. Sinusoidal fenestrations were observed by scanning electronic microscopy and fluorescent immunohistochemistry of PECAM-1 (CD31). Whole liver lysates were detected of α-smooth muscle actin (α-SMA) and type-I collagen by Western blot. Primary rat HSCs-T6 cells were analyzed by detecting α-SMA, F-actin, DNA fragmentation through confocal microscopy, Western blot, terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay and cellomics arrayscan, respectively.</p><p><b>RESULTS</b>Amygdalin and emodin in XYXD were identified. XYXD (993 mg/kg) inhibited Sirius red positive area up to 70.1% (P<0.01), as well as protein levels of α-SMA and type-I collagen by 42.0% and 18.5% (P<0.05) respectively. In vitro, XYXD (12.5 μg/mL, 50 μg/mL) suppressed the activation of HSCs and reversed the myofibroblastic HSCs into quiescent, demonstrated as inhibition of fluorescent F-actin by 32.3% and 46.6% (P<0.05). Besides, XYXD induced the apoptosis of HSC-T6 cells by 20.0% (P<0.05) and 49.5% (P<0.01), evidenced by enhanced TUNEL positivity. Moreover, ultrastructural observation suggested XYXD inhibited defenestration of SECs, which was confirmed by 31.1% reduction of protein level of CD31 (P<0.05).</p><p><b>CONCLUSIONS</b>XYXD inhibited both HSCs activation and SECs defenestration which accompany chronic liver injuries. These data may help to understand the underlying mechanisms of XYXD for prevetion of chronic liver diseases.</p>

Performance of FibroScan in evaluating the curative effects of traditional Chinese medicine on liver fibrosis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To assess the performance of FibroScan in evaluating the curative effects of traditional Chinese medicine (TCM) on liver fibrosis, and to analyze factors influencing the diagnostic accuracy.</p><p><b>METHODS</b>Data of FibroScan values, types of disease, use of drug, liver function indexes, prothrombin time (PT) and international normalized ratio (INR) were collected at both pre- (1 month prior) and post-FibroScan for 102 patients who underwent at least two FibroScan procedures. Patients were subgrouped according to presence of fibrosis, presence of cirrhosis, and TCM formulation and statistically analyzed.</p><p><b>RESULTS</b>The pre- and post-FibroScan mean liver stiffness measurements (LSMs) were significantly different when the variation of LSM was more than or equal to2 kPa for the non-fibrotic group (vs. the fibrotic group), or when the variation wasmore than or equal to4 kPa for the cirrhotic group (vs. the non-cirrhotic group). In addition, the three TCM formulation groups showed significant differences, with the most robust difference exhibited between the FuZheng HuaYu formulation group and the other treatment groups (P = 0.010). No significant differences were observed for the liver function indexes, PT, or INR. However, the post-FibroScan levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyltransferase (GGT) was significantly reduced in patients with reduced LSM.</p><p><b>CONCLUSION</b>FibroScan may be a useful non-invasive clinical tool for evaluating the comprehensive curative effect of treatments for chronic liver diseases, and its performance is not obviously impacted by ALT, AST, GGT, PT, and INR. The criteria for efficacy established by FibroScan are 2 kPa for the patients without liver fibrosis and 4 kPa for patients with liver cirrhosis.</p>

Uniform designed research on the active ingredients assembling of huangqi decoction for inhibition of DMN-induced liver fibrosis / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To screen out effective ingredients of Huangqi Decoction (HQD) on dimethylnitrosamine (DMN) induced liver fibrosis and its assembling actions.</p><p><b>METHODS</b>(1) DMN solution (0. 5%) was peritoneally injected to rats to prepare the liver fibrosis model for 12 times, starting from the 1st day of modeling to the end of the 4th week. Uniform design method with 4-factor 8-level table was used to optimize the proportion of four ingredients from HQD, including astragaloside (AS), astragalus flavonoids (AF), glycyrrhizae acid (GA), and glycyrrhizae flavonoids (GF). Moreover, the changes of hydroxyproline (Hyp) content in the liver issue and the level of alanine aminotransferase (ALT) in serum were observed as screen indices, and the method of regression analysis was used to find out an optimal combination. (2) A further study for comparing and verifying the efficacy of the obtained optimized prescription was conducted by observing the changes of fibrosis pathology, the content of Hyp in the liver tissue and serum enzyme activity after medication.</p><p><b>RESULTS</b>The optimal proportion of AS and GA was 164:48. Compared with the model group, the content of Hyp in the liver tissue and the levels of ALT, aspartate aminotransferase (AST), and alkaline phosphatase (ALP) in serum decreased significantly, indicating the inhibiting effect of HQD and the AS/GA combination group on hepatic fibrosis formation (P<0.05). The AS/GA combination group was better than AS/GA used alone group in reducing the content of Hyp in the liver tissue and the level of ALT in serum. Furthermore, the AS/GA combination group was better than the HQD group in reducing the level of ALT in serum.</p><p><b>CONCLUSIONS</b>AS and GA were effective ingredients of HQD, and the combination of AS and GA had obvious synergistic effect in reducing liver collagen deposition and decreasing serum ALT activity in DMN-induced liver fibrosis.</p>

Structural shifts of gut flora in rat acute alcoholic liver injury and jianpi huoxue decoction's effect displayed by ERIC-PCR fingerprint / 中国结合医学杂志

<p><b>OBJECTIVE</b>To study the structural shifts of gut flora in rats with acute alcoholic liver injury (AALI), and the effect of jianpi huoxue decoction (JPHXD) on the gut flora.</p><p><b>METHODS</b>Thirty-six Sprague-Dawley rats were randomly allocated to the control, AALI and JPHXD groups equally. The rats in the control group were given water and those in AALI and JPHXD groups were given ethanol by intragastric gavage for 5 days, while rats in the JPHXD group were administered JPHXD simultaneously. The blood and liver tissue were collected at the end of the experiment. The activities of serum alkaline aminotransferase (ALT), aspartate aminotransferase (AST), hepatic γ-glutamyltranspetidase (γ-GT) and hepatic triglyceride (TG) levels were determined. Plasma endotoxin level in the portal vein was measured. Pathological changes of liver tissues were determined by hematoxylin and eosin (HE) staining and oil red O staining. The total DNA of gut flora were extracted from fecal samples by Bead-beating method and determined by ERIC-PCR fingerprint method. The similarity cluster analysis and principal component analysis were performed to analyze the ERIC-PCR fingerprint respectively.</p><p><b>RESULTS</b>In the AALI group, the ratio of liver/body weight, activities of ALT, AST and hepatic γ-GT, amount of hepatic TG were elevated significantly compared with those in the control group (all P<0.01). JPHXD decreased the ratio, activities of ALT, AST, γ-GT and TG significantly compared with those in the AALI group (P<0.05 or P<0.01). HE and oil red O staining showed that fat deposited markedly in liver tissue, while JPHXD alleviated pathological changes markedly. Plasma LPS level in rat portal vein in the AALI group increased significantly (P<0.01), but it was decreased significantly in the JPHXD group (P<0.01). The cluster analysis and principal component analysis of ERIC-PCR fingerprint showed that gut flora in the AALI group changed markedly, and JPHXD could recover gut flora to some extent.</p><p><b>CONCLUSIONS</b>The structure of gut flora shifted markedly during acute alcoholic liver injury, JPHXD had prevention effect through the modification of gut flora.</p>

Huangqi decoction inhibits cholangiocyte proliferation and transdifferentiation in cholestatic liver fibrosis induced by BDL in rats / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To elucidate the antifibrotic mechanism of Huangqi decoction in rats with BDL-induced cholestatic liver fibrosis.</p><p><b>METHODS</b>Liver fibrosis model was induced by ligating the common bile duct (BDL) in rats. Sham-operation was performed in control rats. The BDL rats were randomly divided into two groups: the BDL group and the Huangqi decoction group. Huanqi decoction was given intragastrically for 4 weeks. At the end of the fifth week after BDL, animals were sacrificed.</p><p><b>RESULTS</b>Compared with the sham control group, mortality rate in BDL group was 33.3% and incidence rate of ascites was 90%, and hepatic function was abnormal in most of the rats in BDL group. The number of Hepatocytes was decreased and the number of cholangiocytes significantly increased in BDL group. In addition, Hyp content of liver tissue and protein expression of CK 7 and a-SMA were significantly increased. Immunostaining indicated that CK 7 and a-SMA were co-localized in BDL group. These changes were markedly suppressed by the Huangqi decoction.</p><p><b>CONCLUSIONS</b>These observations suggest that Huangqi decoction can inhibit cholangiocyte proliferation and cholangiocyte transdifferentiation.</p>

Study on effects of extracts from Salvia Miltiorrhiza and Curcuma Longa in inhibiting phosphorylated extracellular signal regulated kinase expression in rat's hepatic stellate cells / 中国结合医学杂志

<p><b>OBJECTIVE</b>To study the effect of salvianolic acid B (SAB) and curcumin, the extracts of Salvia Miltiorrhiza and Curcuma Longa, on the proliferation and activation of hepatic stellate cell (HSC), and the extracellular signal regulated kinase (ERK) expression in it.</p><p><b>METHODS</b>Rat's HSC-T6 were cultured and treated by SAB or curcumin. The inhibitory effect on cell proliferation was determined by 3-(4, 5-dimthyl-2-2thiazoly)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) colorimetry, and the expression levels of alpha smooth actin (alpha-SMA), collagen type I, and ERK were determined by Western blot.</p><p><b>RESULTS</b>SAB and curcumin inhibited the proliferation and activation of rat's HSC-T6 in dose-dependent fashion and significantly reduced the expression level of alpha-SMA (P < 0.01). Curcumin significantly reduced the expression of collagen type I (P < 0.05). Both SAB and curcumin showed insignificant effect on the ERK expression level, but they could significantly reduce the level of phosphorylated-ERK expression, showing significant difference as compared with that in the control group (P < 0.01 and P < 0.05 respectively).</p><p><b>CONCLUSION</b>SAB and curcumin could significantly inhibit the proliferation, activation of HSC, and the production of type I collagen in HSC, the mechanism may be associated with their inhibition on ERK phosphorylation.</p>