DNA Methylation and Transcription of HLA-F and Serum Cytokines Relate to Chinese Medicine Syndrome Classification in Patients with Chronic Hepatitis B / 中国结合医学杂志

OBJECTIVE@#To explore the molecular bases of Chinese medicine (CM) syndrome classification in chronic hepatitis B (CHB) patients in terms of DNA methylation, transcription and cytokines.@*METHODS@#Genome-wide DNA methylation and 48 serum cytokines were detected in CHB patients (DNA methylation: 15 cases; serum cytokines: 62 cases) with different CM syndromes, including dampness and heat of Gan (Liver) and gallbladder (CHB1, DNA methylation: 5 cases, serum cytokines: 15 cases), Gan stagnation and Pi (Spleen) deficiency (CHB2, DNA methylation: 5 cases, serum cytokines: 15 cases), Gan and Shen (Kidney) yin deficiency (CHB3, DNA methylation: 5 cases, serum cytokines: 16 cases), CHB with hidden symptoms (HS, serum cytokines:16 cases) and healthy controls (DNA methylation: 6 cases). DNA methylation of a critical gene was further validated and its mRNA expression was detected on enlarged samples. Genome-wide DNA methylation was detected using Human Methylation 450K Assay and furthered verified using pyrosequencing. Cytokines and mRNA expression of gene were evaluated using multiplex biometric enzyme-linked immunosorbent assay (ELISA)-based immunoassay and reverse transcription-quantitative polymerase chain reaction (RT-qPCR), respectively.@*RESULTS@#Totally 28,667 loci, covering 18,403 genes were differently methylated among CHB1, CHB2 and CHB3 (P<0.05 and |Δβ value| > 0.17). Further validation showed that compared with HS, the hg19 CHR6: 29691140 and its closely surrounded 2 CpG loci were demethylated and its mRNA expressions were significantly up-regulated in CHB1 (P<0.05). However, they remained unaltered in CHB2 (P>0.05). Levels of Interleukin (IL)-12 were higher in CHB3 and HS than that in CHB1 and CHB2 groups (P<0.05). Levels of macrophage inflammatory protein (MIP)-1α and MIP-1β were higher in CHB3 than other groups and leukemia inhibitory factor level was higher in CHB1 and HS than CHB2 and CHB3 groups (P<0.05). IL-12, MIP-1α and MIP-1β concentrations were positively correlated with human leukocyte antigen F (HLA-F) mRNA expression (R2=0.238, P<0.05; R2=0.224, P<0.05; R=0.447, P<0.01; respectively). Furthermore, combination of HLA-F mRNA and differential cytokines greatly improved the differentiating accuracy among CHB1, CHB2 and HS.@*CONCLUSIONS@#Demethylation of CpG loci in 5' UTR of HLA-F may up-regulate its mRNA expression and HLA-F expression was associated with IL-12, MIP-1α and MIP-1β levels, indicating that HLA-F and the differential cytokines might jointly involve in the classification of CM syndromes in CHB.@*REGISTRATION NO@#ChiCTR-RCS-13004001.

Key symptoms selection for two major syndromes diagnosis of Chinese medicine in chronic hepatitis B / 中国结合医学杂志

<p><b>OBJECTIVE</b>To identify key symptoms of two major syndromes in chronic hepatitis B (CHB), which can be the clinical evidence for Chinese medicine (CM) doctors to make decisions.</p><p><b>METHODS</b>Standardization scales on diagnosis for CHB in CM were designed including physical symptoms, tongue and pulse appearance. The total of 695 CHB cases with dampness-heat (DH) syndrome or Pi (Spleen) deficiency (SD) syndrome were collected for feature selection and modeling, another 275 CHB patients were collected in different locations for validation. Key symptoms were selected based on modified information gain (IG), and 5 classifiers were applied to assist with models training and validation. Classification accuracy and area under receiver operating characteristic curves (AUC) were evaluated.</p><p><b>RESULTS</b>(1) Thirteen DH syndrome key symptoms and 13 SD syndrome key symptoms were selected from original 125 symptoms; (2) The key symptoms could achieve similar or better diagnostic accuracy than the original total symptoms; (3) In the validation phase, the key symptoms could identify syndromes effectively, especially in DH syndrome, which average prediction accuracy on 5 classifiers could achieve 0.864 with the average AUC 0.772.</p><p><b>CONCLUSION</b>The selected key symptoms could be simple DH and SD syndromes diagnostic elements applied in clinical directly. (Registration N0.: ChiCTR-DCC-10000759).</p>

Regulatory Effect of Qushi Huayu Recipe on Gene Expression Profiles of Fatty Liver Rats / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the intervention and mechanism of Qushi Huayu Recipe (QHR) on gene expression profiles in high lipid diet induced fatty liver rats.</p><p><b>METHODS</b>Fatty liver model was prepared in 20 male SD rats using single high fat diet (88% common forage +2% cholesterol +10% lard). Four weeks after modeling they were divided into the model group and the QHR group according to random digit table, 10 in each group. QHR (at 0. 93 g crude drug/100 g body weight) and distilled water was respectively to rats in the QHR group and the model group by gastrogavage while modeling, once per day. Meanwhile, 10 SD male rats were recruited in a normal group, administered with equal volume of distilled water by gastrogavage. At the end of week 8 all rats were sacrificed, and blood and livers were collected for subsequent analysis. Contents of liver triglyceride (TG) and free fatty acid (FFA) , activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected using biochemical assay. Pathological changes of liver tissue were observed using H&E and oil red O stain. Liver gene expressions were detected by Affymetrix gene expression profiles. Differentially expressed genes were compared between the QHR group and the model group, functions of differentially expressed genes and signal pathways involved analyzed. Ten differentially expressed genes involved in glycolipid metabolism with fold change more than 2 were selected for verification by real-time PCR.</p><p><b>RESULTS</b>(1) Compared with the normal group, contents of liver TG and FFA, and serum activities of ALT and AST obviously increased in the model group (P <0. 01). Compared with the model group, contents of liver TG and FFA, and activities of ALT and AST obviously decreased in the QHR group (P <0. 05, P <0. 01). QHR could reduce high fat induced fatty degeneration of liver cells , alleviate inflammation, and improve pathological changes of liver tissue. (2) Compared with the model group, there were 80 differentially expressed genes (with fold change > 2, P < 0.05) with clear functions and appointed gene names, including 44 up-regulated and 36 down-regulated genes. Eighty genes were involved in 27 signal pathways with statistical difference, including glycerolipid metabolism, adipocytokine signaling pathway, insulin signal pathway, drug metabolism signal pathway, etc (P < 0.05). (3) RT-PCR results of 10 glycolipids metabolism regulating genes such as Gk, Scd1, Gpat2, G6pc, Irs1, and so on showed that all RT-PCR genes were completely coincide with up-regulated or down-regulated tendency in results of gene chips. 80% genes had approximate fold change.</p><p><b>CONCLUSION</b>QHR could regulate gene expressions related to fat metabolism, carbohydrate metabolism, anti-lipid peroxidation, and drug metabolism in high fat diet induced fatty liver rats, and its comprehensive pharmacological actions could be manifested.</p>

Effect of CKJ recipe containing serum on activation of rat primary hepatic stellate cells, TGF-beta1 and its receptors / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the effect of CKJ Recipe (consisting of Cordyceps sinensis polysaccharide, amygdaloside, and gypenosides) containing serum on the activation of rat primary hepatic stellate cells (rHSCs) and to explore its pharmacological mechanism.</p><p><b>METHODS</b>rHSCs were isolated form liver and cultured for four days. Then they were divided into the normal control group, the model group, and the CKJ group. rHSCs in the model group and the CKJ group were treated with 2.5 ng/mL transforming growth factor beta1 (TGF-beta1) in serum-free DMEM for 24 h. Serum free DMEM (containing no TGF-beta1) was taken as the control for the normal control group. rHSCs in the CKJ group were treated with 5% CKJ-containing serum for 24 h. rHSCs in the other two groups were treated with 5% blank serum for 24 h.The protein expression level of a smooth muscle actin (alpha-SMA) was determined using high throughput screening (HCS) and Western blot. mRNA expression levels of alpha-SMA, collagen I (Col-I), platelet-derived growth factor receptor beta (PDGF-betaR), TGF-beta1, transforming growth factor beta receptor 1 (TGF-betaR1), and transforming growth factor beta receptor 2 (TGF-beta R2) were detected using quantitative RT-PCR.</p><p><b>RESULTS</b>Compared with the normal control group, the protein expression level of alpha-SMA, mRNA expression levels of alpha-SMA, Col-I, PDGF-betaR, TGF-beta1, TGF-betaR1, and TGF-betaR2 significantly increased in the model group (P<0.05, P<0.01). Compared with the model group, the protein expression level of alpha-SMA, mRNA expression levels of alpha-SMA, Col-I, PDGF-betaR, TGF-beta1, TGF-beta1, and TGF-beta R2 significantly decreased in the CKJ group (P<0.05, P<0.01).</p><p><b>CONCLUSION</b>CKJ containing serum could inhibit the protein expression level of o-SMA, which was probably related with inhibiting TGF-beta1 and its related receptors.</p>

Effect of cordyceps polysaccharide on lipid peroxidation of rats with dimethylnitrosamine-induced liver fibrosis / 中国中药杂志

<p><b>OBJECTIVE</b>To observe the pharmacological effect of Cordyceps polysaccharide on dimethylnitrosamine (DMN)-induced liver fibrosis in rats.</p><p><b>METHOD</b>DMN rat liver fibrosis model was established and divided into the normal group (N, n = 6), the model group (M, n = 11), the Cordyceps polysaccharide group (C, n = 8) and the colchicine group (Q, n = 9). During the modeling for four weeks, Cordyceps polysaccharide (60 mg x kg(-1)) and colchicine (0.1 mg x kg(-1)) were orally administered for three weeks, while the model and normal groups were given disinfected water of the same amount.</p><p><b>OBSERVATION</b>serum ALT, AST, GGT and Alb, TBil content; content of hydroxyproline (Hyp) in liver tissues; liver pathology and collagen staining; SOD activity and MDA, GSH, GSH-Px in liver tissues; protein expression of proliferating cell nuclear antigen (PCNA) in liver tissues.</p><p><b>RESULT</b>Serum ALT, AST, GGT, TBil significantly increased, and A1b decreased significantly in the model group. Hepatic Hyp significantly increased in the model group, whereas the index remarkably decreased in the Cordyceps polysaccharide group and the colchicine group. HE staining: the structure of normal hepatic lobules was damaged, with hepatocytes tumefaction and proliferation of connective tissues in portal tracts in the model group, while the Cordyceps polysaccharide group and the colchicine group recorded notable reduction in above pathological changes. Collagen staining: the model group showed hepatic lobule fibrous septum and many intact pseudolobules; while the Cordyceps polysaccharide group and the colchicine group witnessed decrease in collagen deposition. The model group showed significant decrease in SOD, GSH-Px and GSH and increase in MDA, whereas the Cordyceps polysaccharide group and the colchicine group recorded notable growth in GSH and GSH-Px. The model group showed significant decrease in protein expression of PCNA in liver tissues, while the Cordyceps polysaccharide group and the colchicine group showed significant reduction.</p><p><b>CONCLUSION</b>Cordyceps polysaccharide can significantly inhibit DMN-induced liver fibrosis and lipid peroxidation in rats.</p>

Dynamic change of lipid peroxidation-related protein expression and the intervention effects of Yiguanjian decoction in a rat model of CCl4-induced liver fibrosis / 中华肝脏病杂志

To investigate the dynamic change of lipid peroxidation-related protein expression and the intervention effects of Yiguanjian (YGJ) Decoction on liver fibrosis induced by CCl4 in rat. Fifty-seven male Wistar rats were randomly divided into a liver fibrosis group (n = 39) and a normal group (n = 18). The liver fibrosis was treated with peritoneal injection of 50% CCl4 for nine weeks. At the end of weeks 3 and 6 of CCl4 treatment, six rats were sacrificed to assess the status of liver fibrosis. At the end of week 7, the remaining -fibrotic rats were randomly divided into an untreated model group (M, n=15) and a YGJ-treated group (n = 12). The YGJ group was administered daily, oral YGJ Decoction for three weeks, concomitant with continued CCl4 treatment. The M group and normal group received the same treatment oral regimen and volume of distilled water. At the end of week 8, four rats in group M were sacrificed to observed the fibrosis status. At the end of week 9, the fibrotic rats were sacrificed for sampling. Liver function, histological changes, contents of hydroxyproline (Hyp) and malondialdehyde (MDA), activity of super oxidase dismutase (SOD) and L-glutathione (GSH), protein expression of heat shock protein (HSP)70, heme oxygenase (HO)-1, transferrin, peroxiredoxin (Prxd) 6 and liver fatty acid binding protein (L-FABP) were detected. Compared with normal group-, the MDA content was increased significantly in M group at week 6 (M: 4.23+/-0.45 nmol/mg vs. normal: 2.22+/-0.59 nmol/mg, F = 60.13, P less than 0.01) and week 9 (M: 6.29+/-1.23 nmol/mg vs. normal: 2.22+/-0.59 nmol/mg, F = 66.99, P less than 0.01), but the SOD activity was decreased significantly at the same time points [week 6: (M: 196.94+/-39.20 U/mg vs. normal: 264.50+/-30.44 U/mg, F = 11.12, P less than 0.01]); [week 9: (M: 152.2+/-51.65 U/mg vs. normal: 264.50+/-30.44 U/mg, F = 23.11, P less than 0.01)], as were the GSH content [week 6: (M: 48.47+/-7.27 nmol/mg vs. 60.74+/-9.04 nmol/mg, F = 6.71, P less than 0.05]]; [week 9: (M: 37.89+/-9.01 nmol/mg vs. 60.74+/-9.04 nmol/mg, F = 24.06, P less than 0.01]]. Compared with group M at week 9, the YGH-treated model group had markedly decreased MDA (YGJ: 4.25+/-0.86 nmol/mg vs. M: 6.29+/-1.23 nmol/mg, F = 19.52, P less than 0.01], but significantly increased SOD (YGJ: 198.35+/-46.48 U/mg vs. 152.21+/-51.65 U/mg, F = 4.65, P less than 0.05] and GSH (YGJ: 53.73+/-7.54 nmol/mg vs. M: 37.89+/-9.01 nmol/mg, F = 19.23, P less than 0.01). Compared to normal rats at week 9, group M had significantly higher protein levels of HSP70 (normal: 1.21+/-0.06 vs. M: 0.58+/-0.07, F = 166.87, P less than 0.01) and HO-1 (normal: 1.11+/-0.06 vs. M: 0.58+/-0.06, F = 123.96, P less than 0.01), but significantly decreased levels of Prxd6 (normal: 0.04+/-0.05 vs. M: 1.49+/-0.05, F = 1215.85, P less than 0.01), transferrin (normal: 0.67+/-0.03 vs. M: 1.67+/-0.04, F = 301.35, P less than 0.01), and L-FABP (normal: 0.24+/-0.02 vs. M: 1.44+/-0.14, F = 219.05, P less than 0.01). Compared to group M at week 9, the YGJ treatment group showed significantly reduced HSP70 (YGJ: 0.82+/-0.04 vs. M: 1.21+/-0.06, F = 92.31, P less than 0.01) and HO-1 (YGJ: 0.90+/-0.04 vs. 1.11+/-0.06, F = 26.89, P less than 0.01), but significantly increased Prxd6 (YGJ: 0.88+/-0.11 vs. 0.04+/-0.05, F = 150.17, P less than 0.01), transferrin (YGJ: 1.36+/-0.13 vs. 0.24+/-0.02, F = 237.19, P less than 0.01), and L-FABP (YGJ: 1.04+/-0.12 vs. 0.67+/-0.03, F = 27.53, P less than 0.01). YGJ treatment of fibrotic liver rats reduces lipid peroxidation damage by preventing generation of oxidizing substances.

Pay attention to the study on active antiliver fibrosis components of Chinese herbal medicine / 中国结合医学杂志

In this review, the researches on Chinese herb components with anti-hepatic fibrosis activity in China in the recent 20 years were generalized. Almost thirty active herb components attracted author's attention, especially, salvianolic acid B and oxymatrine which were investigated comprehensively. Moreover, the author considered that, in view of the complex pathogenesis and the multi-pathway and multi-target superiority of Chinese medicine formula, the effective components formula investigations deserve more attention and probably prompt a potential researching direction.

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>

Research approach for biological basis of Chinese medical syndromes of chronic viral hepatitis B / 中国中西医结合杂志

Chronic viral hepatitis B (CHB) is a major infectious disease greatly harmful to the health of Chinese people. Chinese medicine has its speciality and advantages in treating it depending syndrome-differentiation. The objectified researches regarding Chinese medical syndromes in CHB heretofore were reviewed in this article. Moreover, aiming at existing problems and taking the angle of "disease-syndrome combining" study, authors put forward research approach, and approaches for studying systemic biology based biological basis of Chinese medical syndrome in hepatitis B with reductionism and holism, cybernetics and system theories in combination.

Effect of jianpi huoxue recipe on gut flora in rats with alcoholic fatty liver induced by Lieber-DeCarli liquid diet / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study the effect of Jianpi Huoxue Recipe(JPHXR) on the gut flora in rats with alcoholic fatty liver (AFL) induced by Lieber-DeCarli liquid diet.</p><p><b>METHODS</b>Forty Sprague-Dawley rats were divided into 4 groups: A: normal rats, B: rats fed with non-alcoholic liquid diet, C: rats fed with ethanol liquid diet to make AFL model, and D: AFL model rats intervened by gastrogavage of JPHXR 1.0 mL/100 g per day for 8 successive weeks, 10 rats in each group. Except those in Group D (to them an equal volume normal saline was given for Successive instead), JPXHR was administered to rats in other three groups. At the end of experiment, rats were sacrificed, their blood and liver tissue samples were collected for determining serum activities of alanine transaminase (ALT) and aspartate aminotransferase (AST), endotoxin level in portal vein (expressed by lipopolysacchrides content, abbr. as LPS), and pathological examination of liver with HE staining and oil O red staining. Moreover, total DNA of gut flora were extracted from fresh rat fecal samples by Bead-beating method for determining the ERIC-PCR fingerprint, and a cluster analysis on the fingerprint was performed.</p><p><b>RESULTS</b>Compared with the levels of ALT and AST in Group A (31.15 +/- 7.04 U/L, and 53.23 +/- 10.28 U/L respectively) and Group B (26.96 +/- 8.12 and 52.09 +/- 8.62), the corresponding levels in Group C (92.72 +/- 25.83 and 72.60 +/- 23.31) significantly increased (P < 0.01), while the increments in Group D (65.28 +/- 20.36 and 59.11 +/- 10.32) were decreased (P < 0.01, P < 0.05). Pathological examination showed marked fat deposition in Group C, but which was significantly reduced in Group D. Endotoxin level in the portal vein was (0.033 +/- 0.010, EU/mL) in Group A and 0.043 +/- 0.018 in Group B, which was increased significantly in Group C (0.541 +/- 0.085, P < 0.01) and Group D (0.349 +/- 0.098 EU/mL, P < 0.01), but the increase in Group C was more significant (P < 0.01). The cluster analysis of ERIC-PCR fingerprint showed significant changes in gut flora of Group C and D, which was in Group D partially recovered.</p><p><b>CONCLUSIONS</b>JPHXR had good preventive effect against alcoholic fatty liver in rats, and could modify the structure of gut flora to some extent.</p>

The key target of Chinese medicine treatment on alcoholic and nonalcoholic fatty liver disease: the gut / 中国中西医结合杂志

In recent years, the pathogenesis of "gut-liver axis" in alcoholic and nonalcoholic fatty liver disease has attracted more attention in this field. In this paper, the relationship among fatty liver, gut-permeability, gut-derived endotoxin, and gut microbiota was systematically clarified. Based on the researches of treatment and prevention of fatty liver and gut injury by Chinese medicine, the gut is believed as the curative target for fatty liver disease, which not only is the modern annotation for the Chinese medicine practice, but also might possibly become an important view angle and strategy for fatty liver disease treatment.

Uniform designed research on the active ingredients assembling of Chinese medicine prescription for anti-liver fibrosis / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To explore the method and significance for studying active anti-liver fibrosis ingredients consisted Chinese medicine compound prescription based on Chinese medicine theory.</p><p><b>METHODS</b>Optimized prescription was screened out, adopting uniform block design with 4-factor 8-level table and regression analysis, through applying the four known effective ingredients (cordyceps sinensis polysaccharide, salvianolic acid B, amygdaloside and gypenosides) of Fuzheng Huayu Capsule (FZHYC, a new Chinese medine anti-liver fibrosis drug) to two rat liver fibrosis models established separately by dimethylnitrosamine (DMN) and CCl4, and taking the liver content of hydroxyproline (Hyp) as the screen index. Then a further study for comparing and verifying the efficacy of the obtained optimized prescription was conducted on the two former models respectively by observing the changes of Hyp content in liver, serum ALT activity and fibrosis pathology after medication, controlled by the original FZHYC and the recipe assembled by all the four ingredients.</p><p><b>RESULTS</b>Two optimized prescriptions (OPA and OPB) were screened out separately in the studies conducted on the two models. Both of them were consisted of cordyceps sinensis polysaccharide, Amygdaloside and Gypenosides, but different in constituent ratio, i.e., the ratio in OPA was 60 : 80: 50, and that in OPB, 20: 160: 50. Verifying study showed both OPA and OPA were significantly effective, with the efficacy equivalent to that of FZHYC (P>0.05). However, when they were used in combining with salvianolic acid B (the cutout ingredient in the screening), the efficacy lowered surely.</p><p><b>CONCLUSIONS</b>Uniform design is a valuable method in the compatibility research of Chinese Medicine drugs' composition. To assemble a new compound recipe reasonably based on the prescription of traditional compound recipe could make its effect equivalent to that of the original prescription. Ingredients or constituents in a prescription, either presented synergistic or antagonistic effects, are not randomly stacked together, and they should be orderly assembled in intrinsic rules of qualitative and quantitative changing.</p>

Qushi Huayu Decoction (祛湿化瘀方) inhibits protein and gene expression of cathepsin B in HepG2 cells induced by free fatty acids / 中国结合医学杂志

<p><b>OBJECTIVE</b>To study the experimental efficacy of Qushi Huayu Decoction (祛湿化瘀方,QHD) on, protein and gene expression of cathepsin B (ctsb) in HepG2 cells induced by free fatty acids (FFAs).</p><p><b>METHODS</b>The model of HepG2 steatosis and tumor necrosis factor-α (TNF-α) secretion was induced by long-chain FFAs. HepG2 cells were divided into 4 groups: control group (group C), model group (group M), low-dose QHD group (group L) and high-dose QHD group (group H). Long-chain FFAs were added to groups M, L and H. The 10% blank-control serum was added to group C and M, while 5% and 10% QHD-containing sera were added to group L and H, respectively. The levels of serum TNF-α and cellular triglyceride (TG) were detected. Cellular p-IκB and ctsb expression were detected using Western blot and PCR. The expression and distribution of ctsb were observed by immunofluorescence.</p><p><b>RESULTS</b>After incubating with FFA for 24 h, TG deposition in HepG2, TNF-α content in cell supernatant, the protein expression of cellular ctsb and P-IκB, as well as mRNA expression of ctsb increased markedly in group M compared with group C (P<0.05, P<0.01). Compared with group M, TG deposition, the expression of cellular ctsb, P-IκB and ctsb mRNA in groups L and H, as well as TNF-α content in group H, decreased significantly (P<0.05). Cell immunochemical fluorescence studies showed that ctsb was released from lysosomes and distributed in the cytoplasm extensively and diffusedly after being stimulated with FFA. In this study, these above-mentioned changes were inhibited markedly in groups L and H.</p><p><b>CONCLUSION</b>QHD might have a direct inhibitory effect on the ctsb target in the FFA-ctsb-TNFα pathway of hepatic lipotoxicity.</p>

Effect of Jianpi Huoxue decoction-containing serum on tumor necrosis factor-alpha secretion and gene expression of endotoxin receptors in RAW264.7 cells induced by lipopolysaccharide / 中国结合医学杂志

<p><b>OBJECTIVE</b>To evaluate the effect of Jianpi Huoxue decoction (JHD)-containing serum on tumor necrosis factor-alpha (TNF-alpha) secretion and endotoxin receptor gene expression in RAW264.7 cells induced by lipopolysaccharide (LPS).</p><p><b>METHODS</b>The cytotoxicity of blank-control serum and JHD-containing serum at different concentrations were evaluated through the lactate dehydrogenase (LDH) assay in RAW264.7 cells. RAW264.7 cells were divided into six groups: 5% blank-control serum group (C1, n=3), 5% blank-control serum plus LPS group (L1, n=4), 5% JHD-containing serum plus LPS group (J1, n=4), 10% blank-control serum group (C2, n=3), 10% blank-control serum plus LPS group (L2, n=4), and 10% JHD-containing serum plus LPS group (J2, n=4). After cultured with the corresponding serum for 1 h, cells in L1, L2, J1 and J2 were treated with LPS (0.1 microg/mL) for 12 h without rinse. The supernate, cells, protein and RNA were collected for assay. TNF-alpha in the culture supernate was assayed by the enzyme linked immunosorbent assay (ELISA). Protein expression of TNF-alpha in RAW cells was detected by Western-blot. TNF-alpha, Toll-like receptor 2 (TLR2), TLR4 and CD14 mRNA expression in RAW cells were detected by real-time RT-PCR.</p><p><b>RESULTS</b>The LDH assay supported that cultured for 24 h or less with the JHD-containing serum at the concentration of 10% or lower, RAW264.7 cells showed no cytotoxicity. After stimulation with LPS for 2 h, TNF-alpha in the culture supernate of the 5% blank-control serum plus LPS group (L1, P=0.03), 10% blank-control serum plus LPS group (L2, P=0.002) and in the cell layer (P=0.01) of these groups increased remarkably. After stimulation with LPS for 1 h, the mRNA expression of TNF-alpha (P=0.004), TLR (P=0.03), CD14 (P=0.004) was up-regulated obviously. In the 10% JHD-containing serum plus LPS group (J2), the protein expression of TNF-alpha in both supernate (P=0.04) and cell layer (P=0.04), gene expression of TNF-alpha (P=0.03), TLR4 (P=0.001), CD14 (P=0.001) were all inhibited. On the other hand, the TLR2 mRNA expression was not up-regulated after LPS stimulation in the 10% blank-control serum plus LPS group (L2).</p><p><b>CONCLUSION</b>JHD-containing serum inhibited the LPS-induced cytokines expression in RAW264.7 which was probably associated with its inhibitory effect on the mRNA expression of LPS receptors TLR and CD14.</p>

The role of adiponectin and adiponectin receptor 2 in the pathology of fatty liver / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the role of adiponectin (ADP) and adiponectin receptor 2 (adipoR2) in pathology of fatty liver, and to investigate the effect of Chinese herbal decoction (Qushi Huayu Decoction, QHD) on fatty liver disease.</p><p><b>METHODS</b>Two experimental fatty liver models were used. One was induced with high-fat diet for ten weeks, and the rats were divided into normal, model and QHD group, the QHD group was administrated with QHD during the last four weeks. The other experimental fatty liver model was induced by subcutaneous injection of carbon tetrachloride (CCl4) in combination with high-fat and low-protein diet for four weeks, and the rats were also divided into normal, model and QHD group, the QHD group was administrated with QHD during the last two weeks. The observation items include: (1) hepatic steatosis (H.E. staining); (2) serum ADP, hepatic triglyceride (TG), free fatty acid (FFA) and adipoR2; (3) correlation among serum ADP content, hepatic TG, FFA and adipoR2.</p><p><b>RESULTS</b>(1) Serious hepatic steatosis, increased hepatic TG and FFA, decreased serum ADP and hepatic adipoR2 were observed in the two models (P less than 0.01). QHD administration significantly reduced the hepatic TG and FFA, and increased serum ADP and hepatic adipoR2 (P less than 0.01) in these two models. (2) Inverse correlation was observed between hepatic TG, FFA and serum ADP, hepatic adipoR2 in these two models.</p><p><b>CONCLUSION</b>(1) Decreased serum ADP and hepatic adipR2 may play important roles in pathological process of fatty liver. (2) QHD administration increased the serum ADP and hepatic adipoR2.</p>

Effect of qushl huayu decoction on high-fat diet induced hepatic lipid deposition in rate / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To explore the intervention effect of Qushi Huayu Decoction (QHD) on high-fat diet induced hepatic lipid deposition and its dose-effect relationship in rats.</p><p><b>METHODS</b>Fatty liver model of rats were established simply by 10 weeks of high-fat diet feeding, and starting from the 7th week of modeling, they were gastric perfused respectively with saline (model group), high-dose QHD (QHDh group), low-dose QHD (QHDI group) and polyene phosphatidylcholine (PP group) for successive 4 weeks. Liver pathology by electron microscope observation with HE staining and oil red staining; contents of triglyceride (TG) and free fatty acid (FFA) in liver tissue; and serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), and TG in rats were determined.</p><p><b>RESULTS</b>In the model group, the significant hepatic steatosis and vesicle changes as well as severe accumulation of middle- and micro-sized fatty drops in the hepatocyte plasma were found under electron microscope; with TG and FFA contents in liver tissue elevated to 3.2 and 3.5 multiples of those in normal group respectively, but, the difference between them in serum levels of ALT, AST, TG and TC were not significant. Above-mentioned pathological changes in the QHDh, QHDI and PP groups were all ameliorated significantly with the hepatic TG decreased to 57.55%, 72.32% and 71.07%, and FFA decreased to 48.95%, 65.67%, 55.57% of those in model group respectively, especially the effect of QHDh in reducing TG was superior to that of QHDI and PP (P < 0.05).</p><p><b>CONCLUSION</b>QHD shows an evident fatty liver antagonizing effect in rats induced by high-fat diet in a dose-dependent manner.</p>

Analysis depending uniform design on the major herbs in qushi huayu compound for anti-hepatic lipotoxicity / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To analyze the major herbs in Qushi Huayu Compound (QHC) or its various assembles on the fatty deposition and tumor necrosis factor-alpha (TNF-alpha) secretion induced by free fatty acid (FFA) of human hepatic cancer cell line (HepG2) in vitro, for investigating the analytic methods in seeking the basic material in Chinese compound relevant to the pharmacological effect.</p><p><b>METHODS</b>The HepG2 cellular model of fatty deposition and TNF-alpha secretion induced by FFA and seropharmacological technique were adopted. Taking triglyceride (TG) and TNF-alpha inhibitory effect as the indexes of investigation, the effects of 10 combinations assembled by uniform design U1(11) (11(10)) form with drugs chosen from the five herbs in the QHC (oriental wormwood, cape-jasmine fruit, giant knotweed rhizome, Japanese St. John's wort herb and curcuma) were screened to seek the major herbs or optimal combination, which were then validated by grouping in interval.</p><p><b>RESULTS</b>High dosage combination of oriental wormwood and Japanese St. John's wort herb remarkably reduced the TG and TNF-alpha content in the model cells, with the effect insignificantly different from the whole compound. Moreover, single use of oriental wormwood showed a similar effect.</p><p><b>CONCLUSION</b>Oriental wormwood and its combination with Japanese St. John's wort herb are the major herb/optimum combination in QHC for inhibiting fatty deposition and TNF-alpha secretion in hepatic lipo-toxicity model. The major herb or optimal combination in a certain Chinese compound acted on some sticking point could be discovered by adopting uniform design and pharmacodynamics analytic technique.</p>

Inhibitory effects of Qushi Huayu Decoction on fatty deposition and tumor necrosis factor alpha secretion in HepG2 cells induced by free fatty acid / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study the effects of Qushi Huayu Decoction (QHD) contained serum on fatty deposition and tumor necrosis factor alpha (TNF-alpha) secretion of hepatic lipotoxicity model in vitro, for further investigating the mechanism of the decoction for preventing and treating fatty liver.</p><p><b>METHODS</b>The steatosis with TNF-alpha secretion lipotoxic model of HepG2 induced by long-chain free fatty acid (FFA) was duplicated. Groups of normal, model cells and model cells treated with different concentrations of QHD contained serum were set up to test the content of TNF-alpha in culture supernate and triglyceride (TG) in cells, as well as to observe the ultrastructural change of cells by oil-red staining and the protein expression and gene expression of cellular TNF-alpha.</p><p><b>RESULTS</b>After being stimulated with FFA for 24 h, marked deposition of lipid with high content of TG presented in the cells of model group. Compared with the normal group, not only TNF-alpha content of culture supernate but also the protein expression and mRNA expression of intracellular TNF-alpha increased significantly. Contrast to the model group, the contents of TG in cells and TNF-alpha in supernate as well as the protein and mRNA expression of TNF-alpha in the model cell group treated with 10% QHD were lower significantly (all P < 0.01).</p><p><b>CONCLUSION</b>QHD could significantly inhibit the fatty deposition and TNF-alpha secretion in HepG2 cells induced by free fatty acid.</p>

Intervention effects of Jianpi Liqi Huoxue Decoction on lipid peroxidative liver injury induced by alcohol / 中国结合医学杂志

<p><b>OBJECTIVE</b>To study the intervention effects of Jianpi Liqi Huoxue Decoction ( JLHD) on lipid peroxidative liver injury induced by alcohol.</p><p><b>METHODS</b>The rat alcoholic model of liver disease (ALD) induced by Lieber-DeCarli liquid diet was established. Thirty-two male SD rats were randomly divided into 4 groups: the normal group (n =5), the control group (n =9), the model group (n =9) and the JLHD group (n =9). From the 4th week after modeling, the rats were given JLHD or distilled water by gastrogavage respectively, and the samples of blood and liver tissues were taken out from the rats for determination by the end of the 8th week. The hepatic pathological changes were observed with HE staining; the liver injury related indices, including activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum, Y-glutamyl transpeptidase (Y-GT) activity and triglyceride (TG) content in liver tissues, as well as the lipid peroxidation related indices, including malonaldehyde (MDA) content and nitric oxide synthase (NOS) activity in liver tissue, serum Fe2+ level, and the anti-peroxidation capacity related indices, including superoxide dismutase (SOD) activity, glutathion (GSH) content and reactive oxygen species (anti-ROS) activity in liver tissues were determined.</p><p><b>RESULTS</b>(1) There were obvious figures of fatty degeneration and inflammatory infiltration in liver tissues of the model group. As compared with the control group, in the model group, the activity of ALT and AST, and Fe2+ content in serum, Y-GT and NOS activity, TG and MDA content in liver tissues were significantly higher (P<0. 01), while the activity of SOD, GSH and anti-ROS in liver tissues were significantly lower (P<0.01). (2) The fatty degeneration and inflammatory infiltration of liver tissues in the JLHD group were significantly lessen as compared with those in the model group; and the abnormalities of all the indexes revealed in the model rats were restored to certain extent in the JLHD group, and especially significant were the levels of ALT activity, MDA content and Fe2+ , which were nearly normal.</p><p><b>CONCLUSION</b>JLHD has significant effects against alcoholic liver injury, the acting mechanism of which is likely to be related with its anti-lipid peroxidative effect.</p>

Relationship between alcoholic liver injury and endotoxin leakage from gut and intervention effect of jianpi liqi huoxue decoction / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study the effects and mechanisms of Jianpi Liqi Huoxue Decoction (JLHD) in anti-alcoholic liver injury (ALI) through the pathological relation of ALI with changes of intestinal permeability and endotoxin leakage.</p><p><b>METHODS</b>The liver injury model induced by Lieber-DeCarli alcoholic forage was established. Altogether 42 male SD rats were randomly divided into 4 groups, the normal group (n=6), the control group fed with non-alcohol diet (n=12), the model group fed with alcoholic diet (n=12) and the treated group fed with alcoholic diet and treated with JLHD (n=12). The medicine or distilled water was administered by gavage from the 3rd week to the end of the 6th week. Then after fasting for 5 h all the rats except those in the normal group were given lipopolysaccharide (LPS) 10 mg/kg by gavage, and the blood plasma from portal vein, serum from inferior cava vein as well as tissues of liver and intestine were prepared for detection of plasma LPS level in the portal vein to observe the change of intestinal permeability through LPS content in portal vein blood plasma, the pathological and ultrastructural changes of the small intestine by HE staining, the pathological change of liver and triglyceride (TG) content and gamma glutamyl transpeptidase (GGT) activity in liver, the changes of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity, and plasma tumor necrosis factor-alpha (TNF-alpha) level.</p><p><b>RESULTS</b>In rats after modeling, there were obvious fatty degeneration, significant increase of hepatic TG content and GGT activity, serum ALT and AST activity, as well as plasma TNF-alpha level, with high plasma LPS level indicating increased intestinal permeability, and seriously injured mucosa microvilla of small intestine. However, all the above abnormal changes were milder in the treated group than those in the model group. Meanwhile, the TNF-alpha content, endotoxin level and ALT activity were found to be positively correlated.</p><p><b>CONCLUSION</b>JLHD could alleviate liver injury through inhibiting the alcohol induced increased intestinal permeability and lessening endotoxin leakage.</p>