Screening and Identification of the Biomarkers Applied for the Evaluation of Acute and Chronic Thermal Tolerance Ability in Largemouth Bass (Micropterus salmoides).

Affected by the continuously rising temperature, thermal stress leads to a delinked growth rate and resistance to stress in cultured largemouth bass (Micropterus salmoides, LMB) in China. Identification of LMB with better thermal resistance will benefit the breeding of new varieties. However, there has been limited reporting on the evaluation to identify LMB with better thermal resistance. LMB consists of the northern LMB (Micropterus salmoides salmoides, NLMB) and the Florida LMB (Micropterus salmoides floridanus, FLMB). Due to their different geographical distributions, it has been suggested that FLMB exhibit better thermal resistance compared to NLMB. In this study, NLMB and FLMB were subjected to thermal stress for 3 h (acute) and 60 d (chronic) at 33 °C, respectively. Subsequently, the variations of 12 candidate biomarkers between NLMB and FLMB were analyzed. Exposure to acute thermal stress significantly increased plasma cortisol, blood glucose, and lactate levels; activities of superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT), glucose kinase (GK), pyruvate kinase (PK), lactate dehydrogenase (LDH), and glucose 6 phosphatase (G6Pase); and the expressions of hsp70 and hsp90 in both NLMB and FLMB (p < 0.05). Compared to NLMB, FLMB exhibited a lower plasma cortisol level and a higher expression of hsp90 under acute thermal stress (p < 0.05). Exposure to chronic thermal stress significantly increased plasma cortisol and blood glucose levels, as well as activities of GK, PK, LDH, and G6Pase, as well as expressions of hsp70 and hsp90 in both NLMB and FLMB (p < 0.05). Additionally, FLMB showed a lower expression of hsp70 compared to NLMB (p < 0.05). In conclusion, our results showed that LMB with lower plasma cortisol level and higher expression of hsp90 under acute thermal stress, as well as lower expression of hsp70 under chronic thermal stress were suggested to have better thermal resistance. Our study provides valuable information for identifying and breeding LMB varieties with better thermal resistance in the future.

Development and characterization of 40 InDel markers from the major histocompatibility complex genes of largemouth bass (Micropterus salmoides).

BACKGROUND: The genetic improvement in growth and food habit domestication of largemouth bass (Micropterus salmoides) have made breakthroughs in past decades, while the relevant work on disease resistance were rarely carried out. Major histocompatibility complex (MHC) genes, which are well known as their numbers and high polymorphisms, have been used as candidate genes to mine disease-resistant-related molecular markers in many species. METHODS AND RESULTS: In present study, we developed and characterized 40 polymorphic and biallelic InDel markers from the major histocompatibility complex genes of largemouth bass. The minor allele frequency, observed heterozygosity, expected heterozygosity and polymorphic information content of these markers ranged from 0.0556 to 0.5000, 0.1111 to 0.6389, 0.1064 to 0.5070, and 0.0994 to 0.3750, respectively. Three loci deviated significantly from Hardy-Weinberg equilibrium, while linkage disequilibrium existed at none of these loci. CONCLUSION: These InDel markers might provide references for the further correlation analysis and molecular assisted selection of disease resistance in largemouth bass.

Expression characteristics of the cyp19a1b aromatase gene and its response to 17ß-estradiol treatment in largemouth bass (Micropterus salmoides).

To investigate the regulatory role of the cyp19a1b aromatase gene in the sexual differentiation of largemouth bass (Micropterus salmoides, LMB), we obtained the full-length cDNA sequence of cyp19a1b using rapid amplification of cDNA ends technique. Tissue expression characteristics and feedback with 17-ß-estradiol (E2) were determined using quantitative real-time PCR (qRT-PCR), while gonad development was assessed through histological section observations. The cDNA sequence of LMB cyp19a1b was found to be1950 base pairs (bp) in length, including a 5' untranslated region of 145 bp, a 3' untranslated region of 278 bp, and an open reading frame encoding a protein consisting of 1527 bp that encoded 508 amino acids. The qRT-PCR results indicated that cyp19a1b abundantly expressed in the brain, followed by the gonads, and its expression in the ovaries was significantly higher than that observed in the testes (P < 0.05). After feeding fish with E2 for 30 days, the expression of cyp19a1b in the pseudo-female gonads (XY-F) was significantly higher than that in males (XY-M) (P < 0.05), whereas expression did not differ significantly between XX-F and XY-F fish (P > 0.05). Although the expression of cyp19a1b in XY-F and XX-F fish was not significantly different after 60 days (P>0.05), both exhibited significantly higher levels than that of XY-M fish (P<0.05). Histological sections analysis showed the presence of oogonia in both XY-F and XX-F fish at 30 days, while spermatogonia were observed in XY-M fish. At 60 days, primary oocytes were abundantly observed in both XY-F and XX-F fish, while a few spermatogonia were visible in XY-M fish. At 90 days, the histological sections' results showed that a large number of oocytes were visible in XY-F and XX-F fish. Additionally, the gonads of XY-M fish contained numerous spermatocytes. These results suggest that cyp19a1b plays a pivotal role in the development of ovaries and nervous system development in LMB.

Integrated transcriptomic and proteomic analyses reveal the mechanism of easy acceptance of artificial pelleted diets during food habit domestication in Largemouth bass (Micropterus salmoides).

Acceptance of artificial pelleted diets contributes to increasing the cultured areas and output of carnivorous fish. However, the mechanism of acceptance of artificial pelleted diets remains largely unknown. In this study, the easy acceptance of artificial pelleted diets (EAD) group and the not easy acceptance of artificial pelleted diets (NAD) group of Largemouth bass (Micropterus salmoides) were divided based on the ratios of stomach weight/body weight (SB) after 0.5 h feeding, which was bigger than 18% in the EAD group and ranged from 8 to 12% in the NAD group. Through transcriptome and proteome sequencing, a total of 2463 differentially expressed genes (DEGs) and 230 differentially expressed proteins (DEPs) were identified, respectively. Integrated analyses of transcriptome and proteome data revealed that 152 DEPs were matched with the corresponding DEGs (named co-DEGs-DEPs), and 54 co-DEGs-DEPs were enriched in 16 KEGG pathways, including the metabolic pathways, steroid biosynthesis, fatty acid biosynthesis, etc. Furthermore, 3 terpenoid backbone biosynthesis-related genes (Hmgcr, Hmgcs, and Fdps) in metabolic pathways, 10 steroid biosynthesis-related genes (Fdft1, Sqle, Lss, Cyp51a1, Tm7sf2, Nsdhl, Hsd17b7, Dhcr24, Sc5d, and Dhcr7), and 3 fatty acid biosynthesis-related genes (Acaca, Fasn, and Ascl) were all up-regulated in the EAD group, suggesting that the lipid metabolism pathway and steroid biosynthesis pathway play important roles in early food habit domestication in Largemouth bass. In addition, the detection results of randomly selected 15 DEGs and 15 DEPs indicated that both transcriptome and proteome results in the study were reliable. Our study provides useful information for further research on the mechanisms of food habit domestication in fish.

Eight single nucleotide polymorphisms and their association with food habit domestication traits and growth traits in largemouth bass fry (Micropterus salmoides) based on PCR-RFLP method.

Background: The largemouth bass (Micropterus salmoides), an economically important freshwater fish species widely farmed in China, is traditionally cultured using a diet of forage fish. However, given the global decline in forage fish fisheries and increasing rates of waterbody pollution and disease outbreaks during traditional culturing, there is a growing trend of replacing forage fish with formulated feed in the largemouth bass breeding industry. The specific molecular mechanisms associated with such dietary transition in this fish are, nevertheless, poorly understood. Methods: To identify single nucleotide polymorphisms (SNPs) related to food habit domestication traits and growth traits in largemouth bass fry, we initially genotyped fry using eight candidate SNPs based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, with genetic parameters being determined using Popgen32 and Cervus 3.0. Subsequently, we assessed the associations between food habit domestication traits of largemouth bass fry and these SNPs using the Chi-square test or Fisher's exact test. Furthermore, we used a general linear model to assess the relationships between the growth traits of largemouth bass fry and these SNPs. The Pearson correlation coefficient between growth traits and the SNPs was also determined using bivariate correlation analysis in IBM SPSS Statistics 22. Finally, the phenotypic variation explained (PVE) by the SNPs was calculated by regression analysis in Microsoft Excel. Results: The genotyping results obtained based on PCR-RFLP analysis were consistent with those of direct sequencing. Five SNPs (SNP01, SNP02, SNP04, SNP05, and SNP06) were found to be significantly correlated with the food habit domestication traits of fry (P < 0.05); SNP01 (P = 0.0011) and SNP04 (P = 0.0055) particularly, had showed highly significant associations. With respect to growth traits, we detected significant correlations with the two SNPs (SNP01 and SNP07) (P < 0.05), with SNP01 being significantly correlated with body length, and height (P < 0.05), and SNP07 being significantly correlated with body height only (P < 0.05). Conclusions: Our findings indicated that the PCR-RFLP can be used as a low-cost genotyping method to identify SNPs related to food habit domestication and growth traits in largemouth bass, and that these trait-related SNPs might provide a molecular basis for the future breeding of new varieties of largemouth bass.

Different responses to glucose overload between two strains of largemouth bass (Micropterus salmoides).

In order to improve the glucose utilization capacity of largemouth bass (Micropterus salmoides), responses to glucose overload between two strains (Y: breeding strain; W: wild strain) were compared at 0, 6, 12, and 24 h after glucose injection (1.67 g/kg). The data revealed that plasma glucose in the Y strain (<12 h) recovered faster than in the W strain (12 h), with the Y strain secreted more insulin within 6 h post-injection. Triglyceride (TG) and low-density lipoprotein-cholesterol (VLDL-CH) content in the Y strain increased, peaking at 12 h, then decreased, whereas the W strain's TG content was not affected and VLDL-CH content decreased. The hepatic and muscular fatty acid synthetase, liver x receptor-1, and sterol regulatory element-binding protein expressions were consistent with the TG content change. Both strains' liver and muscle glycogen contents exhibited similar trends to that of the glycogen synthase gene-increasing, then declining, and peaking at 6 and 12 h. The expression levels of hepatic and muscular phosphofructokinase and pyruvate kinase in the Y strain increased, peaking at 12 h. In the W strain, they were suppressed and reached the minimum at 24 h. The mRNA levels of hepatic and muscular phosphoenolpyruvate carboxykinase and glucose-6-phosphatase were enhanced and peaked at 24 h in both strains, hepatic isocitrate dehydrogenase-1, and α-ketoglutarate dehydrogenase complex expression increased after declining, peaking at 12 and 24 h. Two genes in the W strain's muscles showed a similar trend. Both strains' transcriptome results identified seven common functional genes for resistance to hyperglycemia that were involved in the circadian rhythm pathway, which is a suggested key pathway for coping with hyperglycemia. Furthermore, 48 differential genes were identified between the two strains, and these genes were enriched in the TGF-beta and cell cycle signaling pathways, indicating that these pathways may be key factors affecting the differential responses to glucose overload. We conducted a comprehensive comparison of glucose overload molecular responses between two strains of M. salmoides, and the results can provide a promising strategy to improve the glucose utilization capacity of M. salmoides based on advantageous pre-existing traits.

Genetic diversity analysis and development of molecular markers for the identification of largemouth bass (Micropterus salmoides L.) based on whole-genome re-sequencing.

Largemouth bass (Micropterus salmoides L.) is generally considered to comprise two subspecies, Florida bass (M. floridanus) and Northern Largemouth bass (M. salmoides), which have biological characteristic differences because of their geographical distribution. In this study, whole-genome re-sequencing was performed among 10 Florida and 10 Northern largemouth bass, respectively. In total, 999,793 SNPs and 227,797 InDels were finally identified, and 507,401 SNPs (50.75%) and 116,213 InDels (51.01%) were successfully mapped to annotated 18,629 genes and 14,060 genes, respectively. KEGG classification indicated that most of these genes were focused on the pathways including signal transduction, transport and catabolism, and endocrine system. Genetic diversity analysis indicated that Florida largemouth bass had higher genetic diversity than Northern largemouth bass, indicating that the germplasm quality of Northern largemouth bass had markedly reduced in China. To examine the accuracies of the identified markers, 23 SNPs and eight InDels (the insertions or deletions of more than 45 bp) were randomly selected and detected among Florida largemouth bass, Northern largemouth bass, and their F1 hybrids. The detection efficiencies of all the markers were higher than 95%; nineteen SNPs and three InDels could accurately distinguish the two subspecies and their F1 hybrids with 100% efficiencies. Moreover, the three InDel markers could clearly distinguish the two subspecies and their F1 hybrids with a PCR-based agarose gel electrophoresis. In conclusion, our study established a simple PCR-based method for the germplasm identification of largemouth bass, which will be useful in the germplasm protection, management, and hybridization breeding of largemouth bass.

Timing of early gonadal differentiation and effects of estradiol-17ß treatments on the sex differentiation in Largemouth bass (Micropterus salmoides).

In this study, an efficient estradiol-17ß (E2)-induced feminization method was established based on the timing of early gonadal differentiation in Largemouth bass (Micropterus salmoides). Histological section results showed that from 20 days post-hatch (dph) to 30 dph, the germ cells gradually differentiated into oogonium and spermatic deferent, respectively. Moreover, female-biased genes Foxl2 and Cyp19a1a were up-regulated to the first peak at 20 dph, while the male-biased genes Dmrt1 were up-regulated to the first peak at 30 dph. These results indicated that the timing of early gonadal differentiation in Largemouth bass was between 20 and 30 dph. Therefore, 15 dph Largemouth bass with a body length of 15.10 ± 0.09 mm were chosen, and four E2-treated diets were set as 0 (E0, control), 50 mg/kg E2 (E50), 100 mg/kg E2 (E100), and 200 mg/kg E2 (E200). After feeding with E2-treated diets for 60 days, female ratios were 55%, 100%, 100%, and 100% in E0, E50, E100, and E200 groups, respectively. No intersex fish were observed in all the groups. However, 30% of females in the E200 group possessed thinner ovaries, with smaller ovary cavity structures and a decreased number of primary oocyte cells than those in other groups. Besides, the Largemouth bass in the E0 group grew more than those in E50, E100, and E200 groups during the E2 treatments period (P < 0.05). In conclusion, our study suggested that 50-100 mg/kg E2-treated diets could effectively induce the feminization of 15 dph Largemouth bass within 60 days duration time, which provided valuable information for the breeding of the all-male Largemouth bass population.

Fabp4 contributes toward regulating inflammatory gene expression and oxidative stress in Ctenopharyngodon idella.

Fatty acid-binding protein (Fabp)-4 is a member of the FABP family. Mammalian fabp4 has been demonstrated to involve in inflammation and immunity, whereas the related data of fish fabp4 remain limited. Therefore, we further investigated the effects of fabp4 on immunity in Ctenopharyngodon idella. The fabp4 sequence spanned 405 bp was cloned first, sharing high identity to fabp4 from other fish and mammals. Fabp4 expression was the highest in the adipose tissue, followed by the heart, muscle, and liver. In vivo, lipopolysaccharide (LPS) triggered the expression of fabp4, toll-like receptor (tlr)-22, interleukin (il)-1ß, and tumor necrosis factor (tnf)-α in the kidney and spleen. In vitro, exposing C. idella CIK cells to LPS decreased their viability, and the expression of fabp4 was also increased by LPS. However, BMS309403, an inhibitor of FABP4, mitigated these effects. Furthermore, treating the cells with LPS or fabp4 overexpression plasmids resulted in reactive oxygen species (ROS) generation and upregulation of inflammatory genes expression, including tlr22, type-I interferon (ifn-1), interferon regulatory factor (irf)-7, tnfα, il-1ß, and interferon-ß promoter stimulator 1. These effects were ameliorated by preincubation with BMS309403. Moreover, incubating the cells with glutathione reduced the production of ROS and the expression of inflammatory genes that were evoked by LPS and plasmid treatments. These results showed that fabp4 acts as a pro-inflammatory molecule via elevating ROS levels, providing a novel understanding of the molecular regulation of innate immunity in teleosts.

Comparative skin transcriptome of two Oujiang color common carp (Cyprinus carpio var. color) varieties.

Body color variation has long been a hot research topic in evolutionary and functional biology. Oujiang color common carp (Cyprinus carpio var. color) is a well-known economical and ornamental fish. Three main types of pigments and four distinct color patterns are typical characters of Oujiang color common carp, which makes it an excellent fish model to study body coloration. In this study, skin transcriptome assembly and comparisons were conducted in two Oujiang color common carp varieties: whole red and whole white. Transcriptome comparison revealed that more differentially expressed energy metabolism genes were upregulated in whole white compared to whole red. The results indicated that energy metabolism genes might be strongly associated with environmental adaption and growth performance and likely affect the red and white color formation in Oujiang color common carp. Our study provided direct guidance for the aquaculture industrials of Oujiang color common carp and presented valuable genetic resources for body color research in fish.

Ingredients of Huangqi decoction slow biliary fibrosis progression by inhibiting the activation of the transforming growth factor-beta signaling pathway.

BACKGROUND: Huangqi decoction was first described in Prescriptions of the Bureau of Taiping People's Welfare Pharmacy in Song Dynasty (AD 1078), and it is an effective recipe that is usually used to treat consumptive disease, anorexia, and chronic liver diseases. Transforming growth factor beta 1 (TGFß1) plays a key role in the progression of liver fibrosis, and Huangqi decoction and its ingredients (IHQD) markedly ameliorated hepatic fibrotic lesions induced by ligation of the common bile duct (BDL). However, the mechanism of IHQD on hepatic fibrotic lesions is not yet clear. The purpose of the present study is to elucidate the roles of TGFß1 activation, Smad-signaling pathway, and extracellular signal-regulated kinase (ERK) in the pathogenesis of biliary fibrosis progression and the antifibrotic mechanism of IHQD. METHODS: A liver fibrosis model was induced by ligation of 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 IHQD group. IHQD was administrated intragastrically for 4 weeks. At the end of the fifth week after BDL, animals were sacrificed for sampling of blood serum and liver tissue. The effect of IHQD on the TGFß1 signaling pathway was evaluated by western blotting and laser confocal microscopy. RESULTS: Decreased content of hepatic hydroxyproline and improved liver function and histopathology were observed in IHQD rats. Hepatocytes, cholangiocytes, and myofibroblasts in the cholestatic liver injury released TGFß1, and activated TGFß1 receptors can accelerate liver fibrosis. IHQD markedly inhibited the protein expression of TGFß1, TGFß1 receptors, Smad3, and p-ERK1/2 expression with no change of Smad7 expression. CONCLUSION: IHQD exert significant therapeutic effects on BDL-induced fibrosis in rats through inhibition of the activation of TGFß1-Smad3 and TGFß1-ERK1/2 signaling pathways.

[Chinese herbal medicine Xiayuxue Decoction inhibits liver angiogenesis in rats with carbon tetrachloride-induced liver fibrosis].

OBJECTIVE: To evaluate the effects of Xiayuxue Decoction, a compound traditional Chinese medicine, on liver angiogenesis in rats with carbon tetrachloride (CCl(4))-induced liver fibrosis. METHODS: Liver cirrhosis was induced by intraperitoneal injection of 50% CCl(4)-olive oil solution at the dose of 1 mL/kg body weight, twice per week for 9 consecutive weeks. After 3- and 6-week injection, 6 rats in the normal group and 6 rats in the model group were randomly sacrificed for dynamic observation. The survival rats of model group were randomly divided into model group (n=15) and Xiayuxue Decoction group (n=11). Six normal rats were used as a normal control. Xiayuxue Decoction was administered orally starting from the 7th week for 3 weeks. At the end of the ninth week, animals were sacrificed and liver tissues were harvested to measure histological changes, activities of matrix metalloproteinase-2 (MMP-2) and MMP-9 and protein expressions of platelet endothelial cell adhesion molecule-1 (CD31), von Willebrand factor (vWF), vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor-2 (VEGFR2), complement decay-accelerating factor (DAF) and α-smooth muscle actin (α-SMA) in the liver tissues. RESULTS: Compared with the normal group, liver injury, fatty degeneration and collagen deposition were evidently observed in the model group and protein expressions of CD31, vWF, VEGF, VEGFR2, DAF and α-SMA were gradually increased. In addition, the activities of MMP-2 and MMP-9 in liver tissues were enhanced in the model group (P<0.01). Compared with 9-week model group, liver injury, fatty degeneration and collagen deposition were markedly inhibited by Xiayuxue Decoction; protein expressions of CD31, vWF, VEGF, VEGFR2,α-SMA and DAF and activities of MMP-2 and MMP-9 in the liver tissues were decreased in the Xiayuxue Decoction group (P<0.01). CONCLUSION: The angiogenesis is evident and aggravating gradually during the progression of liver cirrhosis induced by CCl(4). Xiayuxue Decoction inhibits the angiogenesis by decreasing the activities of MMP-2 and MMP-9, inhibiting the activation of hepatic stellate cells, and damaging the new vessel integrality.

[Mechanism of hepatocytes transdifferentiation to bile duct epithelial cells and intervention of huangqi decoction].

OBJECTIVE: To investigate the mechanism of hepatocytes transdifferentiation to bile duct epithelial cells (BECs) and intervention of Huangqi decoction (HQD) on hepatic fibrosis formation in rats with secondary cholestasis. METHODS: Seventy-five SD male rats were made into cholestatic hepatic fibrosis model animals by bile duct ligation, and randomized into the control group (n = 50) and the HQD group (n = 15). Starting from one week after modeling, they were administered orally with saline and HQD respectively for four weeks. Besides, a sham-operated group was set up with 10 rats operated by choledochus segregating only and administered after then with saline. Rats were killed in batches at different time points, i.e. each five from the control group and sham-operated group at the end of the 1st week, five from the control group for each time at the end of the 2nd, 3rd and 4th week, and all the remaining rats at the end of the 5th week. Their liver tissues were taken for histological change examination, content of hydroxyproline (Hyp) determination; protein expression of BECs marker cytokeratin 7 (CK7) and the hepatocyte specific antigen HepPar detection by Western blot, and CK7-Hep Par co-localization by laser confocal microscopy. Then IPP software was used to analyze Sirius red stained positive areas of CK7 and Hep Par, as well as the average IOD of CK7/Hep Par co-localization. RESULTS: Hepatocytes in hepatic tissues (Hep Par positive cell) in the model rats decreased gradually along was time went by after modeling (Sham > M1w > M2w > M3w > M4w > M5w), which was in parallel with the increase of BECs (CK7 positive cells), degree of fibrosis, Hyp content and CK7 protein expression. Increasing of co-localized positive cells of CK7/Hep Par began at 1 week and reached the peak 3 weeks after modeling, then it decreased gradually. The Hep Par protein expression was negatively correlated with that of CK7; the Hep Par positive cell expression was negatively correlated with CK7 positive cell expression and collagen deposition; while the CK7 positive cell expression was positively correlated with the collagen deposition in the liver tissue. Compared with the model control group, the mortality, CK7/Hep Par co-localized positive cells, fibrosis degree, Hyp content and CK7 protein expression were lesser obviously (P < 0.01), while Hep Par positive cell and protein expressions were higher significantly in the HQD group. CONCLUSIONS: Hepatocytes transdifferentiation to BECs might be a key pathological element for secondary cholestatic hepatic fibrosis formation; the restraining action of HQD is possibly a major action mechanism of HQD for effectively intervening and treating secondary cholestasis hepatic fibrosis.

[Effects of Yiguanjian Decoction on liver cirrhosis formation:a differential proteomics study in rats].

OBJECTIVE: To investigate the effects of Yiguanjian Decoction, a compound traditional Chinese herbal medicine, on rats with cirrhosis based on the method of differential proteomics. METHODS: Wistar male rats (n=48) were randomly divided into normal control group (n=12) and model-making group (n=36). Rat cirrhosis model was established by intraperitoneal injection of 50% carbon tetrachloride (CCl4) plus olive oil solution (1 mL/kg, twice weekly for 9 weeks). After 3- and 6-week injection, 6 rats each time were sacrificed for dynamic observation before medicine intervention, and the 24 remained rats were randomly divided into untreated group (n=12) and Yiguanjian Decoction group (n=12) at the first day of the 7th week. All animals were sacrificed by the end of the 9th week, and total protein of liver tissue was isolated by two-dimensional gel electrophoresis (2-DE); some differentially expressed protein spots were analyzed and identified by matrix-assisted laser desorption/ionization two-stage time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) and database querying. Protein expressions of Cu/Zn superoxide dismutase (Cu/Zn SOD) and DJ-1 were validated by Western blot and immunohistochemical methods. RESULTS: 2-DE maps with high resolution and good repeatability were obtained. In all 50 protein spots identified by MALDI-TOF/TOF-MS and database querying, there were 5 protein spots related to oxidative stress named Cu/Zn SOD, DJ-1, glutathione synthetase, glutathione S-transferase Yb-1 subunit and aldo-keto reductase family 7, A2 respectively. Compared with the normal control group, expressions of Cu/Zn SOD, DJ-1, glutathione S-transferase Yb-1 subunit and aldo-keto reductase family 7, A2 in the untreated group were decreased significantly. Expressions of Cu/Zn SOD and aldo-keto reductase family 7, A2 were decreased time-dependently. Compared with the untreated group in 9th week, protein expressions of Cu/Zn SOD, DJ-1, glutathione S-transferase Yb-1 subunit and aldo-keto reductase family 7, A2 in the Yiguanjian Decoction groups were increased significantly while expression of glutathione synthetase was decreased notably. Western blot and immunohistochemical results of Cu/Zn SOD and DJ-1 expressions coincided with proteomics results. CONCLUSION: Anti-oxidative depression is a key pathological change of cirrhosis induced by CCl4 in rats, and increasing expression of proteins related to anti-oxidative stress may be a major mechanism of Yiguanjian Decoction in treating cirrhosis induced by CCl4 effectively.

[Huangqi decoction inhibits cholangiocyte proliferation and transdifferentiation in cholestatic liver fibrosis induced by BDL in rats].

OBJECTIVE: To elucidate the antifibrotic mechanism of Huangqi decoction in rats with BDL-induced cholestatic liver fibrosis. METHODS: 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. RESULTS: 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. CONCLUSIONS: These observations suggest that Huangqi decoction can inhibit cholangiocyte proliferation and cholangiocyte transdifferentiation.

Action mechanism of Yi Guan Jian Decoction on CCl4 induced cirrhosis in rats.

AIM OF STUDY: To investigate action mechanism of Yi Guan Jian Decoction on cirrhosis induced by CCl(4) in rats. MATERIAL AND METHODS: CCl(4) (3 mL/kg) for the first time and then olive oil CCl(4) solution 50% (2 mL/kg) was administered hypodermically to rats twice each week for 12 weeks. At the end of 8th week, rats were randomly divided into CCl(4) control group (n=10), Yi Guan Jian Decoction group (n=9) and Xiao Chai Hu Decoction group (n=9). Yi Guan Jian Decoction and Xiao Chai Hu Decoction were oral administrated per day respectively for 4 weeks, concomitantly continued CCl(4) administration. At 12th weekend, the rats were sacrificed for sampling and detection of liver function, histological changes of liver tissue, liver tissue hydroxyproline content and expression of alpha-SMA, CD68, MMP-13, TIMP-1, TIMP-2, Caspase-12, HGFalpha, MMP-2, MMP-9 and hepatocyte apoptotic index. RESULTS AND CONCLUSIONS: (1) Compared with that of normal rats, expression of alpha-SMA, CD68 and TIMP-1 in liver tissue of 8 week model group rats increases significantly (P<0.01), moreover further increased in the 12 week of model group. However, MMP-13, HGFalpha, TIMP-2 content decreases gradually and the statistical difference is seen between each time point (P<0.01). Activity of MMP-2, MMP-9, content of Caspase-12 and hepatocyte apoptotic index increased gradually at 4th, 8th, 12th week. (2) Compared to that of the same time point model group, activity of MMP-9 and contents of MMP-13, TIMP-2 and HGFalpha in Yi Guan Jian Decoction group improves significantly (P<0.01), and activity of MMP-2 and contents of alpha-SMA, TIMP-1, Caspase-12 and hepatocyte apoptotic index decreases significantly (P<0.01). This work suggests that Yi Guan Jian Decoction exerts significant therapeutic effect on CCl(4)-induced cirrhosis in rats, through mechanism of inhibiting hepatocytes apoptosis and hepatic stellate cells activation, and regulating the function of Kupffer cell. ETHNOPHARMACOLOGICAL RELEVANCE: This study investigates the mechanism of Yi Guan Jian against cirrhosis from aspect of heptocytes apoptosis and hepatic stellate cells activation. It suggest that although of unknown bioactive ingredients, mechanism of traditional Chinese medicine recipe against cirrhosis can be disclosed and of profound significance.