Effect of Soothing Gan (Liver) and Invigorating Pi (Spleen) Recipes on TLR4-p38 MAPK Pathway in Kupffer Cells of Non-alcoholic Steatohepatitis Rats.

OBJECTIVE: To investigate the mechanism of inflflammatory-mediated toll-like receptor 4 (TLR4)-p38 mitogen-activated protein kinase (p38 MAPK) pathway in Kupffer cells (KCs) of non-alcoholic steatohepatitis (NASH) rats and the intervention effect of soothing Gan (Liver) and invigorating Pi (Spleen) recipes on this pathway. METHODS: After 1 week of acclimatization, 120 Sprague-Dawley male rats were randomly divided into 8 groups using a random number table (n=15 per group): normal group, model group, low-dose Chaihu Shugan Powder (, CHSG) group (3.2 g/kg), high-dose CHSG group (9.6 g/kg), low-dose Shenling Baizhu Powder (, SLBZ) group (10 g/kg), high-dose SLBZ (30 g/kg) group, and low- and highdose integrated recipe (L-IR, H-IR) groups. All rats in the model and treatment groups were fed with a high-fat diet (HFD). The treatments were administrated by gastrogavage once daily and lasted for 26 weeks. The liver tissues were detected with hematoxylin-eosin (HE) and oil red O staining. Levels of liver lipids, serum lipids and transaminases were measured. KCs were isolated from the livers of rats to evaluate the mRNA expressions of TLR4 and p38 MAPK by real-time flfluorescence quantitative polymerase chain reaction, and proteins expressions of TLR4, p-p38 MAPK and p38 MAPK by Western blot. Levels of inflammatory cytokines including tumor necrosis factor α (TNF-α), interleukin (IL)-1 and IL-6 in KCs were measured by enzyme-linked immunosorbent assay. RESULTS: After 26 weeks of HFD feeding, HE and oil red O staining showed that the NASH model rats successfully reproduced typical pathogenesis and histopathological features. Compared with the normal group, the model group exhibited significant increases in body weight, liver weight, liver index, serum levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol, and aspartate aminotransferase as well as TC and TG levels in liver tissues, and significant decrease in serum level of high-density lipoprotein cholesterol (Plt;0.05 or Plt;0.01), while those indices were significantly ameliorated in the H-IR group (Plt;0.05 or Plt;0.01). Higher levels of TNF-α, IL-1 and IL-6 in KCs were observed in the model group compared with the normal group (Plt;0.01). Significant decreases in TNF-α, IL-1 and IL-6 were observed in the H-SLBZ, H-IR and L-IR groups compared with the model group (Plt;0.05 or Plt;0.01). The mRNA expressions of TLR4 and p38 MAPK and protein expressions of TLR4, p38 MAPK and p-p38 MAPK in KCs in the model group were significantly higher than the normal group (Plt;0.01), while those expression levels in the L-IR and H-IR groups were significantly lower than the model group (Plt;0.05 or Plt;0.01). CONCLUSION: Inflflammation in KCs might play an important role in the pathogenesis of NASH in rats. The data demonstrated the importance of TLR4-p38MAPK signaling pathway in KCs for the anti-inflflammatory effect of soothing Gan and invigorating Pi recipes.

[Expression and significance of Nrf2/ARE pathway ralated factors in the HepG2 cell model of steatosis].

OBJECTIVE: To explore a new method of establishing HepG2 cell model of steatosis and observe the expression and significance of nuclear factor erythroid-2p45-related factor 2(Nrf2)/antioxidative response element (ARE) pathway related factors in HepG2 cells of steatosis. METHODS: HepG2 cells were induced with DMEM containing 25% fetal bovine serum, 0.1% MCT/LCT Fat Emulsion and 0.1 mmol/L free fatty acid (FFA) at different stages and the control group cells were cultured with normal DMEM medium. After the cell models were successfully established, lipid droplets in cytoplasm were observed with Oil Red 0 staining, and the triglyceride (TG) accumulation in HepG2 cells were tested by biochemical assay. Intracellular reactive oxygen species (ROS) concentration were detected by flow cytometry. Nitric oxide (NO), superoxide dismutase(SOD), malonyldialdehyde(MDA) and glutathione peroxidase(GSH-Px) were tested by biological reagent kit, while the protein expression of nuclear factor erythroid-2p45-related factor 2(Nrf2), heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase-1(NQO1) were analyzed by Western blot. RESULTS: Compared with that in the control group, red cytoplasmic lipid droplets were visible in model group; TG,ROS, NO, MDA concentration (P < 0.05, P < 0.01) and the protein expression of Nrf2, HO-1 and NQO1 (P < 0.05, P < 0.01)were significantly higher in model group, while SOD, GSH-Px concentration reduced significantly (P < 0.01). CONCLUSION: The in vitro cell model of steatosis and oxidative stress was successfully established. The activation of Nrf2/ARE pathway related factors maybe relevant to the overreaction of oxidative stress in HepG2 cells of steatosis.

Validation and Comparison of Reference Genes for qPCR Normalization of Celery (Apium graveolens) at Different Development Stages.

A suitable reference gene is an important prerequisite for guarantying accurate and reliable results in qPCR analysis. Celery is one of the representative vegetable in Apiaceae and is widely cultivated and consumed in the world. However, no reports have been previously published concerning reference genes in celery. In this study, the expression stabilities of nine candidate reference genes in leaf blade and petiole at different development stages were evaluated using three statistics algorithms geNorm, NormFinder, and BestKeeper. Our results showed that TUB-B, TUB-A, and UBC were the most reference genes among all tested samples. GAPDH represented the maximum stability for most individual sample, while the UBQ displayed the minimum stability. To further validate the stability of reference genes, the expression pattern of AgAP2-2 was calculated by using the selected genes for normalization. In addition, the expression patterns of several development-related genes were studied using the selected reference gene. Our results will be beneficial for further studies on gene transcription in celery.

Effects of Soothing Liver and Invigorating Spleen Recipes on the IKKß-NF-κB Signaling Pathway in Kupffer Cells of Nonalcoholic Steatohepatitis Rats.

This study investigates the effect of soothing liver and invigorating spleen recipes on steatohepatitis examining the IKKß-NF-κB signaling pathway in KCs of NASH rats. SD male rats were randomly divided into 8 groups, and the NASH model was induced by a high-fat diet (HFD). After 26 weeks, liver tissue was examined in H&E stained sections and liver function was monitored biochemically. KCs were isolated by Seglen's method, with some modifications. The mRNA and protein expression of the IKKß-NF-κB signaling pathway components was examined by quantitative PCR and Western blotting. The results show that the high-fat diet induced NASH in the rats, and the soothing liver recipe and invigorating spleen recipe decreased the levels of TNF-α, IL-1, and IL-6 in KCs, as well as inhibiting the mRNA and protein expression of the IKKß-NF-κB signaling pathway components. In conclusion, the experiment indicated the importance of the IKKß-NF-κB signaling pathway in KCs for the anti-inflammatory effects of the soothing liver and invigorating spleen recipes.