Shenling Baizhu San ameliorates non-alcoholic fatty liver disease in mice by modulating gut microbiota and metabolites.

Purpose: The prevalence of non-alcoholic fatty liver disease (NAFLD) and its related mortality is increasing at an unprecedented rate. Traditional Chinese medicine (TCM) has been shown to offer potential for early prevention and treatment of NAFLD. The new mechanism of "Shenling Baizhu San" (SLBZS) is examined in this study for the prevention and treatment of NAFLD at the preclinical level. Methods: Male C57BL/6J mice were randomly divided into three groups: normal diet (ND), western diet + CCl4 injection (WDC), and SLBZS intervention (WDC + SLBZS). Body weights, energy intake, liver enzymes, pro-inflammatory factors, and steatosis were recorded in detail. Meanwhile, TPH1, 5-HT, HTR2A, and HTR2B were tested using qRT-PCR or ELISA. Dynamic changes in the gut microbiota and metabolites were further detected through the 16S rRNA gene and untargeted metabolomics. Results: SLBZS intervention for 6 weeks could reduce the serum and liver lipid profiles, glucose, and pro-inflammatory factors while improving insulin resistance and liver function indexes in the mice, thus alleviating NAFLD in mice. More importantly, significant changes were found in the intestinal TPH-1, 5-HT, liver 5-HT, and related receptors HTR2A and HTR2B. The 16S rRNA gene analysis suggested that SLBZS was able to modulate the disturbance of gut microbiota, remarkably increasing the relative abundance of probiotics (Bifidobacterium and Parvibacter) and inhibiting the growth of pro-inflammatory bacteria (Erysipelatoclostridium and Lachnoclostridium) in mice with NAFLD. Combined with metabolomics in positive- and negative-ion-mode analyses, approximately 50 common differential metabolites were selected via non-targeted metabolomics detection, which indicated that the targeting effect of SLBZS included lipid metabolites, bile acids (BAs), amino acids (AAs), and tryptophan metabolites. In particular, the lipid metabolites 15-OxEDE, vitamin D3, desoxycortone, and oleoyl ethanol amide were restored by SLBZS. Conclusion: Integrating the above results of multiple omics suggests that SLBZS ameliorates NAFLD via specific gut microbiota, gut-derived 5-HT, and related metabolites to decrease fat accumulation in the liver and inflammatory responses.

High-cholesterol diet promotes depression- and anxiety-like behaviors in mice by impact gut microbe and neuroinflammation.

Neuropsychiatric disorders, including anxiety and depression, are one of the most common mental illnesses worldwide. A growing body of evidence shows that there is a complex relationship between dietary patterns and mental health. In our study, C57BL/6J mice were divided into three groups: control diet group (CON, 10 % kcal fat), high-cholesterol diet model group (HCD, 42.0 % kcal fat + 1.25 % kcal Cholesterol), and chronic restraint stress group (CRS, 10 % kcal fat) which as a positive control group for the depression model. Six weeks later, depressive- and anxiety-like behavior were evaluated for using the OFT, SPT and TST. Glucose intolerance and liver fat were detected by IGTT and liver lipid kit. The expression of peripheral and central inflammation was detected by LEGEND plex kits. 5-HT (also named 5-hydroxytryptamine, 5-HT) and related receptors expression were monitored by ELISA, RT-PCR and Western blot. Meantime, gut microbe of stool samples was performed by 16S rRNA gene sequencing. Similar to CRS model, short-term HCD intervention induced anxiety and depression-like behavior behavioral abnormalities in mice. HCD consumption resulted in significantly increased body weight, liver fat (LDL-C, TC, TG), peripheral inflammation (IL-1ß, MCP-1, IL-17A) and neuroinflammation (MCP-1). The concentration of 5-HT increased in the hippocampus, meanwhile, the expression of 5-HT receptor HTR2A was distinct in different regions of the brain tissue. More importantly, we found that compared with the CON diet, HCD induced the decrease of intestinal flora diversity, especially the decrease the relative abundance of Akkermansia_muciniphila, which was statistically significant. Further, Pearson correlation analysis showed that Akkermansia_muciniphila was significantly negatively correlated with the concentration of MCP-1, IL-17A in serum and 5-HT in hippocampus. Therefore, we speculated that the disorder of neuroinflammation induced by HCD consumption promotes depression- and anxiety-like behaviors in mice through the gut microbe.

A Meta-Analysis of Sleep Disorders and Nonalcoholic Fatty Liver Disease: Potential Causality and Symptom Management.

Nonalcoholic fatty liver disease is a type of metabolic disease, and recent research indicates that it may be associated with sleep disorders. We conducted a meta-analysis of current studies to estimate the associations between nonalcoholic fatty liver disease and sleep situation, including sleep duration, daytime sleepiness, and sleep disorder. This study follows the checklist of the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA). Articles in the recent 10 years were searched from five databases. Eighteen articles, which met the eligibility criteria, were included in this meta-analysis. The results show that patients with nonalcoholic fatty liver disease have a shorter sleep duration and higher Epworth Sleepiness Scale score. Patients with short sleep duration (≤6 hours per night) or with obstructive sleep apnea have a higher risk of nonalcoholic fatty liver disease. In conclusion, there is a significant association between nonalcoholic fatty liver disease and sleep disorders in the included studies. In addition, patients with nonalcoholic fatty liver disease may have more severe daytime sleepiness and shorter sleep duration. More attention should be paid to the sleep situation of nonalcoholic fatty liver disease patients to potentially slow the disease progression.

Dietary and metabolic risk of neuropsychiatric disorders: insights from animal models.

Neuropsychiatric disorders are major causes of the global burden of diseases, frequently co-occurring with multiple co-morbidities, especially obesity, type 2 diabetes mellitus, non-alcoholic fatty liver disease and its various risk factors in the metabolic syndrome. While the determining factors of neuropsychiatric disorders are complex, recent studies have shown that there is a strong link between diet, metabolic state and neuropsychiatric disorders, including anxiety and depression. There is no doubt that rodent models are of great value for preclinical research. Therefore, this article focuses on a rodent model of chronic consumption of high-fat diet (HFD), and/or the addition of a certain amount of cholesterol or sugar, meanwhile, summarising the pattern of diet that induces anxiety/depressive-like behaviour and the underlying mechanism. We highlight how dietary and metabolic risk influence neuropsychiatric behaviour in animals. Changes in dietary patterns, especially HFD, can induce anxiety- or depression-like behaviours, which may vary by diet exposure period, sex, age, species and genetic background of the animals used. Furthermore, dietary patterns significantly aggravate anxiety/depression-like behaviour in animal models of neuropsychiatric disorders. The mechanisms by which diet induces anxiety/depressive-like behaviour may involve neuroinflammation, neurotransmitters/neuromodulators, neurotrophins and the gut-brain axis. Future research should be focused on elucidating the mechanism and identifying the contribution of diet and diet-induced metabolic risk to neuropsychiatric disorders, which can form the basis for future clinical dietary intervention strategies for neuropsychiatric disorders.

Gut Microbiome and Nonalcoholic Fatty Liver Disease (NAFLD): Implications for Nursing Care of Individuals Living With Chronic Metabolic Diseases.

At present, the incidence of nonalcoholic fatty liver disease (NAFLD) in adults is increasing year by year and at a younger age. Evidence-based healthcare has confirmed that NAFLD is closely related to obesity, cardiovascular disease, type 2 diabetes, metabolic syndrome, and other chronic metabolic diseases. Despite the growing prevalence of NAFLD, little is known about symptoms for patients at risk of NAFLD progression, thus preventing healthcare providers from intervening at an early stage. In addition, these symptoms usually cause problems for patients to cope with other chronic metabolic diseases. Symptoms may have a biological basis; especially as the changes of gut microbes may affect the symptoms of metabolic diseases. This article aims to describe the new role of gut microbes in the development of NAFLD, focusing on the potential relationship between gut microbes and symptoms of NAFLD, as well as the mechanism of action of the "gut-liver-brain" axis. This information can be useful in developing precise nursing interventions for NAFLD patients, restoring the "health" of gut microbes, and alleviating the symptom burden of chronic metabolic disease in NAFLD.

Berberine Ameliorates High-Fat Diet-Induced Non-Alcoholic Fatty Liver Disease in Rats via Activation of SIRT3/AMPK/ACC Pathway.

This study aimed to verify the effects of berberine (BBR) on the fat metabolism proteins involved in the sirtuin 3 (SIRT3)/adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC) pathway in the liver tissues of rats with high-fat diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD). Forty-eight rats were randomly divided into the normal control (NC) group, HFD group or BBR group, with 16 rats in each group. After 8 and 16 weeks of treatment, serum and liver samples were collected. Subsequently, body parameters, biochemical parameters and liver pathology were examined. The expression levels of proteins involved in the SIRT3/AMPK/ACC pathway in the liver were detected by Western blotting. After 8 and 16 weeks of a HFD, the successful establishment of rat models with different degrees of NAFLD was confirmed by hematoxylin and eosin (H&E) and Oil Red O staining. NAFLD rat models exhibited obesity and hyperlipidemia, and the protein expression levels of SIRT3, p-AMPK, p-ACC, and CPT-1A in the liver were significantly decreased compared to those in the NC group. The concurrent administration of BBR with the HFD effectively improved serum and liver lipid profiles and ameliorated liver injury. Furthermore, the protein expression levels of SIRT3, p-AMPK, p-ACC, and CPT-1A in the liver were significantly increased in the BBR group as compared with those in the HFD group. In conclusion, our data suggest that the mechanism by which BBR ameliorates HFD-induced hepatic steatosis may be related to the activation of the SIRT3/AMPK/ACC pathway in the liver.

Traditional Chinese Medicine and Intestinal Microbiota: A Complementary and Integrative Health Approach to Ameliorate Obesity-Related Diseases.

The incidence of obesity and obesity-related diseases, such as type 2 diabetes mellitus and nonalcoholic fatty liver disease, is increasing worldwide, which threatens quality of life and human health. "The toxins in the stool enter the body and blood and then cause a variety of diseases"; this quote illustrates that the Chinese ancients recognized the negative effects of harmful intestinal metabolites on the body. As the largest microecosystem in the human body, intestinal microbiota and their metabolites affect the nutrition, metabolism, and immune function of the host, which is an important pathogenic factor in obesity and obesity-related diseases. Herbal-based supplements are used for many years in the treatment of obesity, type 2 diabetes mellitus, and nonalcoholic fatty liver disease in China. Traditional herbal medicine contains fiber, polyphenols, and polysaccharides that exert prebiotics-like activities in the prevention and treatment of obesity-related diseases. This article provides a systematic mini-review of the literature concerning traditional Chinese medicine for modulation of the intestinal microbiota to ameliorate obesity, type 2 diabetes mellitus, and nonalcoholic fatty liver disease in China. Initially, the relationship between traditional Chinese medicine and intestinal microbiota was introduced, followed by specific research results on this relationship based on 25 original articles. Therefore, this mini-review will provide a complementary and integrative approach for the treatment of these obesity-related diseases.

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.

Oral Administration of Compound Probiotics Ameliorates HFD-Induced Gut Microbe Dysbiosis and Chronic Metabolic Inflammation via the G Protein-Coupled Receptor 43 in Non-alcoholic Fatty Liver Disease Rats.

The aim of this study was to investigate how the effects of compound probiotics modulate the gut microbiota, short-chain fatty acid (SCFA), body composition, serum and liver lipids, and inflammatory markers in non-alcoholic fatty liver disease (NAFLD) rats. Twenty-four male SD rats were randomly divided into 3 groups: normal control group (standard feed), high-fat diet (HFD) feeding group (83% standard feed + 10% lard oil + 1.5% cholesterol + 0.5% cholate + 5% sucrose), and compound probiotics intervention group (HFD + 0.6 g × kg-1 × d-1 compound probiotics). The microbial population was assessed by 16S rDNA amplification and sequence analysis. Body composition, serum and liver lipids, serum inflammatory markers, colonic SCFAs, and relative proteins were assessed. The results showed that compound probiotics significantly reduced body weight, visceral and total fat mass, and the levels of hepatic TC and TG and serum TG, FFA, ALT, LPS, IL-1ß, and IL-18 (P < 0.05). The proportions of TM7 phylum (0.06 vs 1.57%, P < 0.05) clearly increased, while that of Verrucomicrobia phylum (5.69 vs 2.61%, P < 0.05) clearly decreased. Compound probiotics also increased the representation of Ruminococcus genus (0.95 vs 1.83%, P < 0.05), while the proportion of Veillonella genus decreased (0.10 vs 0.03%, P < 0.05). The levels of colonic SCFAs and GPR43, NLRP3, ASC, and CASPASE-1 proteins also changed significantly (P < 0.05). Compound probiotics modulated gut microbiota, SCFAs, and their receptor GPR43 in NAFLD rats. These changes might inhibit lipid deposition and chronic metabolic inflammation in response to the insult of HFD.

Chaihu-Shugan-San Decoction Modulates Intestinal Microbe Dysbiosis and Alleviates Chronic Metabolic Inflammation in NAFLD Rats via the NLRP3 Inflammasome Pathway.

We evaluate the effects of the Chaihu-Shugan-San decoction on intestinal microbe dysbiosis and chronic metabolic inflammation via the NLRP3 pathway in NAFLD rats that were fed a high-fat diet. Twenty-four SD rats (male, six weeks old, 200 ± 20 g) were randomly divided into three groups: normal control group (NC group), high-fat diet-fed group (HFD group), and Chaihu-Shugan-San decoction intervention group (CH group). The NC group rats were given standard feed, the HFD group rats were all fed a high-fat diet (83% standard feed + 10% lard oil + 5% sucrose + 1.5% cholesterol + 0.5% cholate), and the CH group rats were given a HFD plus Chaihu-Shugan-San at 9.6 g•kg-1•d-1. Body composition, serum and liver lipids, inflammatory markers, intestinal microbial population, and the NLRP3 pathway-associated protein were assessed. The results showed that Chaihu-Shugan-San decoction significantly reduced body weight and total fat mass and the levels of serum LPS, TG, TNF-α, IL-1ß, and IL-18, as well as liver TC, TG, TNF-α, IL-1ß, and IL-18 (P < 0.05). The abundance of Enterobacteriaceae (0.375% versus 0.064%, P < 0.05), Staphylococcaceae families (0.049% versus 0.016%, P < 0.05) and Veillonella genus (0.096% versus 0.009%, P < 0.01) significantly decreased, whereas the abundance of Anaeroplasma genus (0.0005% versus 0.0178%, P < 0.01) significantly increased. The expression levels of NLRP3, ASC, and Caspase-1 were changed significantly (P < 0.05). In summary, the Chaihu-Shugan-San decoction modulated intestinal microbe dysbiosis, reduced fat accumulation, and alleviated inflammatory factor expression, which are all processes related to the NLRP3 inflammasome pathway in NAFLD rats.

Probiotic mixture of Lactobacillus and Bifidobacterium alleviates systemic adiposity and inflammation in non-alcoholic fatty liver disease rats through Gpr109a and the commensal metabolite butyrate.

AIMS: The study explored the systemic adiposity and inflammation through Gpr109a and the commensal metabolite butyrate during the treatment of non-alcoholic fatty liver disease rats with the probiotic mixture of Lactobacillus and Bifidobacterium for 16 weeks. METHODS: Fifteen male SD rats were randomly divided into three groups of five rats each: normal control group (basal feed), high-fat diet (HFD) feeding group (83% basal feed + 10% lard oil + 5% sucrose + 1.5% cholesterol + 0.5% cholate), and probiotic mixture intervention group (HFD + 0.6 g kg-1 day-1 probiotic mixture). Body composition, serum lipids, serum inflammatory markers, Gpr109a, and the commensal metabolite butyrate were assessed. RESULTS: Compared with HFD group, probiotic mixture significantly reduced body weight and the levels of serum FFA, TG, ALT, IL-1ß, and IL-18 (P < 0.05). The levels of Gpr109a and the commensal metabolite butyrate also changed significantly (P < 0.05). CONCLUSIONS: Probiotic mixture might inhibit systemic adiposity and inflammation through Gpr109a and the commensal metabolite butyrate in response to the insult of HFD.

The Potential Effect of Chinese Herbal Formula Hongqijiangzhi Fang in Improving NAFLD: Focusing on NLRP3 Inflammasome and Gut Microbiota.

The present study investigates the potential therapeutic mechanism underlying the effects of the Chinese herbal formula Hongqijiangzhi Fang (HJF) on nonalcoholic fatty liver disease (NAFLD) in rats. Male Sprague Dawley (SD) rats were randomly divided into 4 groups (n = 8): control group was fed a normal diet, three other groups were fed high-fat diets (HFD), and the two treatment groups were intragastrically given a compound probiotic or HJF during the molding time. After 16 w, related indices were detected. The results showed that HJF significantly reduced abdominal aorta serum cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), IL-1ß, and IL-18, portal venous serum lipopolysaccharide (LPS), and liver TC and TG levels in HFD-fed rats. HJF ameliorated hepatic steatosis in the liver and improved the intestinal barrier in HFD-fed rats. Activation of the NLRP3 inflammasome was reduced by HJF in HFD-fed rats. Additionally, the abundances of A. muciniphila (Verrucomicrobiaceae), F. rappini (Helicobacteraceae), and Enterobacteriaceae bacteria significantly decreased in HJF-treated HFD-fed rats. In conclusion, these result suggested that the Chinese herbal formula HJF reduced hepatic steatosis maybe through decreasing certain gut bacteria (such as Enterobacteriaceae bacteria and F. rappini), alleviating intestinal endotoxemia and reducing NLRP3 inflammasome activation.

Effects of extracts from soothing-liver and invigorating-spleen formulas on the injury induced by oxidative stress in the hepatocytes of rats with non-alcoholic fatty liver disease induced by high-fat diet.

OBJECTIVE: To investigate effects of the extracts from soothing-liver and invigorating-spleen formulas on the injury due to oxidative stress, mediated by the Nuclear factor-like 2 (Nrf2)-Antioxidant response element (ARE) pathway, in the hepatocytes of rats with non-alcoholic fatty liver disease (NAFLD) induced by high-fat diet. METHODS: Soothing-liver and invigorating-spleen formula mixtures were prepared for five groups: normal, model, soothing-liver formula group (SLG), invigorating-spleen formula group (ISG), integrated formula group (IG). The rat model of NAFLD was induced by feeding rats a high-fat diet (HFD). After 16 weeks, the hepatic tissue was examined following Haematoxylin-Eosin (H&E) staining and with Transmission electron microscopy (TEM). Levels of hepatic lipids, serum lipids and hepatic functions were measured using a biochemical analyser. Hepatocytes were isolated from the livers of rats and were identified by cellar immunohistochemistry, cellular immunofluorescence and flow cytometry. The expression levels of Nrf2, Kelch-like epichlorohydrin-associated protein 1 (Keap-1), haeme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) mRNAs were assessed by real-time fluorescence quantitative PCR. Nrf2, Keap-1, HO-1 and NQO1 proteins were measured by Western blotting. Finally, the levels of oxidative stress factors Superoxide Dismutase (SOD), malonaldehyde (MDA) and Glutathione peroxidase (GSH-Px) in hepatocytes were measured by WST-1, TBA and colorimetry. RESULTS: The H & E and TEM results showed that the NAFLD model rats successfully reproduced typical pathogenetic and histopathological features. The liver function and levels of hepatic lipids and serum lipids from the model rats were dramatically increased. Compared with the model group, the levels of hepatic lipids, serum lipids and hepatic function in the treatment groups were ameliorated to different degrees. The yields of purified hepatocytes in each rat were 4-5 ¡Á 108. The viability of the isolated hepatocytes was higher than 95%, with a purity over 93.2%. Cellular immunohistochemistry analysis showed that the hepatocytes were brown, while in the cellular immunofluorescence analysis, the hepatocytes showed green fluorescence. The expression levels of Nrf2, Keap-1, HO-1 and NQO1 mRNA and protein in the hepatocytes were significantly higher in the model group than in the normal group (P < 0.05, P < 0.01). Compared with the model group, the expression of Nrf2, Keap-1, HO-1 and NQO-1 mRNAs and proteins in all treatment groups increased, especially in the IG (P < 0.01). CONCLUSION: The extracts from soothing-liver and invigorating-spleen formulas may protect the liver against the injury induced by oxidative stress in hepatocytes by influencing the Nrf2-ARE pathway, which may be the mechanism having the potential for prevention and treatment of NAFLD.

[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.

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.

[Intervention of berberine on lipid deposition in liver cells of non-alcoholic fatty liver disease rats induced by high fat diet].

OBJECTIVE: To explore the effect of berberine on lipid metabolism disorder and lipid deposition in liver cells of non-alcoholic fatty liver disease (NAFLD) rats induced by high fat diet. METHODS: After one week adaptable feeding, 45 SPF level male SD rats were randomly divided into 3 groups, the normal control group, the model group, and the berberine group, 15 in each group. Except those in the normal control group, all rats were fed with high fat diet to prepare NAFLD model. As for rats in the berberine group, Berberine Hydrochloride was administered by gastrogavage. HE staining and oil red O staining were performed to identify the model after 8 weeks. Hepatocytes were isolated, and their activities and purities were tested by Typan blue staining and flow cytometry (FCM). Serum levels of TC, TG, HDL-C, and LDL-C were detected using automatic biochemical analyzer. mRNA expression levels of LXRα and FAS in liver cells were analyzed by Real-time quantitative polymerase chain reaction (PCR). Protein levels of LXRα and FAS in liver cells were examined by Western blot. RESULTS: The NAFLD rat model was successfully established by high fat diet. The yields of purified liver cells in each rat were (6.0-7.5) x 10(8). The viability of isolated liver cells with purity over 90% (tested by FCM analysis) was higher than 95%. Compared with the normal control group,the expression of LXRα and FAS at mRNA and protein levels was higher in the model group (P < 0.01). Compared with the model group, the expression of LXRα and FAS at mRNA and protein levels was obviously down-regulated in the berberine group (P < 0.01). CONCLUSIONS: LXRα/FAS signaling pathway was one of important signaling pathways of NAFLD lipid metabolism disorders. Berberine could recover hepatocyte fatty deposits in NAFLD rats by adjusting the LXR/FAS signaling pathway of hepatocytes, which might be one of important mechanisms for fighting against NAFLD.

Effects of Chaihu-Shugan-San and Shen-Ling-Bai-Zhu-San on p38 MAPK Pathway in Kupffer Cells of Nonalcoholic Steatohepatitis.

This study aimed to investigate the effects of Chaihu-Shugan-San (CSS), Shen-Ling-Bai-Zhu-San (SLBZS), and integrated recipe of the above two recipes on inflammatory markers and proteins involved in p38 MAPK pathway in Kupffer cells of NASH rats induced by high fat diet (HFD). Rats were administered at low or high dose of CSS, SLBZS, and integrated recipe except normal group and model group for 16 weeks. The levels of hepatic lipid, TNF- α , IL-1, and IL-6 in liver tissues were measured. Kupffer cells were isolated from livers to evaluate expressions of TLR4, p-p38 MAPK, and p38 MAPK by Western blotting. The results showed that the NASH model rats successfully reproduced typical pathogenetic and histopathological features. Levels of hepatic lipid and liver tissues inflammatory factors in high-dose SLBZS group and integrated recipe group were all lower than that of model group decreased observably. Expressions of TLR4, p-p38 MAPK, and p38 MAPK in Kupffer cells were decreased in all treatment groups, but there was no significant difference between treatment groups. The high-dose SLBZS group had the lowest expression levels of TLR4, and the most visible downtrend in the expression levels of p-p38 MAPK and p38 MAPK was found in the high-dose integrated recipe group. The ratio of p-p38 MAPK to total p38 MAPK protein was obviously increased in all treatment groups. Therefore, our study showed that the activation of p38 MAPK pathway in Kupffer cells might be related to the release of inflammatory factors such as TNF- α , IL-1, and IL-6 in NASH rats. High dose of SLBZS and integrated recipe might work as a significant anti-inflammatory effect in Kupffer cells of NASH rats induced by HFD through suppression of p38 MAPK pathway. It indicated that p38 MAPK pathway may be the possible effective target for the recipes.

[Effects of soothing liver and invigorating spleen recipes on LPS-induced hepatocytes injury of rats and TLR4/p38MAPK signal pathway].

OBJECTIVE: To explore the effects of soothing liver and invigorating spleen recipes on lipopolysaccharide(LPS) induced hepatocyte inflammation of rats and TLR4/p38MAPK signal pathway. METHOD: The hepatocytes of SD rats were cultured and identified in vitro. The medicated serum of soothing liver and invigorating spleen recipes was prepared. The hepatocytes were treated with soothing liver and invigorating spleen recipes. Then Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) expression in cultural supernatants were assayed by ELISA. The expressions of Toll-Like 4 (TLR4), p38 mitogen activated protein kinases (p38MAPK) and p-p38 mitogen-activated protein kinase (p-p38MAPK) were detected by Western blot. RESULT: The rat medicated serum of soothing liver and invigorating spleen recipes was extracted for 2-3 mL. The purified rat hepatocytes were 1.5 x 10(8)-2.0 x 10(8). The cell viability was above 95% detected by Typan blue staining. The hepatocytes were identified by immumofluorescence assay. The detection of hepatocyte cultural supernatants: compared with that of the control group, IL-6 and TNF-α expression were increased in the LPS group (P < 0.01). While compared with that of the LPS group, the expressions of IL-6 and TNF-α were decreased after soothing liver and invigorating spleen recipes intervention (P < 0.01). The detection of hepatocyte proteins: compared with that of the control group, the protein expressions of p38MAPK, p-p38MAPK and TLR4 were all increased significantly in the LPS group (P < 0.01). Compared with that of the LPS group, the protein expressions of p38MAPK was decreased significantly in SB239063 group and it was also decreased in the soothing liver and invigorating spleen recipes group, but with no significant difference. Compared with that of the LPS group, p38MAPK expression was reduced significantly in the soothing liver and invigorating spleen recipes group and the SB239063 (p38MAPK pathway inhibitor) group (P < 0.01). TLR4 protein expression was decreased markedly in the soothing liver and invigorating spleen recipes group (P < 0.01) but had no difference between the SB239063 group and the LPS group. CONCLUSION: The soothing liver and invigorating spleen recipes may regulate hepatocyte inflammatory injury of rats through TLR4/p38MAPK signaling pathway.

R280T mutation of p53 gene promotes proliferation of human glioma cells through GSK-3ß/PTEN pathway.

p53 mutation is associated with "gain-of-function" capabilities of human cancers. We aim to identify p53 mutations in human glioma cells and to explore the potential mechanism for mutant p53-promoted cellular growth. Whole genomic DNA was isolated from SWO-38, a human glioma cell line and amplified for the region of exons 5, 6, and 8 in p53 gene using polymerase chain reaction (PCR). By means of direct sequencing of PCR products and alignment analysis using BLAST database, a mutation of G to C transition at codon 280 of p53 exon 8 (AGA→ACA), i.e. R280T was detected in SWO-38 cells. Knockdown of R280T mutant p53 by RNA interference inhibited the GSK-3ß/PTEN associated cell proliferation, and PI3K/Akt but not Wnt/ß-catenin signaling pathway was involved in this process. Furthermore, depletion or overexpression of PTEN alone did not affect cell proliferation and cell cycle, implicating the impairment of PTEN function in SWO-38 cells. However, knockdown of both PTEN and p53 mutation could significantly rescue the p53 depletion-mediated growth inhibition, suggesting that the R280T mutation in glioma may promote the proliferation through an underlying mechanism related to PTEN. Our observations indicate that the R280T mutation of p53 regulates the proliferation of human glioma cells related to the GSK-3ß/PTEN pathway. These findings provide valuable insights for better understanding the molecular mechanism of uncontrolled growth of glioma cells.

Sprouting of nervous fibers and upregulation of C-X-C chemokine receptor type 4 expression in hippocampal formation of rats with enhanced spatial learning and memory.

Hippocampal mossy fiber sprouting following training in the Morris water maze (MWM) is associated with spatial learning, memory and neural plasticity. The C-X-C chemokine receptor type 4 (CXCR4) is the main receptor for stromal cell-derived factor-1 (SDF-1), which is a chemokine that can regulate axonal elongation. This study aimed to investigate the relationship between the morphological plasticity of hippocampal formation and CXCR4 expression. A model of spatial learning and memory was established in rats by training using the MWM. Mossy fiber sprouting in the striatum oriens of the CA3 area of the hippocampus was found in trained rats by Neo-Timm's method. As shown by immunohistochemistry, the CXCR4 immunopositive neurons were distributed in all layers and areas of hippocampal formation. There were no differences among groups regarding the distribution or shape of the immunopositive neurons. However, the immunoreactive staining intensity was increased in trained rats as compared with the control rats. Both CXCR4 gene transcription and translation were significantly upregulated in the trained group as compared with the control group (P < 0.01). Morphological plasticity in the form of axonal sprouting in the hippocampal formation can be induced by enhanced spatial learning and memory activity, and CXCR4 mRNA and protein expression is upregulated, indicating a positive correlation between CXCR4 expression and axonal sprouting.