Non-invasive diagnosis of non-alcoholic fatty liver disease: Current status and future perspective.

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease throughout the world. Hepatocellular carcinoma (HCC) and liver cirrhosis can result from nonalcoholic steatohepatitis (NASH), the severe stage of NAFLD progression. By some estimates, NAFLD affects almost one-third of the world's population, which is completely new and serious public health issue. Unfortunately, NAFLD is diagnosed by exclusion, and the gold standard for identifying NAFLD/NASH and reliably measuring liver fibrosis remains liver biopsy, which is an invasive, costly, time-consuming procedure and involves variable inter-observer diagnosis. With the progress of omics and imaging techniques, numerous non-invasive serological assays have been generated and developed. On the basis of these developments, non-invasive biomarkers and imaging techniques have been combined to increase diagnostic accuracy. This review provides information for the diagnosis and assessment of NAFLD/NASH in clinical practice going forward and may assist the clinician in making an early and accurate diagnosis and in proposing a cost-effective patient surveillance. We discuss newly identified and validated non-invasive diagnostic methods from biopsy-confirmed NAFLD patient studies and their implementation in clinical practice, encompassing NAFLD/NASH diagnosis and differentiation, fibrosis assessment, and disease progression monitoring. A series of tests, including 20-carboxy arachidonic acid (20-COOH AA) and 13,14-dihydro-15-keto prostaglandin D2 (dhk PGD2), were found to be potentially the most accurate non-invasive tests for diagnosing NAFLD. Additionally, the Three-dimensional magnetic resonance imaging (3D-MRE), combination of the FM-fibro index and Liver stiffness measurement (FM-fibro LSM index) and the machine learning algorithm (MLA) tests are more accurate than other tests in assessing liver fibrosis. However, it is essential to use bigger cohort studies to corroborate a number of non-invasive diagnostic tests with extremely elevated diagnostic values.

Efficacy and Mechanism of a Chinese Classic Prescription of Yueju in Treating Nonalcoholic Steatohepatitis and Protecting Hepatocytes from Apoptosis.

Yueju, a famous classic Chinese prescription, has been extensively used in treating depression syndromes for hundreds of years. Recent studies have reported that Yueju showed good effects in treating metabolic diseases, such as obesity and hyperlipidemia. Nonalcoholic steatohepatitis (NASH), which leads to cirrhosis and severe cardiovascular diseases, is closely linked to obesity and abnormal lipid metabolism. In this study, Yueju could decrease the levels of alanine aminotransferase, aspartate transaminase, triglyceride, cholesterol, and low-density lipoprotein-C but increase the high-density lipoprotein-C in the serum of the NASH rat model induced by high-fat and high-cholesterol diet. Yueju could alleviate hepatosteatosis by increasing the phosphorylation of acetyl-CoA carboxylase and inhibiting the expression of fatty acid synthase and stearoyl-CoA desaturase 1. Yueju downregulated the expression of α-smooth muscle actin and collagen type 1A1, ameliorating the liver fibrilization. Yueju could also protect the hepatocytes from apoptosis by upregulating antiapoptosis protein Bcl-2 and X-linked inhibitor of apoptosis protein and downregulating apoptotic proteins Bax and cleaved poly ADP-ribose polymerase. Thus, Yueju could improve liver function, regulate lipid metabolism, alleviate hepatosteatosis and fibrosis, and protect hepatocytes from apoptosis against NASH. Yueju may be used as an alternative effective medicine for NASH treatment.

Intestinal Microecology: An Important Target for Chinese Medicine Treatment of Non-alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) has become one of the most common chronic liver diseases worldwide, causing serious economic and medical burdens. Currently, Chinese medicine (CM) has become an important means in treating NAFLD in China. Intestinal microecology (IM) is an important part of the internal environment in the human body and is involved in the occurrence and development of NAFLD. In this paper, the authors systematically discuss the significance of IM in the pathogenesis of NAFLD and the current status of research on the CM treatment of NAFLD via IM regulation. In combination with our own research practice, we propose that IM is an important target for the treatment of NAFLD with CM and formulate plans for future research to target limitations existing in current studies.

Salidroside and Curcumin Formula Prevents Liver Injury in Nonalcoholic Fatty Liver Disease in Rats.

INTRODUCTION AND AIM: Salidroside and curcumin (SC) formula could alleviate lipid deposition in high fat diet-induced nonalcoholic fatty liver disease (NAFLD). However, the mechanisms are still unknown, and the magnitude of potential therapeutic benefit remains understudied. MATERIAL AND METHODS: The rats were treated with high fat diet for 14 weeks to induce NAFLD. The experiment was divided into control, model (NAFLD), SC formula and rosiglitazone groups (n = 7 in each group). Hematoxylin-eosin (H&E) staining was applied to detect liver morphological changes. Biochemical, metabolic indices and inflammation factors in liver tissue and serum were detected. Additionally, the activities of related enzymes were detected by enzyme-linked immunosorbent assay. RESULTS: In the established rat model, typical lipid deposition and liver steatosis were observed. Liver triglyceride, free fatty acids, sera alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transferase, fasting insulin, fasting blood glucose and homeostasis model assessment of insulin resistance were elevated in model group. Liver malondialdehyde was significantly elevated, while superoxide dismutase was significantly decreased in model group, compared with control. Moreover, tumor necrosis factor-α and Interleukin-1 were significantly produced in model group, compared with control. As a mechanism, high fat diet decreased tissue AMP-activated protein kinase (AMPK), phosphorylated AMPK, carnitine palmitoyltransferase 1 and increased inacetyl-CoA carboxylase (ACCase), phosphorylated ACCase. Importantly, these abnormal changes caused by high fat diet were reduced by SC formula administration. CONCLUSION: SC formula could ameliorate the injury caused by high fat diet. The effect was likely mediated via its influence on insulin resistance, lipid peroxidation injury and AMPK signaling pathway.

[Effects of berberine on serum inflammatory factors and carotid atherosclerotic plaques in patients with acute cerebral ischemic stroke].

This study aims to analyze the effect of berberine on serum inflammatory factors and carotid atherosclerotic plaques in ppatients with acute cerebral ischemic stroke(AIS). In the study, 120 patients with AIS were randomly divided into berberine group(n=60) and general group (n=60). The 60 cases in the general group were provided with general therapy according to the latest guidelines of diagnosis and treatment of AIS. The berberine group received berberine 300 mg(tid) in addition to the therapy of the general group. The levels of serum inflammatory factors, the nerve function defect grades and the indexes of carotid atherosclerosis plaques [including the total plaque area(TPA), intima-media thickness(IMT) and the number of unstable carotid atherosclerotic plaques] were measured and compared. The results indicated that the levels of serum inflammatory factors, the NIHSS(national institute of health stroke scales) cores and the indexes of carotid atherosclerosis plaques were not significantly different between the berberine groups of general group, with positive correlation between serum inflammatory factors and NIHSS scores(P<0.05). The levels of serum inflammatory factors and NIHSS scores of the berberine groups on 14 d were significantly lower than those on 1 d(P<0.05). The levels of serum inflammatory factors and NIHSS scores of the berberine group on 14 d were significantly lower than those of the general group(P<0.05). The TPA and the number of unstable carotid atherosclerotic plaques of the berberine groups on 90 d were significantly lower than those of general group, with significant differences(P<0.05). The IMT showed a downward trend, but with significant difference.The mRS(modified rankin scale) scores of the berberine group on 90 d were significantly lower, with a higher rate of short-term favorable prognosis (P<0.05). There was no significant difference in the incidence of adverse reactions between the two groups. This study showed that berberine in addition to the general therapy can significantly lower the levels of serum MIF and IL-6, reduce the degree of carotid atherosclerosis to some extent and improve neurological impairment and the prognosis of patients with AIS.

[The relationship of ultrastructure and function of hypothalamus-pituitary-adrenal axis in early stage of sepsis in rats].

OBJECTIVE: To observe the changes in ultrastructure and function of hypothalamic-pituitary-adrenal axis (HPAA), and to approach the relationship between them in early stage of sepsis in rats. METHODS: Thirty male Sprague-Dawley (SD) rats were randomly divided into normal control group, sham group, sepsis group. The sepsis model was reproduced by cecal ligation and puncture (CLP). The rats were sacrificed after collection of blood at 6 hours after CLP, and the levels of adrenocorticotropic hormone (ACTH) and corticosterone (CORT) in the plasma, and the corticotropin release hormone (CRH) in the tissue of hypothalamus were detected. The histopathological changes in HPAA were observed with transmission electron microscopy. RESULTS: The levels of ACTH and CORT in plasma, and the CRH in hypothalamus tissue of sepsis group were increased in the early stage of sepsis compared with the normal control group or sham group [ACTH (pmol/L): 5.78±0.36 vs. 1.94±0.31, 2.51±0.10; CORT (nmol/L): 88.48±4.47 vs. 22.02±1.62, 34.20±2.51; CRH (µg/L): 101.92±6.61 vs. 61.65±6.05, 66.65±4.03, P<0.05 or P<0.01]. The changes in ultrastructure of the hypothalamus, pituitary and adrenal were also found. In sepsis group, the ultrastructure of hypothalamus was as follows. Rough endoplasmic reticulum expansion and degranulation of rough endoplasmic reticulum, and swelling of Golgi complex were found. A large number of endocrine granules could be seen in ATCH cells in the pituitary with depletion of adrenal lipid droplets. CONCLUSION: In septic rats, the HPAA was excessively activated, and ACTH and CORT in plasma, and CRH in hypothalamus were significantly increased in early stage of sepsis. The changes in ultrastructure of HPAA were obvious, and the change in function was closely related to the ultrastructural changes.

[Effect of hepatitis B virus X gene on the expression of spastic paraplegia 21].

OBJECTIVE: To investigate the effect of hepatitis B virus(HBV) X gene on the expression of SPG21. METHODS: The expressions of SPG21 mRNA and protein in HepG2 and HepG2.2.15 cells were tested by RT-PCR and western blot. HepG2 cells were co-transfected with reporter plasmid pGL3-SPG21 and plasmids carrying individual genes of HBV, the luciferase activity was measured and the expressions of SPG21 were detected by RT-PCR and western blot. RESULTS: The expressions of SPG21 mRNA and protein were higher in HepG2.2.15 cells than in HepG2 cells (0.36+/-0.06 vs 0.21+/-0.05, P value is less than 0.05). The activity of SPG21 in HepG2 cells transfected with pCMV-X was higher (875+/-27 vs 67+/-12, P value is less than 0.01) as compared to blank control group (transfected with pCMV-tag2B). HBV X gene enhanced SPG21 gene promoter activity, SPG21 mRNA expression and SPG21 protein production in HepG2 cells in a dose-dependent manner. CONCLUSION: HBV X gene can specially activate SPG21 expression.

[The role of adiponectin and adiponectin receptor 2 in the pathology of fatty liver].

OBJECTIVE: 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. METHODS: 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. RESULTS: (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. CONCLUSION: (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.

[Effects of Qushi Huayu Decoction in prevention and treatment of fatty liver in rats based on adiponectin-free fatty acid pathway].

OBJECTIVE: To explore the effects of Qushi Huayu Decoction (QSHYD), a compound traditional Chinese herbal medicine, in prevention and treatment of non-alcoholic fatty liver disease (NAFLD) in rats. METHODS: Forty Wistar male rats were used to establish the NAFLD model by subcutaneous injection of carbon tetrachloride (CCl(4)) for 4 weeks (twice weekly) along with high-fat and low-protein diet for 2 weeks. After two-week administration, the rats were randomly divided into four groups: untreated group, high-dose QSHYD group, medium-dose QSHYD group and low-dose QSHYD group. Another six rats were used as normal control. After 2-week treatment, the following indexes were detected: (1) liver pathology; (2) contents of serum adiponectin (ADP) and liver triglyceride (TG); (3) concentrations of liver FFA, adiponectin receptor 2 (AdipoR2), malonyl-coenzyme A (malony1-CoA), AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACCase), fatty acid synthase (FAS) and carnitine palmitoyl transferase-1 (CPT-1). RESULTS: Compared with the normal group, there were physiological changes associated with hepatic steatosis and inflammation in liver tissues in the untreated group as observed by oil red O staining and HE staining. The TG, FFA, malony1-CoA, FAS, and ACCase concentrations in liver tissues in the untreated group were elevated significantly. While the contents of ADP in serum and AdipoR2, CPT-1 and AMPK in liver tissues in the untreated group were decreased markedly. The pathological damages in each QSHYD-treated group were significantly less than those in the untreated group. The TG and FFA contents in liver tissues in each QSHYD-treated group were significantly decreased. The FAS, ACCase and malonyl-CoA concentrations in liver tissues of the high QSHYD-treated group were reduced markedly as compared with the untreated group. High- and medium-dose of QSHYD could significantly increase ADP content in serum and AMPK, CPT-1 and AdipoR2 contents in liver tissues. CONCLUSION: QSHYD can affect the ADP-FFA pathway by increasing the content of serum ADP, which may be one of its important mechanisms in preventing and treating NAFLD in rats.

[Effect of qushl huayu decoction on high-fat diet induced hepatic lipid deposition in rate].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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). CONCLUSION: QHD shows an evident fatty liver antagonizing effect in rats induced by high-fat diet in a dose-dependent manner.

[Effects of Qushi Huayu Decoction on cathepsin B and tumor necrosis factor-alpha expression in rats with non-alcoholic steatohepatitis].

OBJECTIVE: To study the mechanism of Qushi Huayu Decoction (QHD), a compound of traditional Chinese herbal medicine, in prevention and treatment of non-alcoholic steatohepatitis (NASH). METHODS: Thirty-five Wistar male rats were randomly divided into normal group, untreated group, QHD group and Ganle (diisopropylamine dichloroacetate) group. The rats except those in normal group were subcutaneously injected with carbon tetrachloride (CCl(4)) for 4 weeks (twice per week) and simultaneously fed with high-fat and low-protein diet for 2 weeks to induce NASH. Then, the rats were administrated with QHD, Ganle, or distilled water for 2 weeks, respectively. After harvest, alanine aminotransferase (ALT) activity and tumor necrosis factor-alpha (TNF-alpha) content in serum as well as triglyceride (TG) and free fatty acid (FFA) in liver tissue were evaluated, and relativity analysis among these parameters was performed. Cathepsin B (Ctsb), phospho-inhibitor kappa B (P-IkappaB), TNF-alpha protein expressions in liver tissue were assayed with western-blot. The expression and distribution of ctsb in liver tissue were observed with immunohistochemical method. RESULTS: The contents of TG, FFA and activity of ALT were significantly decreased in QHD group. While in the Ganle group, only the activity of ALT in serum was decreased significantly. Expressions of Ctsb, P-IkappaB and TNF-alpha proteins in liver tissues and serum TNF-alpha level were all enhanced in untreated group which, however, were significantly inhibited in the QHD group. And as expected, there were significant relativities among contents of TG in liver tissues and the content of FFA in liver tissue and activity of ALT in serum, content of TNF-alpha in serum and content of FFA in liver tissue and activity of ALT in serum. CONCLUSION: The inhibiting effects of QHD on fat deposition and inflammation in liver are related with its inhibition on the "FFA-Ctsb-TNF-alpha" pathway of lipo-toxicity.

Effect of JIANPI HUOXUE decoction on inflammatory cytokine secretion pathway in rat liver with lipopolysaccharide challenge.

AIM: To evaluate the effect of Chinese traditional medicinal prescription, JIANPI HUOXUE decoction (JHD) on cytokine secretion pathway in rat liver induced by lipopolysaccharide (LPS). METHODS: Twenty-four male SD rats were divided into normal group (n = 4), model group (n = 10) and JHD group (n = 10) randomly. Rats in model group and JHD group were administrated with normal saline or JHD via gastrogavage respectively twice a day for 3 d. One hour after the last administration, rats were injected with LPS via tail vein, 50 mug/kg. Simultaneously, rats in normal group were injected with equivalent normal saline. After LPS stimulation for 1.5 h, serum and liver tissue were collected. Pathological change of liver tissues was observed through hematoxylin-eosin (H.E.) staining. Tumor necrosis factor alpha (TNF-alpha) in serum were assayed by enzyme linked immunosorbent assay (ELISA). The protein expression of TNF-alpha, phosphorylated inhibit-kappaB (p-IkappaB) and CD68 in liver were assayed by Western blot. The distribution of CD68 protein in liver was observed through immunohistochemical staining. The mRNA expression of TNF-alpha, interleukin-6 (IL-6), CD14, toll-like receptor 2 (TLR2) and TLR4 in liver were assayed by real-time RT-PCR. RESULTS: Predominant microvesicular change, hepatocyte tumefaction and cytoplasm dilution were observed in liver tissues after LPS administration as well as obvious CD68 positive staining in hepatic sinusoidal. After LPS stimulation, serum TNF-alpha (31.35 +/- 6.06 vs 12225.40 +/- 9007.03, P < 0.05), protein expression of CD68 (1.13 +/- 0.49 vs 3.36 +/- 1.69, P < 0.05), p-IkappaB (0.01 +/- 0.01 vs 2.07 +/- 0.83, P < 0.01) and TNF-alpha (0.27 +/- 0.13 vs 1.29 +/- 0.37, P < 0.01) in liver and mRNA expression of TNF-alpha (1.96 +/- 2.23 vs 21.45 +/- 6.00, P < 0.01), IL-6 (4.80 +/- 6.42 vs 193.50 +/- 36.36, P < 0.01) and TLR2 (1.44 +/- 0.62 vs 4.16 +/- 0.08, P < 0.01) in liver were also increased significantly. These pathological changes were all improved in JHD group. On the other hand, TLR4 mRNA (1.22 +/- 0.30 vs 0.50 +/- 0.15, P < 0.05) was down-regulated and CD14 mRNA increased but not significantly after LPS stimulation. CONCLUSION: JHD can inhibit cytokine secretion pathway induced by LPS in rat liver, which is probably associated with its regulation on CD68, p-IkappaB and endotoxin receptor TLR2.