Comparing hepatic steatosis distribution patterns between non-alcoholic fatty liver disease and fatty liver disease with chronic hepatitis B by second-harmonic generation/two-photon excited fluorescence method.

INTRODUCTION AND OBJECTIVES: Hepatitis B virus (HBV) might be an etiological factor modulating fat distribution in steatotic livers. We aim to compare hepatic steatosis distribution patterns between NAFLD and FL&CHB patients with second-harmonic generation (SHG)/two-photon excited fluorescence (TPEF) method. PATIENTS AND METHODS: 42 patients with NAFLD, 46 with FL&CHB and 55 without steatosis were enrolled in the study. Overall and regional steatosis in liver sections were quantified by SHG/TPEF method. The accuracy of which was validated by pathologist evaluation and magnetic resonance spectroscopy (MRS). Difference in degree of overall and regional steatosis between NAFLD and FL&CHB groups was analyzed by Mann-Whitney U test. Multivariable linear regression analysis was used to model factors contributing to steatosis distribution. RESULTS: The hepatic steatosis measured by SHG/TPEF method was highly correlated with pathologist grading (r=0.83, p<0.001) and MRS measurement (r=0.82, p<0.001). The level of overall steatosis in FL&CHB group is significantly lower than that in NAFLD group (p<0.001). In NAFLD group, periportal region has significantly lower steatosis percentage than lobule region and overall region (p<0.001); while in FL&CHB group there is no difference among regions. The ratio of steatosis at periportal region to lobule region is significantly higher in FL&CHB group than that in NAFLD group (p<0.05). Multivariable linear regression analysis shows that HBV infection is the major contributing factor (ß=0.322, p<0.01). CONCLUSIONS: SHG/TPEF method is an accurate and objective method in hepatic steatosis quantification. By quantifying steatosis in different histological regions, we found steatosis distribution patterns are different between FL&CHB and NAFLD patients.

Chronic moderate alcohol consumption relieves high-fat high-cholesterol diet-induced liver fibrosis in a rat model.

Nonalcoholic fatty liver disease is a worldwide health issue and chronic alcohol consumption may have different effects on this disease. This study explored the role of chronic moderate alcohol consumption on high-fat high-cholesterol (HFHC) diet-induced liver fibrosis in a rodent model. Male Sprague-Dawley rats were divided into five groups: standard chow group, standard chow plus Er Guo Tou (EGT, a Chinese spirit made from fermented cereals) group, HFHC group, HFHC plus EGT group, and HFHC plus pure ethanol (EtOH) group. Rats were fed standard chow or HFHC chow for 12 weeks. EGT or pure ethanol was administrated at a daily dose of 4 g/kg body weight via intra-gastric gavage from week 4. At the end of week 12, hematoxylin and eosin staining, Sirius red and immunohistochemistry of liver sections were examined. The hepatic expression of F4/80, TNF-α, IL-1ß, IL-6, CXCL1, CXCL2, α-SMA, Collagen, TGF-ß, MMP2, MMP9, and TIMP1 was calculated. Both moderate EGT and pure ethanol did not increase plasma endotoxin in the portal vein comparing with the FHFC group. EGT and pure ethanol did not improve hepatic inflammation, but ameliorated liver fibrosis in histology. Moderate EGT and pure ethanol ameliorated HFHC diet-induced activation of Kupffer cells and hepatic stellate cells. In conclusion, chronic moderate EGT and pure ethanol could ameliorate HFHC diet-induced liver fibrosis.

Relationship of serum uric acid level with non-alcoholic fatty liver disease and its inflammation progression in non-obese adults.

AIM: This study aimed to evaluate the relationship between serum uric acid (SUA) level and non-alcoholic fatty liver disease (NAFLD) in non-obese adults. METHODS: A cross-sectional study was carried out among 4098 adults, including 1936 non-obese and 2162 obese individuals. An additional 93 non-obese adults with biopsy-proven NAFLD were also included. RESULTS: The overall prevalence of NAFLD was 39.51% in the study group, and 14.88% in non-obese adults. The NAFLD patients had significantly higher SUA levels than controls in both men and women. The non-obese group had a higher NAFLD risk with increased SUA levels than the obese group, with odd ratios (95% confidence interval) of 2.559 (1.870-3.503) and 1.692 (1.371-2.087), respectively. In 93 non-obese adults with biopsy-proven NAFLD, SUA levels were significantly higher in those with non-alcoholic steatohepatitis. The prevalence of non-alcoholic steatohepatitis and lobule inflammation tended to increase to 57.58% and 66.67% as the SUA level increased to the fourth quartile. Subjects with hyperuricemia had significantly higher NAFLD activity scores and more serious lobule inflammation than the normal group. CONCLUSION: Non-obese adults have higher NAFLD risk with increased SUA levels than obese individuals, and the inflammation progression of NAFLD is associated with increased SUA level in non-obese subjects.

Increased ratio of neutrophil elastase to α1-antitrypsin is closely associated with liver inflammation in patients with nonalcoholic steatohepatitis.

An imbalance between neutrophil elastase (NE) and its inhibitor α1-antitrypsin (A1 AT) is known to contribute to the development of obesity-related inflammation. This study aimed to investigate the role of the NE-A1 AT system in the histological progression of non-alcoholic fatty liver disease (NAFLD), and to evaluate the ability of it to predict nonalcoholic steatohepatitis (NASH). A total of 252 adults (NAFLD group, n = 202; healthy group, n = 50) were recruited. Clinical biochemical characteristics, NE and A1 AT concentrations were measured in all subjects. Among the NAFLD group, 86 patients had previously undergone liver biopsy and information on histological characteristics was consequently available. The area under the receiver operating characteristic curve (AUC) was used to determine the predictive accuracy of the NE-A1 AT system for NASH. NAFLD patients had an elevated serum NE concentration and a reduced A1 AT level with consequent NE/A1 AT imbalance. NE increased in the early stage of steatosis, preceding the decline in A1 AT, dating from the onset of NASH (NAS 3-4), and subsequently NE/A1 AT increased in the presence of NASH. Nonetheless, this increase began to resolve as the disease state progressed to advanced fibrosis. A1 AT had a sensitivity (SEN) of 83.8% and a specificity (SP) of 83.3% with the optimal cut-off of -1459.43, NE/A1 AT had a SEN of 88.8% and a SP of 83.3% with cut-off of 0.363 to predict NASH. An increased NE: A1 AT ratio is closely associated with liver Inflammation in patients with NASH and could serve as a novel marker to predict NASH in humans.

[Effect of Compound Zhajin Granule on Toll-like Receptor 4 Signaling Pathway in Nonalcoholic Steatohepatitis Mice].

OBJECTIVE: To observe the effect of Compound Zhajin Granule (CZG) on Toll-like re-ceptor 4 (TLR4) signaling pathway in high-fructose corn syrup induced NASH mice. METHODS: Thirty 6-week-old male C3H mice were divided into the high fat and high fructose (HFHFr) group (n = 20) and the control group (n = 10) according to body weight. Mice in the HFHFr group ate high fat diet and drank 20% fructose water, while those in the control group ate common diet and drank common water. After 8 weeks mice in the HFHFr group were divided into two group according to body weight, the HFHFr group and the CZG group, 10 in each group. Mice in the CZG group were fed with high fat forage and 20% fructose water, and administered with 50 mL/kg 12. 8% CZG (prepared by hawthorn, Radix Curcumae, Alisma Orientale, Fritillaria Thunbergii, Silybum Marianum, peach seed in the ratio of 3:1.5:1.5:2:1.5:2:1) by gastrogavage. Mice in the HFHFr group were fed in the same way and daily administered with equal volume of distilled water by gastrogavage. Sixteen weeks later all mice were sacrificed. Body weight, liver wet weight, liver function, and lipid metabolism were detected. Pathological changes of liver tissues were assessed by HE staining, oil red O staining, and Masson staining. Expressions of TLR4, myeloid differentiation factor 88 (MyD88), tumor necrosis factor-alpha (TNF-α) were detected using immunohistochemical staining and real-time fluorescent quantitative PCR. RESULTS: Body weight, alanine aminotransferase (ALT), aspartate aminotransferase (AST) were obviously lower in the CZG group than in the HFHFr group (P < 0.05); oil red O stained area and density were decreased more in the CZG group than in the control group. HE staining showed ballooning inflammation was reduced more in the CZG group than in the HFHFr group. Masson staining was negative. Positive rates of TLR4 and MyD88 and mRNA expressions were significantly lower in the CZG group than in the HFHFr group (all P < 0.05). CONCLUSION: CZG could significantly inhibit TLR4 signaling pathway of liver in NASH mice.

The N termini of a-subunit isoforms are involved in signaling between vacuolar H+-ATPase (V-ATPase) and cytohesin-2.

Previously, we reported an acidification-dependent interaction of the endosomal vacuolar H(+)-ATPase (V-ATPase) with cytohesin-2, a GDP/GTP exchange factor (GEF), suggesting that it functions as a pH-sensing receptor. Here, we have studied the molecular mechanism of signaling between the V-ATPase, cytohesin-2, and Arf GTP-binding proteins. We found that part of the N-terminal cytosolic tail of the V-ATPase a2-subunit (a2N), corresponding to its first 17 amino acids (a2N(1-17)), potently modulates the enzymatic GDP/GTP exchange activity of cytohesin-2. Moreover, this peptide strongly inhibits GEF activity via direct interaction with the Sec7 domain of cytohesin-2. The structure of a2N(1-17) and its amino acids Phe(5), Met(10), and Gln(14) involved in interaction with Sec7 domain were determined by NMR spectroscopy analysis. In silico docking experiments revealed that part of the V-ATPase formed by its a2N(1-17) epitope competes with the switch 2 region of Arf1 and Arf6 for binding to the Sec7 domain of cytohesin-2. The amino acid sequence alignment and GEF activity studies also uncovered the conserved character of signaling between all four (a1-a4) a-subunit isoforms of mammalian V-ATPase and cytohesin-2. Moreover, the conserved character of this phenomenon was also confirmed in experiments showing binding of mammalian cytohesin-2 to the intact yeast V-ATPase holo-complex. Thus, here we have uncovered an evolutionarily conserved function of the V-ATPase as a novel cytohesin-signaling receptor.

Non-alcoholic fatty liver disease (NAFLD) fibrosis score predicts 6.6-year overall mortality of Chinese patients with NAFLD.

The non-alcoholic fatty liver disease (NAFLD) fibrosis score (NFS) has emerged as a useful predictor of long-term outcome in NAFLD patients. We evaluated the predictive performance of the NFS for overall mortality in a Chinese population with NAFLD. All NAFLD patients diagnosed ultrasonographically at Xixi Hospital of Hangzhou between 1996 and 2011 were retrospectively recruited to the study. Outcome was determined by interview and causes of death were confirmed by medical records. The area under the receiver operating characteristic curve (AUCROC ) was used to determine the predictive accuracy of the NFS, BARD (body mass index, aspartate aminotransferase (AST)/alanine aminotransferase (ALT) ratio, diabetes) score, FIB-4 index and the AST/platelet ratio index (APRI) for mortality. Data from a total of 180 eligible patients (median age 39 years; 96 men) were analysed, with 12 deaths over a median follow-up period of 6.6 years (range 0.5-14.8 years). Using Cox model analysis, the NFS as a continuous variable was identified as the only predictor for all-cause mortality (hazard ratio 2.743, 95% confidence interval (CI) 1.670-4.504). The NFS yielded the highest AUCROC of 0.828 (95% CI 0.728-0.928, P < 0.05), followed by the FIB-4 index, APRI and BARD score (AUCROC 0.806 (P < 0.05), 0.732 (P < 0.05) and 0.632, respectively). The data indicated that the NFS is a useful predictor of 6.6-year all-cause mortality for Chinese patients with NAFLD.

Toll-like receptor-4 signalling in the progression of non-alcoholic fatty liver disease induced by high-fat and high-fructose diet in mice.

The aim of the present study was to investigate Toll-like receptor-4 (TLR4) signalling at different stages of non-alcoholic fatty liver disease (NAFLD) induced by a high-fat, high-fructose (HFHFr) diet in mice. Both TLR4 wild-type (WT) and mutant (TLR4(mut) ) mice were fed either standard chow (SC) or the HFHFr diet for different periods of time from 4 to 16 weeks. Pathological characteristics and function of the liver were assessed. Simple steatosis, steatohepatitis and hepatic fibrosis occurred sequentially in Week 4, 8 and 16 in WT mice fed with the HFHFr. Expression of TLR4, myeloid differentiation factor 88 (MyD88), interferon regulatory factor (IRF) 3 and IRF7 started to increase at Week 4, peaked at Week 8 and then declined to basal levels at Week 16. This pattern was consistent with changes in inflammation in the liver revealed by haematoxylin and eosin staining. However, lipid accumulation, inflammation and fibrosis in livers of TLR4(mut) mice fed the HFHFr diet were significantly alleviated. In addition, the expression of activin A in WT mice fed the HFHFr diet increased at Week 16. The data suggest that TLR4 signalling mediates non-alcoholic steatohepatitis before fibrosis and that activin A is subsequently involved in NAFLD.

[Development and evaluation of a high-fat/high-fructose diet-induced nonalcoholic steatohepatitis mouse model].

OBJECTIVE: To develop and evaluate a mouse model of nonalcoholic steatohepatitis (NASH) induced by a high-fat and high-fructose (HFHFr) diet. METHODS: Six-week-old C3H mice were randomly divided into groups for HFHFr diet experimental modeling, high fat-only (HF) diet controls, high fructose-only (HFr) diet controls, and standard chow (SC) diet controls. The standard HFHFr diet was modified so that it consisted of 76.5% standard chow, 12% lard, 1% cholesterol, 5% egg yolk powder, 5% whole milk powder, and 0.5% sodium cholate, along with 20% fructose drinking water. At the end of experimental weeks 4, 8, and 16, measurements were taken for the NASH-related parameters of body mass, serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), lipid profile, and wet liver weight (upon sacrifice). In addition, histological changes in the liver were evaluated by hematoxylin-eosin (HE) and oil red O staining. The significance of differences between groups was assessed by statistical analysis, using the METHODS: of t-test, Wilcoxon rank sum test, x2 test, F test or Fisher's test as appropriate. RESULTS: As compared to the mice in the SC group at the corresponding time points, the mice in the HFHFr and HF groups showed significantly higher body mass and wet liver weight, as well as more extensive and robust lipid disposition in hepatic tissues as evidenced by oil red O staining. However, HE staining indicated that the HFHFr and HF groups had different degrees of macrosteatosis accompanied with intralobular inflammatory foci, with the former showing more remarkable NASH-related histological changes. Analysis at the end of week 16 showed that about 80% of the mice in the HFHFr group had developed NASH [nonalcoholic fatty liver disease (NAFLD) activity score (NAS): less than 5]. The levels of low-and high-density lipoprotein (LDL and HDL) cholesterol, as well as the levels of ALT and AST, were increased from the end of week 4 to the end of week 8 for the HFHFr and HF groups. At the end of week 16, the two groups differed in the extent of increase in total cholesterol and LDL and HDL cholesterol, with only the HFHFr group showing statistically significant changes. Specifically, at the end of week 16, the HFHFr group showed ALT levels of 108.5 +/- 93.34 U/L (F=5.099, P =0.005 vs. HF group: 44.30 +/- 35.71 U/L, HFr group: 46.70 +/- 17.95 U/L, SC group: 24.70 +/- 6.57 U/L), AST levels of 316.30 +/- 208.98 U/L (F=6.654, P=0.001 vs. HF: 132.12 +/- 75.43 U/L, HFr: 143.30 +/- 38.53 U/L, SC: 122.60 +/- 12.76 U/L), total cholesterol levels of 5.18 +/- 0.58 mmol/L (F=72: 470, P =0.000 vs. HF: 3.94 +/- 0.75 mmol/L, HFr: 2.30 +/- 0.50 mmol/L, SC: 2.02 +/- 0.24 mmol/L), HDL cholesterol levels of 3.05 +/- 0.49 mmol/L (F=25.413, P =0.000 vs. HF: 2.65 +/- 0.54 mmol/L HFr: 1.77 +/- 0.47 mmol/L, SC: 1.58 +/- 0.16 mmol/L), LDL cholesterol levels of 1.11 +/- 0.23 mmol/L (F =83.297, P =0.000 vs. HF: 0.72 +/- 0.17 mmol/L, HFr: 0.27 +/- 0.04 mmol/L, SC: 0.20 +/- 0.05 mmol/ L). CONCLUSION: The present study suggests that a mouse model of NASH can be successfully induced by a 16-week modified HFHFr diet.

Characteristics of intestinal flora in nonobese nonalcoholic fatty liver disease patients and the impact of ursodeoxycholic acid treatment on these features.

The study aimed to investigate the alterations in gut microbiota among nonobese individuals with nonalcoholic fatty liver disease (NAFLD) and their response to treatment with ursodeoxycholic acid (UDCA). A total of 90 patients diagnosed with NAFLD and 36 healthy subjects were recruited to participate in this study. Among them, a subgroup of 14 nonobese nonalcoholic steatohepatitis (NASH) were treated with UDCA. Demographic and serologic data were collected for all participants, while stool samples were obtained for fecal microbiome analysis using 16S sequencing. In nonobese NAFLD patients, the alpha diversity of intestinal flora decreased (Shannon index, p < 0.05), and the composition of intestinal flora changed (beta diversity, p < 0.05). The abundance of 20 genera, including Fusobacterium, Lachnoclostridium, Klebsiella, etc., exhibited significant changes (p < 0.05). Among them, nine species including Fusobacterium, Lachnoclostridium, Klebsiella, etc. were found to be associated with abnormal liver enzymes and glucolipid metabolic disorders. Among the 14 NASH patients treated with UDCA, improvements were observed in terms of liver enzymes, CAP values, and E values (p < 0.05), however, no improve the glucolipid metabolism. While the alpha diversity of intestinal flora did not show significant changes after UDCA treatment, there was a notable alteration in the composition of intestinal flora (beta diversity, p < 0.05). Furthermore, UCDA treatment led to an improvement in the relative abundance of Alistipes, Holdemanella, Gilisia, etc. among nonobese NASH patients (p < 0.05). Nonobese NAFLD patients exhibit dysbiosis of the intestinal microbiota. UDCA can ameliorate hepatic enzyme abnormalities and reduce liver fat content in nonobese NASH patients, potentially through its ability to restore intestinal microbiota balance.

Morus alba L. (Sangzhi) alkaloids mitigate atherosclerosis by regulating M1/M2 macrophage polarization.

BACKGROUND: Atherosclerosis (AS) is an important cause of cardiovascular disease, posing a substantial health risk. Recognized as a chronic inflammatory disorder, AS hinges on the pivotal involvement of macrophages in arterial inflammation, participating in its formation and progression. Sangzhi alkaloid (SZ-A) is a novel natural alkaloid extracted from the mulberry branches, has extensive pharmacological effects and stable pharmacokinetic characteristics. However, the effects and mechanisms of SZ-A on AS remain unclear. PURPOSE: To explore the effect and underlying mechanisms of SZ-A on inflammation mediated by macrophages and its role in AS development. METHODS: Atherosclerosis was induced in vivo in apolipoprotein E-deficient mice through a high-fat and high-choline diet. We utilized macrophages and vascular endothelial cells to investigate the effects of SZ-A on macrophage polarization and its anti-inflammatory properties on endothelial cells in vitro. The transcriptomic analyses were used to investigate the major molecule that mediates cell-cell interactions and the antiatherogenic mechanisms of SZ-A based on AS, subsequently validated in vivo and in vitro. RESULTS: SZ-A demonstrated a significant inhibition in vascular inflammation and alleviation of AS severity by mitigating macrophage infiltration and modulating M1/M2 macrophage polarization in vitro and in vivo. Moreover, SZ-A effectively reduced the release of the proinflammatory mediator C-X-C motif chemokine ligand (CXCL)-10, predominantly secreted by M1 macrophages. This reduction in CXCL-10 contributed to improved endothelial cell function, reduced recruitment of additional macrophages, and inhibited the inflammatory amplification effect. This ultimately led to the suppression of atherogenesis. CONCLUSION: SZ-A exhibited potent anti-inflammatory effects by inhibiting macrophage-mediated inflammation, providing a new therapeutic avenue against AS. This is the first study demonstrating the efficacy of SZ-A in alleviating AS severity and offers novel insights into its anti-inflammatory mechanism.

Mechanistic insights into Yifei Sanjie pill's regulation of EMT to enhance gefitinib treatment effect in NSCLC by in silico analysis and experimental validation.

ETHNOPHARMACOLOGICAL RELEVANCE: The Yi-Fei San-Jie pill (YFSJ) is a well-known Chinese medicine that has been used to treat non-small cell lung cancer in China for decades. AIM OF THE STUDY: Previous studies have shown that YFSJ combined with gefitinib can effectively inhibit the proliferation of gefitinib-resistant non-small cell lung cancer (NSCLC) cell lines by promoting apoptosis and autophagy, but the molecular biological mechanisms involved and whether YFSJ combined with gefitinib can have synergistic effects still need to be further explored. Thus, the present study aimed to establish an in silico and experimental framework to decipher the underlying mechanism by which YFSJ augments the efficacy of gefitinib in treating NSCLC. MATERIALS AND METHODS: Integrated approaches, including microarray analysis, network pharmacology, RNA sequencing, bioinformatics algorithm analysis and in vivo and in vitro experiments, were applied to elucidate the underlying mechanism. RESULTS: Analysis of microarray datasets indicated that gefitinib may play a role in the regulation of the epithelial-mesenchymal transition (EMT) of PC9 cells. EMT-related Gene Ontology (GO) terms and the MAPK pathway were found to be enriched in the differentially expressed genes (DEGs), and a decreasing trend was observed in the EMT score. Network pharmacology analysis revealed that the potential NSCLC-related targets of YFSJ also showed enrichment in EMT-related GO terms and the MAPK pathway. Experimental findings demonstrated that combined YFSJ-treated serum and gefitinib treatment significantly inhibited PC9 cell migration and invasion. In addition, the combined treatment dramatically reduced the tumour volume in an animal model. The effectiveness of the combination treatment surpassed that of gefitinib alone in both cell and animal experiments. RNA sequencing analysis revealed significant enrichment of DEGs in EMT-related GO terms for the gefitinib treatment group, YFSJ treatment group, and combination treatment group compared to the control group. Notably, the negative regulation of EMT showed significant enrichment in the DEGs of the combination treatment group. The MAPK pathway was significantly enriched among the different groups. Moreover, combined treatment with YFSJ and gefitinib may exert synergistic anti-NSCLC effects by inhibiting the p-p38 MAPK/GSK3ß signalling axis, subsequently suppressing downstream EMT processes. CONCLUSION: Combined treatment with YFSJ and gefitinib could enhance the sensitivity of NSCLC cells to gefitinib by suppressing EMT through the EGFR/p-p38 MAPK/GSK3ß signalling axis. YFSJ may serve as an important adjunctive medication for NSCLC patients receiving gefitinib treatment in clinical practice.

Ginsenoside Rg3, a promising agent for NSCLC patients in the pandemic: a large-scale data mining and systemic biological analysis.

Introduction: Non-small cell lung cancer (NSCLC) patients are particularly vulnerable to the Coronavirus Disease-2019 (COVID-19). Currently, no anti-NSCLC/COVID-19 treatment options are available. As ginsenoside Rg3 is beneficial to NSCLC patients and has been identified as an entry inhibitor of the virus, this study aims to explore underlying pharmacological mechanisms of ginsenoside Rg3 for the treatment of NSCLC patients with COVID-19. Methods: Based on a large-scale data mining and systemic biological analysis, this study investigated target genes, biological processes, pharmacological mechanisms, and underlying immune implications of ginsenoside Rg3 for NSCLC patients with COVID-19. Results: An important gene set containing 26 target genes was built. Target genes with significant prognostic value were identified, including baculoviral IAP repeat containing 5 (BIRC5), carbonic anhydrase 9 (CA9), endothelin receptor type B (EDNRB), glucagon receptor (GCGR), interleukin 2 (IL2), peptidyl arginine deiminase 4 (PADI4), and solute carrier organic anion transporter family member 1B1 (SLCO1B1). The expression of target genes was significantly correlated with the infiltration level of macrophages, eosinophils, natural killer cells, and T lymphocytes. Ginsenoside Rg3 may benefit NSCLC patients with COVID-19 by regulating signaling pathways primarily involved in anti-inflammation, immunomodulation, cell cycle, cell fate, carcinogenesis, and hemodynamics. Conclusions: This study provided a comprehensive strategy for drug discovery in NSCLC and COVID-19 based on systemic biology approaches. Ginsenoside Rg3 may be a prospective drug for NSCLC patients with COVID-19. Future studies are needed to determine the value of ginsenoside Rg3 for NSCLC patients with COVID-19.

Mechanisms of tumor-associated macrophages affecting the progression of hepatocellular carcinoma.

Tumor-associated macrophages (TAMs) are essential components of the immune cell stroma of hepatocellular carcinoma. TAMs originate from monocytic myeloid-derived suppressor cells, peripheral blood monocytes, and kupffer cells. The recruitment of monocytes to the HCC tumor microenvironment is facilitated by various factors, leading to their differentiation into TAMs with unique phenotypes. TAMs can directly activate or inhibit the nuclear factor-κB, interleukin-6/signal transducer and signal transducer and activator of transcription 3, Wnt/ß-catenin, transforming growth factor-ß1/bone morphogenetic protein, and extracellular signal-regulated kinase 1/2 signaling pathways in tumor cells and interact with other immune cells via producing cytokines and extracellular vesicles, thus affecting carcinoma cell proliferation, invasive and migratory, angiogenesis, liver fibrosis progression, and other processes to participate in different stages of tumor progression. In recent years, TAMs have received much attention as a prospective treatment target for HCC. This review describes the origin and characteristics of TAMs and their mechanism of action in the occurrence and development of HCC to offer a theoretical foundation for further clinical research of TAMs.

Systems biology strategy and experimental validation to uncover the pharmacological mechanism of Xihuang Pill in treating non-small cell lung cancer.

BACKGROUND: Non-small cell lung cancer (NSCLC) accounts for almost 85% of lung cancer-related deaths worldwide. Xihuang Pill (XHP) is a representative anticancer Chinese patented medicine used to treat NSCLC in China. However, to date, a systematic analysis of XHP's antitumour effects and its impact on the immune microenvironment has not been performed. PURPOSE: Based on the systems biology strategy and experimental validation, the present study aimed to investigate the pharmacological mechanisms involved in treating NSCLC with XHP. METHODS: A subcutaneous tumour model was established to evaluate XHP's tumour-inhibitory effect in BALB/c nude mice. RNA sequencing (RNA-seq) and bioinformatics analysis were conducted to identify differentially expressed genes (DEGs) and signalling pathways related to XHP treatment. Network analysis based on network pharmacology and protein-to-protein networks was applied to identify the compounds and genes targeted by XHP. External data from the TCGA-NSCLC cohort were used to verify the clinical significance of XHP-targeted genes in NSCLC. The expression of survival-related candidate genes after XHP treatment was verified via qPCR. The protein expression of calcium voltage-gated channel subunit alpha 1C (CACNA1C) in different NSCLC cell lines was analysed in the Human Protein Atlas database (HPA) and DepMap Portal. Using the Estimation of STromal and Immune cells in MAlignant Tumour tissues using Expression data (ESTIMATE) algorithm and the single-sample gene set enrichment analysis (ssGSEA) algorithm uncovered the role of CACNA1C in the NSCLC tumour microenvironment (TME). RESULTS: XHP (2 g/kg/d) significantly inhibited the growth of transplanted A549 tumours. RNA-seq identified a total of 529 DEGs (189 upregulated and 340 downregulated). In addition, 542 GO terms, 41 significant KEGG pathways, 9 upregulated hallmarks pathways, and 18 downregulated hallmark pathways were enriched. These GO terms and signalling pathways were closely related to cell proliferation, immunity, energy metabolism, and the inflammatory response of NSCLC. In addition, XHP's network pharmacology analysis identified 301 compounds and 1,432 target genes. A comprehensive strategic analysis identified CACNA1C as a promising gene by which XHP targets and regulates the TME of NSCLC, benefiting patient survival. CACNA1C expression was positively correlated with both the immune score and stromal score but negatively correlated with the tumour purity score. Additionally, CACNA1C expression was significantly correlated with the infiltration levels of 15 types of immune cells and the expression levels of 6 well-known checkpoint genes. CONCLUSIONS: Our results show that by regulating the pathways associated with cell proliferation and immunity, XHP can suppress cancer cell growth in NSCLC. Additionally, XHP may increase the expression of CACNA1C to suppress immune cell infiltration and regulate the expression of checkpoint-related genes, thereby improving the overall survival of NSCLC patients.

Bioinformatics and System Biology Approach to Reveal the Interaction Network and the Therapeutic Implications for Non-Small Cell Lung Cancer Patients With COVID-19.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the leading cause of coronavirus disease-2019 (COVID-19), is an emerging global health crisis. Lung cancer patients are at a higher risk of COVID-19 infection. With the increasing number of non-small-cell lung cancer (NSCLC) patients with COVID-19, there is an urgent need of efficacious drugs for the treatment of COVID-19/NSCLC. Methods: Based on a comprehensive bioinformatic and systemic biological analysis, this study investigated COVID-19/NSCLC interactional hub genes, detected common pathways and molecular biomarkers, and predicted potential agents for COVID-19 and NSCLC. Results: A total of 122 COVID-19/NSCLC interactional genes and 21 interactional hub genes were identified. The enrichment analysis indicated that COVID-19 and NSCLC shared common signaling pathways, including cell cycle, viral carcinogenesis, and p53 signaling pathway. In total, 10 important transcription factors (TFs) and 44 microRNAs (miRNAs) participated in regulations of 21 interactional hub genes. In addition, 23 potential candidates were predicted for the treatment of COVID-19 and NSCLC. Conclusion: This study increased our understanding of pathophysiology and screened potential drugs for COVID-19 and NSCLC.

Aldolase directly interacts with ARNO and modulates cell morphology and acidic vesicle distribution.

Previously, we demonstrated that the vacuolar-type H(+)-ATPase (V-ATPase) a2-subunit functions as an endosomal pH sensor that interacts with the ADP-ribosylation factor (Arf) guanine nucleotide exchange factor, ARNO. In the present study, we showed that ARNO directly interacts not only with the a2-subunit but with all a-isoforms (a1-a4) of the V-ATPase, indicating a widespread regulatory interaction between V-ATPase and Arf GTPases. We then extended our search for other ARNO effectors that may modulate V-ATPase-dependent vesicular trafficking events and actin cytoskeleton remodeling. Pull-down experiments using cytosol of mouse proximal tubule cells (MTCs) showed that ARNO interacts with aldolase, but not with other enzymes of the glycolytic pathway. Direct interaction of aldolase with the pleckstrin homology domain of ARNO was revealed by pull-down assays using recombinant proteins, and surface plasmon resonance revealed their high avidity interaction with a dissociation constant: K(D) = 2.84 × 10(-10) M. MTC cell fractionation revealed that aldolase is also associated with membranes of early endosomes. Functionally, aldolase knockdown in HeLa cells produced striking morphological changes accompanied by long filamentous cell protrusions and acidic vesicle redistribution. However, the 50% knockdown we achieved did not modulate the acidification capacity of endosomal/lysosomal compartments. Finally, a combination of small interfering RNA knockdown and overexpression revealed that the expression of aldolase is inversely correlated with gelsolin levels in HeLa cells. In summary, we have shown that aldolase forms a complex with ARNO/Arf6 and the V-ATPase and that it may contribute to remodeling of the actin cytoskeleton and/or the trafficking and redistribution of V-ATPase-dependent acidic compartments via a combination of protein-protein interaction and gene expression mechanisms.

Is caveolin involved in normal proximal tubule function? Presence in model PT systems but absence in situ.

Kidney proximal tubule (PT) cells are specialized for the uptake and transport of ions, solutes, peptides, and proteins. These functions are often regulated by hormones that signal at the cell surface and are internalized by clathrin-mediated endocytosis. However, the caveolin/caveolae pathway has also been implicated in normal PT function, often based on data from isolated PTs or PT cells in culture. Although we reported previously that caveolae and caveolin 1 are not detectable in PTs in vivo, reports of caveolin expression and function in PT cells appear periodically in the literature. Therefore, we reexamined caveolin expression in PTs in vivo, in isolated "purified" PTs following collagenase digestion, and in cultured PT cells. Caveolin 1 and 2 protein, mRNA, or immunofluorescence was undetectable in PTs in vivo, but PT cell cultures expressed caveolin 1 and/or 2. Furthermore, caveolin 1 and 2 mRNAs were detected in isolated PTs along with the endothelial markers CD31 and ICAM1. In contrast, no caveolin or endothelial marker mRNAs were detectable in samples isolated from snap-frozen kidneys by laser cut microdissection, which eliminates contamination by other cell types. We conclude 1) caveolin 1 and 2 are not normally expressed by PT cells in situ, 2) caveolin expression is "activated" in cultured PT cells, 3) contamination with non-PT, caveolin-expressing cells is a potential source of caveolin 1 and 2 that must be taken into account when isolated PTs are used in studies to correlate expression of these proteins with PT function.

Dragon enhances BMP signaling and increases transepithelial resistance in kidney epithelial cells.

The neuronal adhesion protein Dragon acts as a bone morphogenetic protein (BMP) coreceptor that enhances BMP signaling. Given the importance of BMP signaling in nephrogenesis and its putative role in the response to injury in the adult kidney, we studied the localization and function of Dragon in the kidney. We observed that Dragon localized predominantly to the apical surfaces of tubular epithelial cells in the thick ascending limbs, distal convoluted tubules, and collecting ducts of mice. Dragon expression was weak in the proximal tubules and glomeruli. In mouse inner medullary collecting duct (mIMCD3) cells, Dragon generated BMP signals in a ligand-dependent manner, and BMP4 is the predominant endogenous ligand for the Dragon coreceptor. In mIMCD3 cells, BMP4 normally signaled through BMPRII, but Dragon enhanced its signaling through the BMP type II receptor ActRIIA. Dragon and BMP4 increased transepithelial resistance (TER) through the Smad1/5/8 pathway. In epithelial cells isolated from the proximal tubule and intercalated cells of collecting ducts, we observed coexpression of ActRIIA, Dragon, and BMP4 but not BMPRII. Taken together, these results suggest that Dragon may enhance BMP signaling in renal tubular epithelial cells and maintain normal renal physiology.

Exploring the Potential Mechanism of Shufeng Jiedu Capsule for Treating COVID-19 by Comprehensive Network Pharmacological Approaches and Molecular Docking Validation.

OBJECTIVE: Shufeng Jiedu capsule (SFJDC) is a well-known Chinese patent drug that is recommended as a basic prescription and applied widely in the clinical treatment of COVID-19. However, the exact molecular mechanism of SFJDC remains unclear. The present study aims to determine the potential pharmacological mechanisms of SFJDC in the treatment of COVID-19 based on network pharmacology. METHODS: The network pharmacology-based strategy includes collection and analysis of active compounds and target genes, network construction, identification of key compounds and hub target genes, KEGG and GO enrichment, recognition and analysis of main modules, as well as molecule docking. RESULTS: A total of 214 active chemical compounds and 339 target genes of SFJDC were collected. Of note, 5 key compounds (ß -sitosterol, luteolin, kaempferol, quercetin, and stigmasterol) and 10 hub target genes (TP53, AKT1, NCOA1, EGFR, PRKCA, ANXA1, CTNNB1, NCOA2, RELA and FOS) were identified based on network analysis. The hub target genes mainly enriched in pathways including MAPK signaling pathway, PI3K-Akt signaling pathway and cAMP signaling pathway, which could be the underlying pharmacological mechanisms of SFJDC for treating COVID-19. Moreover, the key compounds had high binding activity with three typical target proteins including ACE2, 2OFZ, and 1SSK. CONCLUSION: By network pharmacology analysis, SFJDC was found to effectively improve immune function and reduce inflammatory responses based on its key compounds, hub target genes, and the relevant pathways. These findings may provide valuable evidence for explaining how SFJDC exerting the therapeutic effects on COVID-19, providing a holistic view for further clinical application.