Under normal conditions, insulin promotes hepatic de novo lipogenesis (DNL). However, during insulin resistance (IR), when insulin signalling is blunted and accompanied by hyperinsulinaemia, the promotion of hepatic DNL continues unabated and hepatic steatosis increases. Here, we show that WD40 repeat-containing protein 6 (WDR6) promotes hepatic DNL during IR. Mechanistically, WDR6 interacts with the beta-type catalytic subunit of serine/threonine-protein phosphatase 1 (PPP1CB) to facilitate PPP1CB dephosphorylation at Thr316, which subsequently enhances fatty acid synthases transcription through DNA-dependent protein kinase and upstream stimulatory factor 1. Using molecular dynamics simulation analysis, we find a small natural compound, XLIX, that inhibits the interaction of WDR6 with PPP1CB, thus reducing DNL in IR states. Together, these results reveal WDR6 as a promising target for the treatment of hepatic steatosis.

Thyroid-stimulating hormone (TSH) treatment activates IκB/NFκB (p65) and ERK, P38 in macrophages, but how these pathways are activated, and how they contribute to the pro-inflammatory effect of TSH on macrophages remain unknown. TSH receptor (TSHR) is coupled to four subfamilies of G proteins (Gs, Gi/o, Gq/11 and G12/13) for its downstream signalling. This study investigated the G protein subtypes responsible for the pro-inflammatory effect of TSH on macrophages. qPCR showed that Gi2, Gi3, Gas, Gq, G11, G12, G13, G15 are abundantly expressed by macrophages. The contribution of different G-protein pathways to the pro-inflammatory effect was studied by the corresponding inhibitors or siRNA interference. While TSH-induced IκB phosphorylation was not inhibited by Gs inhibitor NF449, Gi inhibitor pertussis toxin, Gq or G11 siRNA, it was blocked by phospholipase C inhibitor U73122 or G15 siRNA interference. TSH-induced ERK and P38 phosphorylation was blocked by G13 but not G12 siRNA interference. Interfering either G13 or G15 was able to block the pro-inflammatory effect of TSH on macrophages. The present study demonstrate that TSH activates macrophage inflammation by G13/ERK-P38/Rho GTPases and G15/PLC/PKCs/IκB pathways.

As a progressive chronic disease, the effective treatment for non‐alcoholic fibre liver disease (NAFLD) has not yet been thoroughly explored at the moment. The widespread use of Gynostemma pentaphyllum (Thunb) for its anti‐insulin resistance effect indicates that potential therapeutic value may be found in Thunb for NAFLD. Hence, this research aims to discover the latent mechanism of Thunb for NAFLD treatment. To achieve the goal of discovering the latent mechanism of Thunb for NAFLD treatment, molecular docking strategy integrated a network phamacology was adopted in the exploration. We acquire Thunb compounds with activeness from TCMSP database. We collect the putative targets of Thunb and NAFLD to generate the network. Key targets and mechanism are screened by PPI analysis, GO and KEGG pathway enrichment analyses. Molecular docking simulation is introduced into the study as assessment method. Through network analysis and virtual screening based on molecular docking, 2 targets (AKT 1 and GSK3B) are identified as key therapeutic targets with satisfying binding affinity. Main mechanism is believed to be the biological process and pathway related to insulin resistance according to the enrichment analyses outcomes. Particularly, the P13K–AKT signalling pathway is recognized as a key pathway of the mechanism. In conclusion, the study shows that Thunb could be a potential treatment against NAFLD and may suppress insulin resistance through the P13K–AKT signalling pathway. The result of the exploration provides a novel perspective for approaching experimental exploration.

Background. gp91^phox, the catalytic core of NADPH oxidase (NOX) and biomarker of NOX activation, has been recently recognized as a parameter of systemic oxidative stress in several studies. Subclinical hypothyroidism (SH) is characteristic of elevated level of serum thyroid stimulating hormone (TSH) and is frequently accompanied with cholesterolemia. In this study, the levels of serum soluble gp91^phox were measured to assess the oxidative stress in patients with SH. And the relationship among gp91^phox, low-density lipoprotein-C (LDL-C), and TSH was also investigated. Methods. A total of 51 subjects were enrolled and categorized into four groups: the healthy controls subjects (n = 13), controls with high level of LDL-C alone (n = 12), SH with normal level of LDL-C (n = 11), and SH with high level of LDL-C (n = 15). The related clinical and laboratory data were collected for statistical analysis. All the patients were newly diagnosed and did not take any medication. The information of lipid profile and thyroid function was extracted, and the concentrations of gp91phox were obtained with ELISA. Results. The levels of serum soluble gp91phox evidently increased in the patients with SH with a high level of LDL-C (81.52 ± 37.00 ug/mL) as compared to the healthy controls (54.98 ± 1.83ug/mL, p<0.001), controls with high level of LDL-C (61.21 ± 4.48 ug/mL, p=0.038) and SH with a normal level of LDL-C (62.82 ± 11.67ug/mL, p=0.027). Additionally, the levels of gp91^phox showed a significant positive correlation with both the levels of LDL-C (r = 0.595, p<0.001) and TSH (r = 0.346, p=0.013) by the Spearman correlation analyses. The correlation remained significant even when the effect of another factor was controlled (TSH: when the effect of LDL-C was controlled, r = 0.453, p=0.001; LDL-C: when the effect of TSH was controlled, r = 0.291, p=0.040). The main effect analysis showed an independent main effect of either LDL-C (p = 0.041) or TSH (p=0.022) on gp91^phox without interaction (p=0.299). Conclusions. Our work demonstrated that the levels of gp91^phox, a novel biomarker for measuring the oxidative stress, were significantly elevated in the patients with SH. And LDL-C and TSH were both independent predictors of gp91^phox. Abbreviations. BMI : Body mass index; TC : Total cholesterol; LDL-C : Low-density lipoprotein cholesterol; HDL-C : High-density lipoprotein cholesterol; TG : Triglyceride; FBG : Fasting blood glucose; FT3 : Free triiodothyronine; FT4 : Free thyroxine; TSH: Thyroid stimulating hormone; SBP : Systolic blood pressure; DBP : Diastolic blood pressure; SD : Standard deviation; LSD: Least significant difference.

Subclinical hypothyroidism (SCH) is becoming a global health problem due to its increasing prevalence and potential adverse effects, including cardiovascular diseases and nonalcoholic fatty liver disease (NAFLD). However, the association between SCH and NAFLD remains controversial. MicroRNAs (miRNAs/miRs) have been reported to be implicated in lipid metabolism disorders; however, how miRNAs regulate hepatic lipid metabolism in SCH mice remains unknown. The present study investigated miRNA alterations and proteome profiles in an SCH mouse model, which was generated by methimazole administration in mice for 16 weeks. Next, the profiles of 17 miRNAs that are critical to hepatic lipid metabolism and the proteome were investigated using reverse transcription‑quantitative polymerase chain reaction and iTRAQ labeling in the liver specimens of SCH (n=9) and control (n=7) mice. Putative target prediction of miRNAs was also conducted using TargetScan and miRanda. Compared with the control mice, SCH mice had 8 miRNAs and 36 proteins with significantly different expression in the liver tissues. Furthermore, a regulatory module containing 3 miRNAs (miR‑34a‑5p, miR‑24‑3p and miR‑130a‑3p) and 4 proteins (thioredoxin, selenium‑binding protein 2, elongation factor 1β and prosaposin) was identified. Overall, integrated analysis of miRNAs and the proteome highlighted a regulatory module between miRNAs and proteins, which, to a certain extent, may contribute to a better understanding of hepatic lipid metabolism disorders in SCH mice.

Objective. Astragalus mongholicus Bunge [Fabaceae] (AMB), a traditional Chinese medicine (TCM), has been widely used to treat liver diseases in the clinic. However, the efficacy and mechanism of AMB in the treatment of nonalcoholic fatty liver disease (NAFLD) remain unclear. The purpose of this study was to systematically investigate the active components and mechanisms of AMB against NAFLD based on network pharmacology, molecular docking, and experimental verification. Methods. First, the bioactive components and relevant targets of AMB were screened from the Traditional Chinese Medicine Systematic Pharmacology (TCMSP) database, and NAFLD-related targets were obtained from the GeneCards database. Then, the AMB-NAFLD protein target interaction network was built by the STRING database. GO and KEGG pathway enrichment analyses were performed using the DAVID database. The component targets were visualized using Cytoscape software. Finally, molecular docking and experiments were used to verify the results of network pharmacological prediction. Results. Network pharmacology predicted that quercetin may be the main active component in AMB, and the TNF and MAPK signaling pathways may be the key targets of AMB against NAFLD. Molecular docking validation results demonstrated that quercetin, as the main active component of AMB, had the highest binding affinity with TNF. Furthermore, quercetin played a distinct role in alleviating NAFLD through in vitro experiments. Quercetin upregulated the phosphorylation levels of AMPK and inhibited the expression of p-MAPK and TNF-α. In addition, we further discovered that quercetin could increase ACC phosphorylation and CPT1α expression in PA-induced HepG2 cells. Conclusions. Our results indicated that quercetin, as the main active component in AMB, exerts an anti-NAFLD effect by regulating the AMPK/MAPK/TNF-α and AMPK/ACC/CPT1α signaling pathways to inhibit inflammation and alleviate lipid accumulation.