MAPK p38/Ulk1 pathway inhibits autophagy and induces IL-1β expression in hepatic stellate cells

Background: In the past, hepatic stellate cells (HSCs) were considered to be noninflammatory cells and contribute to liver fibrosis by producing extracellular matrix. Recently, it was found that HSCs can also secrete cytokines and chemokines and therefore participate in hepatic inflammation. Autophagy participates in many immune response processes in immune cells. It is unclear whether autophagy is involved in inflammatory cytokine induction in HSCs. Methods: MAPK p38, Ulk1 phosphorylation and the Ulk1-Atg13 complex were analyzed in HSC-T6 cells after LPS treatment. The relationship between autophagy inhibition and inflammation was investigated in primary rat HSCs. Results: We discovered that LPS inhibited autophagy through MAPK p38. The activation of MAPK p38 induced Ulk1 phosphorylation, which disrupted the Ulk1-Atg13 complex and therefore inhibited autophagy. Furthermore, in primary rat HSCs, we demonstrated that autophagy inhibition regulated IL-1β induction, which depended on the MAPK p38/Ulk1 pathway. Conclusions: Our results reveal a continuous signaling pathway, MAPK p38-Ulk1 phosphorylation-Ulk1/Atg13 disruption, which inhibits autophagy and induces IL-1β expression in HSCs.

Heme Oxygenase-1 Inhibits the Proliferation of Hepatic Stellate Cells by Activating PPARγ and Suppressing NF-κB

Background. Hepatic stellate cells (HSCs) are reported to play significant roles in the development of liver fibrosis. Heme oxygenase-1 (HO-1) is a key rate-limiting enzyme, which could decrease collagen synthesis and liver damage. Nevertheless, it was yet elusive towards the function and mechanism of HO-1. Methods. An HO-1 inducer Hemin or an HO-1 inhibitor ZnPP-IX was used to treat the activated HSC-T6, respectively. MTT assay was adopted to detect cell proliferation. Immunocytochemical staining was employed to test the levels of alpha-smooth muscle actin (α-SMA), peroxisome proliferator-activated receptor-γ (PPARγ), and nuclear factor-kappa B (NF-kappa B) levels in HSC-T6. HO-1, PPARγ, and NF-κB expression levels were measured by qRT-PCR and Western blotting. ELISA was then used to detect the levels of transforming growth factor- (TGF-) beta 1 (TGF-β1), interleukin-6 (IL-6), serum hyaluronic acid (HA), and serum type III procollagen aminopeptide (PIIIP). Results. HSC-T6 proliferation was inhibited in Hemin-treated HSCs. The levels of α-SMA, HA, and PIIIP and the production of ECM were lower in Hemin-treated HSCs, whereas those could be rescued by ZnPP-IX. NF-κB activation was decreased, but PPARγ expression was increased after HO-1 upregulation. Furthermore, the levels of TGF-β1 and IL-6, which were downstream of activated NF-κB in HSC-T6, were reduced. The PPAR-specific inhibitor GW9662 could block those mentioned effects. Conclusions. Our data demonstrated that HO-1 induction could inhibit HSC proliferation and activation by regulating PPARγ expression and NF-κB activation directly or indirectly, which makes it a promising therapeutic target for liver fibrosis.

Chemerin Overexpression in the Liver Protects against Inflammation in Experimental Non-Alcoholic Steatohepatitis

Non-alcoholic steatohepatitis (NASH) is marked by macrophage infiltration and inflammation. Chemerin is a chemoattractant protein and is abundant in hepatocytes. The aim of this study was to gain insight into the role of hepatocyte-produced prochemerin in NASH. Therefore, mice were infected with adeno-associated virus 8 to direct hepatic overexpression of prochemerin in a methionine–choline deficient dietary model of NASH. At the end of the study, hepatic and serum chemerin were higher in the chemerin-expressing mice. These animals had less hepatic oxidative stress, F4/80 and CC-chemokine ligand 2 (CCL2) protein, and mRNA levels of inflammatory genes than the respective control animals. In order to identify the underlying mechanisms, prochemerin was expressed in hepatocytes and the hepatic stellate cells, LX-2. Here, chemerin had no effect on cell viability, production of inflammatory, or pro-fibrotic factors. Notably, cultivation of human peripheral blood mononuclear cells (PBMCs) in the supernatant of Huh7 cells overexpressing chemerin reduced CCL2, interleukin-6, and osteopontin levels in cell media. CCL2 was also low in RAW264.7 cells exposed to Hepa1–6 cell produced chemerin. In summary, the current study showed that prochemerin overexpression had little effect on hepatocytes and hepatic stellate cells. Of note, hepatocyte-produced chemerin deactivated PBMCs and protected against inflammation in experimental NASH.

Forsythiaside A Regulates Activation of Hepatic Stellate Cells by Inhibiting NOX4-Dependent ROS

Hepatic stellate cells (HSCs) activation is an important step in the process of hepatic fibrosis. NOX4 and reactive oxygen species expressed in HSCs play an important role in liver fibrosis. Forsythiaside A (FA), a phenylethanoid glycoside extracted and isolated from Forsythiae Fructus, has significant antioxidant activities. However, it is not clear whether FA can play a role in inhibiting the HSCs activation through regulating NOX4/ROS pathway. Therefore, our purpose is to explore the effect and mechanism of FA on HSCs activation to alleviate liver fibrosis. LX2 cells were activated by TGF-β1 in vitro. MTT assay and Wound Healing assay were used to investigate the effect of FA on TGF-β1-induced LX2 cell proliferation and migration. Elisa kit was used to measure the expression of MMP-1 and TIMP-1. Western blot and RT-qPCR were used to investigate the expression of fibrosis-related COLI, α-SMA, MMP-1 and TIMP-1, and inflammation-related TNF-α, IL-6 and IL-1β. The hydroxyproline content was characterized using a biochemical kit. The mechanism of FA to inhibit HSCs activation and apoptosis was detected by DCF-DA probe, RT-qPCR, western blot and flow cytometry. NOX4 siRNA was used to futher verify the effect of FA on NOX4/ROS pathway. The results showed that FA inhibited the proliferation and migration of LX2 cells and adjusted the expression of MMP-1, TIMP-1, COLI, α-SMA, TNF-α, IL-6 and IL-1β as well as promoted collagen metabolism to show potential in anti-hepatic fibrosis. Mechanically, FA down-regulated NOX4/ROS signaling pathway to improve oxidation imbalances, and subsequently inhibited PI3K/Akt pathway to suppress proliferation. FA also promoted the apoptosis of LX2 cells by Bax/Bcl2 pathway. Furthermore, the effects of FA on TGF-β1-induced increased ROS levels and α-SMA and COLI expression were weaken by silencing NOX4. In conclusion, FA had potential in anti-hepatic fibrosis at least in part by remolding of extracellular matrix and improving oxidation imbalances to inhibit the activation of HSCs and promote HSCs apoptosis.

Nano delivery of simvastatin targets liver sinusoidal endothelial cells to remodel tumor microenvironment for hepatocellular carcinoma

Background Hepatocellular carcinoma (HCC) developed in fibrotic liver does not respond well to immunotherapy, mainly due to the stromal microenvironment and the fibrosis-related immunosuppressive factors. The characteristic of liver sinusoidal endothelial cells (LSECs) in contributing to fibrosis and orchestrating immune response is responsible for the refractory to targeted therapy or immunotherapy of HCC. We aim to seek a new strategy for HCC treatment based on an old drug simvastatin which shows protecting effect on LSEC. Method The features of LSECs in mouse fibrotic HCC model and human HCC patients were identified by immunofluorescence and scanning electron microscopy. The effect of simvastatin on LSECs and hepatic stellate cells (HSCs) was examined by immunoblotting, quantitative RT-PCR and RNA-seq. LSEC-targeted delivery of simvastatin was designed using nanotechnology. The anti-HCC effect and toxicity of the nano-drug was evaluated in both intra-hepatic and hemi-splenic inoculated mouse fibrotic HCC model. Results LSEC capillarization is associated with fibrotic HCC progression and poor survival in both murine HCC model and HCC patients. We further found simvastatin restores the quiescence of activated hepatic stellate cells (aHSCs) via stimulation of KLF2-NO signaling in LSECs, and up-regulates the expression of CXCL16 in LSECs. In intrahepatic inoculated fibrotic HCC mouse model, LSEC-targeted nano-delivery of simvastatin not only alleviates LSEC capillarization to regress the stromal microenvironment, but also recruits natural killer T (NKT) cells through CXCL16 to suppress tumor progression. Together with anti-programmed death-1-ligand-1 (anti-PD-L1) antibody, targeted-delivery of simvastatin achieves an improved therapeutic effect in hemi-splenic inoculated advanced-stage HCC model. Conclusions These findings reveal an immune-based therapeutic mechanism of simvastatin for remodeling immunosuppressive tumor microenvironment, therefore providing a novel strategy in treating HCC. Graphical Abstract

Dendropanoxide, a Triterpenoid from Dendropanax morbifera, Ameliorates Hepatic Fibrosis by Inhibiting Activation of Hepatic Stellate Cells through Autophagy Inhibition

Hepatic fibrosis results from chronic liver damage and is characterized by excessive accumulation of extracellular matrix (ECM). In this study, we showed that dendropanoxide (DPX), isolated from Dendropanax morbifera, had anti-fibrotic effects on hepatic fibrosis by inhibiting hepatic stellate cell (HSC) activation. DPX suppressed mRNA and protein expression of α-SMA, fibronectin, and collagen in activated HSCs. Moreover, DPX (40 mg/kg) treatment significantly lowered levels of liver injury markers (aspartate aminotransferase and alanine transaminase), expression of fibrotic markers, and deposition of ECM in a carbon tetrachloride-induced mouse model. Anti-fibrotic effects of DPX were comparable to those of silymarin in a hepatic fibrosis mouse model. As a possible mechanism of anti-fibrotic effects, we showed that DPX inhibited autophagosome formation (LC3B-II) and degradation of p62, which have important roles in HSC activation. These findings suggest that DPX inhibits HSC activation by inhibiting autophagy and can be utilized in hepatic fibrosis therapy.

The Critical Modulatory Role of Spiny Stellate Cells in Seizure Onset Based on Dynamic Analysis of a Neural Mass Model

Growing evidence suggests that excitatory neurons in the brain play a significant role in seizure generation. Nonetheless, spiny stellate cells are cortical excitatory non-pyramidal neurons in the brain, whose basic role in seizure occurrence is not well understood. In the present research, we study the critical role of spiny stellate cells or the excitatory interneurons (EI), for the first time, in epileptic seizure generation using an extended neural mass model inspired by a thalamocortical model originally introduced by another research group. Applying bifurcation analysis on this modified model, we investigated the rich dynamics corresponding to the epileptic seizure onset and transition between interictal and ictal states caused by EI connectivity to other cell types. Our results indicate that the transition between interictal and ictal states (preictal signal) corresponds to a supercritical Hopf bifurcation, and thus, the extended model suggests that before seizure onset, the amplitude and frequency of neural activities gradually increase. Moreover, we showed that (1) the altered function of GABAergic and glutamatergic receptors of EI can cause seizure, and (2) the pathway between the thalamic relay nucleus and EI facilitates the transition from interictal to ictal activity by decreasing the preictal period. Thereafter, we considered both sensory and cortical periodic inputs to study model responses to various harmonic stimulations. Bifurcation analysis of the model, in this case, suggests that the initial state of the model might be the main cause for the transition between interictal and ictal states as the stimulus frequency changes. The extended thalamocortical model shows also that the amplitude jump phenomenon and non-linear resonance behavior result from the preictal state of the modified model. These results can be considered as a step forward to a deeper understanding of the mechanisms underlying the transition from normal activities to epileptic activities.

Hepatic Stellate Cell: A Double-Edged Sword in the Liver

Hepatic stellate cells (HSCs) are located in the space of Disse, between liver sinusoidal endothelia cells (LSECs) and hepatocytes. They have surprised and excited hepatologists for their biological characteristics. Under physiological quiescent conditions, HSCs are the major vitamin A-storing cells of the liver, playing crucial roles in the liver development, regeneration, and tissue homeostasis. Upon injury-induced activation, HSCs convert to a pro-fibrotic state, producing the excessive extracellular matrix (ECM) and promoting angiogenesis in the liver fibrogenesis. Activated HSCs significantly contribute to liver fibrosis progression and inactivated HSCs are key to liver fibrosis regression. In this review, we summarize the comprehensive understanding of HSCs features, including their roles in normal liver and liver fibrosis in hopes of advancing the development of emerging diagnosis and treatment for hepatic fibrosis.