MicroRNAs as Biomarkers and Therapeutic Targets for Nonalcoholic Fatty Liver Disease: A Narrative Review.

PURPOSE: Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the world. However, biomarkers for NAFLD diagnosis and liver-specific drugs for treatment are lacking. This article reviews the possibility of circulating miRNAs in the diagnosis and treatment of NAFLD diseases and focuses on several well-studied miRNAs to provide preclinical data for subsequent related studies. METHODS: Related articles were identified through searches of the PubMed database for literature published from 2010 to December 2022. Search terms included NAFLD, microRNA, biomarker, diagnosis, and therapy. FINDINGS: Current research data indicate that some key circulating miRNAs may be used as diagnostic biomarkers of NAFLD and the combination of several miRNAs improves diagnostic performance. In addition, some preclinical trials using cell and mouse models provide a basis for some miRNAs as potential therapeutic targets. IMPLICATIONS: Current evidence suggests that circulating miRNAs are potential noninvasive biomarkers for clinical diagnosis of NAFLD, which needs to be validated in more heterogeneous and larger cohorts. In addition, several miRNAs regulate multiple downstream pathways related to the pathophysiology of NAFLD in a cell- and tissue-specific manner, making them attractive drug therapeutic targets for NAFLD. However, more preclinical and clinical trials are needed for these miRNAs to become therapeutic targets of NAFLD.

Profile of 5-HT2A receptor involved in signaling cascades associated to intracellular inflammation and apoptosis in hepatocytes and its role in carbon tetrachloride-induced hepatotoxicity.

Previously, we found that the 5-HT2A receptor plays a key role in cell injury. However, the mechanism by which the 5-HT2A receptor mediates intracellular processes remains unclear. In this study, we aimed to clarify this intracellular process in hepatocyte LO2 cells and evaluate its role in CCl4-induced hepatotoxicity in mice. In vitro, both the agonist and overexpression of 5-HT2A receptor could promote 5-HT degradation by upregulating the expression of 5-HT synthases and monoamine oxidase-A (MAO-A) to cause overproduction of ROS in mitochondria. We refer to this as the activation of the 5-HT degradation system (5DS) axis, which leads to the phosphorylation of JNK, p38 MAPK, STAT3, and NF-κB; upregulation of Bax, cleaved-caspase3, and cleaved-caspase9; and downregulation of Bcl-2, followed by apoptosis and oversecretion of TNF-α and IL-1ß in cells. This phenomenon could be markedly blocked by the 5-HT2A receptor antagonist, MAO-A inhibitor, or gene-silencing MAO-A. Through protein kinases C epsilon (PKCε) agonist treatment and gene silencing of the PKCε and 5-HT2A receptor, we demonstrated that the 5-HT2A receptor controls 5-HT synthases and MAO-A expression via the PKCε pathway in cells. Unexpectedly, we discovered that PKCε-mediated phosphorylation of the AKT/mTOR pathway is also a consequence of the activation of the 5DS axis. Furthermore, we confirmed that the inhibition of the 5DS axis using the 5-HT2A receptor antagonist could prevent hepatotoxicity induced by CCl4 both in vitro and in vivo, inhibiting the aforementioned signaling cascades, inflammation, and apoptosis, and that the 5DS activation area overlapped the necrotic area of mouse liver. Taken together, we revealed a 5DS axis in hepatocytes that controls the signaling cascades associated with inflammation and apoptosis and confirmed its role in CCl4-induced hepatotoxicity.

Carbon tetrachloride induced mitochondrial division, respiratory chain damage, abnormal intracellular [H+] and apoptosis are due to the activation of 5-HT degradation system in hepatocytes.

Previously we found that acute liver injury (ALI) with inflammation caused by carbon tetrachloride (CCl4) was associated with the activation of the 5-HT degradation system (5DS), which includes monoamine oxidase A (MAO-A), the 5-HT2A receptor, and 5-HT synthases in hepatocytes. This study aimed to determine the role of 5DS in mitochondrial damage and apoptosis. In hepatocyte LO2 cells, CCl4 activated 5-HT2A receptor at the gene level, and then 5-HT2A receptor mediated the expression of 5-HT synthase and MAO-A at the gene level. Suppression of 5DS with the 5-HT2A receptor antagonist, MAO-A inhibitor, or gene silencing MAO-A significantly reduced the CCl4-induced production of mitochondrial reactive oxygen species (ROS). The ROS-associated upregulation of mitochondrial division proteins (FIS1 and DRP1); downregulation of mitochondrial fusion-associated protein 1, respiratory chain proteins (ND1 and CYTB), and ATP6; and decrease of ATP levels were reversed. Moreover, ROS-associated abnormal levels of caspase pathway-associated proteins (Bcl-2, Bax, cleaved-caspase3 and cleaved-caspase9) and apoptosis were suppressed. Notably, a combination of 5-HT2A receptor antagonist and MAO-A inhibitor almost abolished CCl4 cytotoxicity; abolished mitochondrial membrane potential (MMP) depolarization, mitochondrial structural abnormality, and high mitochondrial pH, with low pH states of the nucleus and cytoplasm. The effects of both were more significant than either alone. LO2 cells exposed to H2O2 or depleted mitochondrial ROS showed that ROS induced mitochondrial division and apoptosis and inhibited the levels of respiratory chain proteins. CCl4-induced abnormalities of ATP generation and MMP were dependent on both ROS and other 5DS-associated factors, probably NH3. Investigation of CCl4-induced ALI mice showed that hepatic injury and apoptosis occur at the site of 5DS activation and are significantly inhibited by the 5-HT2A receptor antagonist and 5-HT synthetic inhibitor in a synergistic manner, as well as mitochondrial damage. Together, we revealed the close relationship between CCl4-induced activation of 5DS and mitochondrial damage, abnormal intracellular [H+], and apoptosis in hepatocytes.

5-HT2A Receptor and 5-HT Degradation Play a Crucial Role in Atherosclerosis by Modulating Macrophage Foam Cell Formation, Vascular Endothelial Cell Inflammation, and Hepatic Steatosis.

AIM: Previously, we found that diabetes-related liver dysfunction is due to activation of the 5-HT2A receptor (5-HT2AR) and increased synthesis and degradation of 5-HT. Here, we investigated the role of 5-HT in the development of atherosclerosis. METHODS: The study was conducted using high-fat diet-fed male ApoE-/- mice, THP-1 cell-derived macrophages, and HUVECs. Protein expression and biochemical indexes were determined by Western blotting and quantitative analysis kit, respectively. The following staining methods were used: oil red O staining (showing atherosclerotic plaques and intracellular lipid droplets), immunohistochemistry (showing the expression of 5-HT2AR, 5-HT synthase, and CD68 in the aortic wall), and fluorescent probe staining (showing intracellular ROS). RESULTS: In addition to improving hepatic steatosis, insulin resistance, and dyslipidemia, co-treatment with a 5-HT synthesis inhibitor and a 5-HT2AR antagonist significantly suppressed the formation of atherosclerotic plaques and macrophage infiltration in the aorta of ApoE-/- mice in a synergistic manner. Macrophages and HUVECs exposed to oxLDL or palmitic acid in vitro showed that activated 5-HT2AR regulated TG synthesis and oxLDL uptake by activating PKCε, resulting in formation of lipid droplets and even foam cells; ROS production was due to the increase of both intracellular 5-HT synthesis and mitochondrial MAO-A-catalyzed 5-HT degradation, which leads to the activation of NF-κB and the release of the inflammatory cytokines TNF-α and IL-1ß from macrophages and HUVECs as well as MCP-1 release from HUVECs. CONCLUSION: Similar to hepatic steatosis, the pathogenesis of lipid-induced atherosclerosis is associated with activation of intracellular 5-HT2AR, 5-HT synthesis, and 5-HT degradation.

Renal ischemia/reperfusion injury in rats is probably due to the activation of the 5-HT degradation system in proximal renal tubular epithelial cells.

AIMS: To explore the relationship between renal ischemia/reperfusion injury (RIRI) and the activation of the renal 5-HT degradation system, including 5-HT2A receptor (5-HT2AR), 5-HT synthases and monoamine oxidase-A (MAO-A). MAIN METHODS: Rat RIRI was induced by removing the right kidney, causing ischemia of the left kidney for 45 min and reperfusion for different times. RIRI model (ischemia for 45 min and reperfusion for 24 h) was pretreated with 5-HT2AR antagonist sarpogrelate hydrochloride (SH) and the 5-HT synthase inhibitor carbidopa. In HK-2 cells, cellular damage was induced by hypoxia (24 h)/reoxygenation (12 h) (H/R) and treated with SH, carbidopa or the MAO-A inhibitor clorgyline. Hematoxylin-eosin, immunohistochemistry, TUNEL and fluorescent probe staining, RT-qPCR, western blotting, ELISA, etc. were used in the tests. KEY FINDINGS: The development of RIRI and the emergence of the RIRI peak were consistent with renal 5-HT degradation system activation. The highest expression regions of the 5-HT degradation system overlapped with those of the most severe lesions in the kidney, which were in proximal renal tubules. Rat RIRI and HK-2 cell damage, including oxidative stress, inflammation and apoptosis, could be almost abolished by synergistic inhibition of SH and carbidopa. Clorgyline also abolished the cellular damage induced by H/R. H/R-induced production of mitochondrial ROS in HK-2 cells was due to MAO-A-catalyzed 5-HT degradation, and 5-HT2AR was involved by mediating the expression of 5-HT synthases and MAO-A. SIGNIFICANCE: These findings revealed a close association between RIRI and activation of the renal 5-HT degradation system.

Nephrotoxicity induced by cisplatin is primarily due to the activation of the 5-hydroxytryptamine degradation system in proximal renal tubules.

As a widely used anticancer drug in the clinic, cisplatin has obvious side effects, especially nephrotoxicity. Previous studies have suggested that the accumulation of intracellular reactive oxygen species (ROS) is a hallmark of cisplatin-induced acute kidney injury. This study aimed to investigate the relationship between ROS accumulation induced by cisplatin and 5-HT degradation. In vivo, by HE and TUNEL staining, we found that cisplatin-induced renal lesions and apoptotic regions, which were located in proximal tubular epithelial cells, were also the regions in which tryptophan hydroxylase 1 (Tph1), aromatic l-amino acid decarboxylase (AADC), 5-HT2A receptor (5-HT2AR) and monoamine oxidase A (MAO-A) were overexpressed, as determined by immunohistochemistry. Notably, the 5-HT2AR antagonist sarpogrelate hydrochloride (SH) and the AADC inhibitor carbidopa (CDP) significantly attenuated cisplatin-induced increases in serum creatinine and blood urea nitrogen levels, renal ROS levels, oxidative stress (SOD activity and MDA), proinflammatory cytokine levels (NF-κB, TNF-α and IL-1ß), proapoptotic factor levels (Bax, Bcl-2, C-caspase 3 and C-caspase 9) and the phosphorylation of p38 and STAT3, as well as renal lesions and apoptosis. The combination of SH and CDP could almost abolish the effects of cisplatin challenge. In vitro, the effects of cisplatin challenge and the inhibitory effects of SH and CDP were also observed in HK-2 cells. Additionally, similar to the combination of SH and CDP, the MAO-A inhibitor clorgyline could also abolish the effects of cisplatin challenge. More importantly, by western blotting, we detected that the upregulation of Tph1, AADC and MAO-A expression induced by cisplatin both in vivo and in vitro could be obviously suppressed by SH to decrease 5-HT synthesis and mitochondrial 5-HT degradation. Altogether, these findings suggested that cisplatin-induced nephrotoxicity is due to the activation of the 5-HT degradation system in proximal tubular epithelial cells, including 5-HT2AR and 5-HT synthesis and degradation. 5-HT2AR plays a role by mediating the expression of MAO-A and the 5-HT synthases Tph1 and AADC.

Role of 5-HT degradation in acute liver injury induced by carbon tetrachloride.

5-hydroxytryptamine (5-HT) is involved in the pathological processes of several liver diseases. Acute liver injury underlies the development of many liver diseases, but the mechanism remains unclear. We aimed to investigate the role of 5-HT in carbon tetrachloride (CCl4)-induced acute liver injury. Acute liver injury was induced with CCl4 (10 mg/kg) in mice pretreated with the 5-HT2A receptor antagonist sarpogrelate hydrochloride (SH) and the 5-HT synthesis inhibitor carbidopa (CDP). LO2 cells were treated with CCl4, 5-HT or 2,5-dimethoxy-4-idopametamine and pretreated with SH, CDP or the monoamine oxidase A (MAO-A) inhibitor clorgyline. Hematoxylin-eosin staining, immunohistochemistry, Real-time quantitative PCR, western blotting, fluorescent probe and biochemical markers were used to evaluate liver compromise. 5-HT2A receptor, 5-HT synthetase and MAO-A were expressed in hepatocytes; their gene and protein expression were upregulated by CCl4, which led to the degradation of mitochondrial 5-HT and overproduction of reactive oxygen species (ROS). Hepatic injury may be aggravated by ROS, which induce oxidative stress and the phosphorylation of p38 mitogen-activated protein kinase, Jun N-terminal kinase, extracellular regulated protein kinase, signal transducer and activator of transcription 3 and nuclear factor kappa-B. 5-HT2A receptor may contribute to acute liver injury by modulating 5-HT synthase and MAO-A expression. The synergistic action of SH and CDP treatment may inhibit CCl4-induced acute liver injury in a dose-dependent manner. Hence, CCl4-induced acute liver injury is due to an increase in mitochondrial ROS production caused by increased 5-HT degradation and probably involves increases in 5-HT2A receptor expression and 5-HT synthesis.