The genus Ruta in the family Rutaceae includes about 40 species, such as the well-known plants R. graveolens L. (common rue) or R. chalepensis L. (fringed rue), but also much lesser-known species such as R. angustifolia Pers. (narrow-leaved fringed rue). This rue specie, originating from the Mediterranean region, is well-distributed in Southeast Asia, notably in the Indo-Chinese peninsula and other territories. In some countries, such as Malaysia, the plant is used to treat liver diseases and cancer. Extracts of R. angustifolia display antifungal, antiviral and antiparasitic effects. Diverse bioactive natural products have been isolated from the aerial parts of the plant, notably quinoline alkaloids and furocoumarins, which present noticeable anti-inflammatory, antioxidant and/or antiproliferative properties. The present review discusses the main pharmacological properties of the plant and its phytoconstituents, with a focus on the anticancer activities evidenced with diverse alkaloids and terpenoids isolated from the aerial parts of the plant. Quinoline alkaloids such as graveoline, kokusaginine, and arborinine have been characterized and their mode of action defined. Arborinine stands as a remarkable inhibitor of histone demethylase LSD1, endowed with promising anticancer activities. Other anticancer compounds, such as the furocoumarins chalepin and rutamarin, have revealed antitumor effects. Their mechanism of action is discussed together with that of other bioactive natural products, including angustifolin and moskachans. Altogether, R. angustifolia Pers. presents a rich phytochemical profile, fully consistent with the traditional use of the plant to treat cancer. This rue species, somewhat neglected, warrant further investigations as a medicinal plant and a source of inspiration for drug discovery and design.

https://www.mdpi.com/2223-7747/12/4/827/pdf?version=1676277724

Ruta angustifolia Pers. (Narrow-Leaved Fringed Rue): Pharmacological Properties and Phytochemical Profile

ABSTRACT ATG4B, a cysteine protease promoting autophagosome formation by reversibly modifying Atg8-family proteins, plays a vital role in controlling macroautophagy/autophagy initiation in response to stress. However, the molecular mechanism underlying the regulation of ATG4B activity is far from well elucidated. In the current study, we firstly revealed that the acetylation level of ATG4B at lysine residue 39 (K39) is strongly involved in regulating its activity and autophagy. Specifically, SIRT2 deacetylates ATG4B K39, enhancing ATG4B activity and autophagic flux, which can be antagonized by EP300/p300. Starvation treatment contributes to EP300 suppression and SIRT2 activation, promoting the deacetylation of ATG4B K39, which leads to the elevation of ATG4B activity and finally autophagy initiation. Mechanistic investigation showed that starvation reduces CCNE (cyclin E), resulting in the downregulation of the CCNE-CDK2 protein complex, decreasing the phosphorylation of SIRT2 Ser331 and finally activating SIRT2. In addition, we confirmed that SIRT2 promotes autophagy via suppressing acetylation of ATG4B at K39 using sirt2 gene knockout (sirt2 −/−) mice. Collectively, our results have revealed the acetylation-mediated regulation of ATG4B cysteine protease activity in autophagy initiation in response to nutritional deficiency.

Involvement of acetylation of ATG4B in controlling autophagy induction

Alcohol abuse, reported by 1/8th critically ill patients, is an independent risk factor for death in sepsis. Sepsis kills over 270,000 patients/year in the US. We reported that the ethanol-exposure suppresses innate-immune response, pathogen clearance, and decreases survival in sepsis-mice via sirtuin 2 (SIRT2). SIRT2 is an NAD+-dependent histone-deacetylase with anti-inflammatory properties. We hypothesized that in ethanol-exposed macrophages, SIRT2 suppresses phagocytosis and pathogen clearance by regulating glycolysis. Immune cells use glycolysis to fuel increased metabolic and energy demand of phagocytosis. Using ethanol-exposed mouse bone marrow- and human blood monocyte-derived macrophages, we found that SIRT2 mutes glycolysis via deacetylating key glycolysis regulating enzyme phosphofructokinase-platelet isoform (PFKP), at mouse lysine 394 (mK394, human: hK395). Acetylation of PFKP at mK394 (hK395) is crucial for PFKP function as a glycolysis regulating enzyme. The PFKP also facilitates phosphorylation and activation of autophagy related protein 4B (Atg4B). Atg4B activates microtubule associated protein 1 light chain-3B (LC3). LC3 is a driver of a subset of phagocytosis, the LC3-associated phagocytosis (LAP), which is crucial for segregation and enhanced clearance of pathogens, in sepsis. We found that in ethanol-exposed cells, the SIRT2-PFKP interaction leads to decreased Atg4B-phosphorylation, decreased LC3 activation, repressed phagocytosis and LAP. Genetic deficiency or pharmacological inhibition of SIRT2 reverse PFKP-deacetylation, suppressed LC3-activation and phagocytosis including LAP, in ethanol-exposed macrophages to improve bacterial clearance and survival in ethanol with sepsis mice. Graphical Abstract

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SIRT2-PFKP interaction dysregulates phagocytosis in macrophages with acute ethanol-exposure

As one of the major acetyltransferases in mammalian cells, p300 (also known as EP300) and its highly related protein CBP (also known as CREBBP), collectively termed p300/CBP, is characterized as a key regulator in gene transcription by modulating the acetylation of histones. In recent decades, proteomic analyses have revealed that p300 is also involved in the regulation of various cellular processes by acetylating many non-histone proteins. Among the identified substrates, some are key players involved in different autophagy steps, which together establish p300 as a master regulator of autophagy. Accumulating evidence has shown that p300 activity is controlled by many distinct cellular pathways to regulate autophagy in response to cellular or environmental stimuli. In addition, several small molecules have been shown to regulate autophagy by targeting p300, suggesting that manipulation of p300 activity is sufficient for controlling autophagy. Importantly, dysfunction of p300-regulated autophagy has been implicated in a number of human disorders, such as cancer, aging and neurodegeneration, highlighting p300 as a promising target for the drug development of autophagy-related human disorders. Here, we focus on the roles of p300-mediated protein acetylation in the regulation of autophagy and discuss implications for autophagy-related human disorders.

Emerging roles of p300/CBP in autophagy and autophagy-related human disorders.

Bergenin (BER), a natural component of polyphenols, has a variety of pharmacological activities, especially in improving drug metabolism, reducing cholestasis, anti-oxidative stress and inhibiting inflammatory responses. The aim of this study was to investigate the effects of BER on liver injury induced by isonicotinic acid hydrazide (INH) and rifampicin (RIF) in mice. The mice model of liver injury was established with INH (100 mg/kg)+RIF (100 mg/kg), and then different doses of BER were used to intervene. The pathological morphology and biochemical indicators of mice were detected. Meanwhile, RNA sequencing was performed to screen the differentially expressed genes and signaling pathways. Finally, critical differentially expressed genes were verified by qRT-PCR and Western blot. RNA sequencing results showed that 707 genes were significantly changed in the INH+RIF group compared with the Control group, and 496 genes were significantly changed after the BER intervention. These differentially expressed genes were mainly enriched in the drug metabolism, bile acid metabolism, Nrf2 pathway and TLR4 pathway. The validation results of qRT-PCR and Western blot were consistent with the RNA sequencing. Therefore, BER alleviated INH+RIF-induced liver injury in mice. The mechanism of BER improving INH+RIF-induced liver injury was related to regulating drug metabolism enzymes, bile acid metabolism, Nrf2 pathway and TLR4 pathway.

The Effect of Bergenin on Isonicotinic Acid Hydrazide and Rifampicin-Induced Liver Injury Revealed by RNA Sequencing

Eukaryotes initiate autophagy when facing environmental changes such as a lack of external nutrients. However, the mechanisms of autophagy initiation are still not fully elucidated. Here, we showed that deacetylation of ATG4B plays a key role in starvation-induced autophagy initiation. Specifically, we demonstrated that ATG4B is activated during starvation through deacetylation at K39 by the deacetylase SIRT2. Moreover, starvation triggers SIRT2 dephosphorylation and activation in a cyclin E/CDK2 suppression–dependent manner. Meanwhile, starvation down-regulates p300, leading to a decrease in ATG4B acetylation at K39. K39 deacetylation also enhances the interaction of ATG4B with pro-LC3, which promotes LC3-II formation. Furthermore, an in vivo experiment using Sirt2 knockout mice also confirmed that SIRT2-mediated ATG4B deacetylation at K39 promotes starvation-induced autophagy initiation. In summary, this study reveals an acetylation-dependent regulatory mechanism that controls the role of ATG4B in autophagy initiation in response to nutritional deficiency.

Deacetylation of ATG4B promotes autophagy initiation under starvation

Scoparone (SCO) has a wide range of pharmacological activities, especially antioxidant and lipid-lowering ones. The purpose of this study was to investigate the effect of SCO on alleviating liver injury induced by alcohol and high-fat diet (HFD) in mice. The pathomorphology, biochemical indices, lipid accumulation, alcohol metabolism, oxidative stress and inflammatory response were examined. RNA sequencing analysis was performed on liver tissues to identify differentially expressed genes (DEGs) and signaling pathways, thus elucidating the mechanism of SCO in protecting the liver. Finally, some of the DEGs were validated by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. The results showed that SCO had significant hepatoprotective effects, which could inhibit lipid accumulation, improve alcohol metabolism, reduce oxidative stress and inhibit inflammatory response. RNA sequencing results showed that 1208 genes were differentially expressed in liver tissues of mice treated with alcohol and HFD, while 2143 genes were significantly changed after SCO intervention. These DEGs were mainly involved in metabolism of xenobiotics by cytochrome P450, fatty acid (triglyceride) metabolism, and cholesterol synthesis pathways. In addition, the results of qRT-PCR and Western blot were consistent with the RNA sequencing. SCO can alleviate liver injury induced by alcohol and HFD in mice, and its mechanism may be related to regulating alcohol metabolism and lipid metabolism pathways. 

The effects of scoparone on alcohol and high-fat diet-induced liver injury revealed by RNA sequencing.

Porcine epidemic diarrhea virus (PEDV) is a threat to the health of newborn piglets and has a significant impact on the swine industry. Short-chain fatty acids (SCFAs) are gut microbial metabolites that regulate intestinal function through different mechanisms to enhance the intestinal barrier and immune function. In this study, we aimed to determine whether butyrate displayed a better effect than other SCFAs on limiting PEDV replication in porcine intestinal epithelial cells. Mechanistically, butyrate treatment activated the interferon (IFN) response and interferon-stimulated gene (ISG) expression. Further experiments showed that inhibition of GPR43 (free fatty acid receptor 2) in intestinal epithelial cells increased virus infection and reduced antiviral effects through IFN λ response. Our findings revealed that butyrate exerts its antiviral effects by inducing GPR43-mediated IFN production in intestinal epithelial cells.

Butyrate limits the replication of porcine epidemic diarrhea virus in intestine epithelial cells by enhancing GPR43-mediated IFN-III production

Effective treatment of liver fibrosis remains a challenging medical problem. Taraxasterol (TAR) has anti-inflammatory, anti-tumor and hepatoprotective effects. Studies have shown that TAR has good biological activity against liver injury induced by various factors. However, the anti-fibrotic effect of TAR and its mechanism are never clarified. The purpose of this study was to investigate the effects of TAR in liver fibrosis and to reveal its possible mechanism by RNA sequencing. Our results suggested that TAR attenuated CCl4-induced hepatocyte necrosis, inflammatory infiltration and ECM deposition. TAR inhibited the levels of ALT, AST, ALP, γ-GT, LN, HA, PC III and IV-C in serum and TNF-α, IL-6, IL-1β and MDA in liver. In addition, TAR increased the activities of SOD and GSH-Px in liver. RNA sequencing analysis of liver tissues revealed that CCl4 and TAR significantly altered 4,155 genes and 2,675 genes, respectively. TAR reversed changes in ECM-related genes. More specifically, TAR mediated the expression of genes related to the activation of the Hippo pathway, while inhibiting the expression of genes related to the activation of HIF-1α, TGF-β/Smad, and Wnt pathways. In the validation experiments, the qRT-PCR results showed that the expression levels of Yap1, Tead3, Hif1α, Vegfa, Tgfβ1, Want3a, and Ctnnb1 mRNA were consistent with the RNA sequencing results. The Western blot results showed that TAR inhibited the levels of TGF-β1 and p-Smad2. In addition, the results in vitro were consistent with those in vivo. Therefore, we concluded that TAR improved CCl4-induced liver fibrosis by regulating Hippo, HIF-1α, TGF-β/Smad and Wnt pathways.

The effects of taraxasterol on liver fibrosis revealed by RNA sequencing.

Animal and human health are severely threatened by coronaviruses. The enteropathogenic coronavirus, porcine epidemic diarrhea virus (PEDV), is highly contagious, leading to porcine epidemic diarrhea (PED), which causes large economic losses in the world's swine industry. Piglets are not protected from emerging PEDV variants; therefore, new antiviral measures for PED control are urgently required. Herein, the anti-PEDV effects and potential mechanisms of fangchinoline (Fan) were investigated. Fan dose-dependently inhibited a PEDV infection at 24 h post-infection (EC50 value = 0.67 μM). We found that Fan mainly affected the PEDV replication phase but also inhibited PEDV at the attachment and internalization stages of the viral life cycle. Mechanistically, Fan blocked the autophagic flux in PEDV-infected cells by regulating the expression of autophagy-related proteins and changing PEDV virus particles. In summary, Fan inhibits PEDV infection by blocking the autophagic flux in cells. Our findings will help develop new strategies to prevent and treat PEDV infection.

https://www.frontiersin.org/articles/10.3389/fmicb.2023.1164851/pdf

Haiyan He

Papers

Deacetylation of ATG4B promotes autophagy initiation under starvation

The effects of scoparone on alcohol and high-fat diet-induced liver injury revealed by RNA sequencing.

Butyrate limits the replication of porcine epidemic diarrhea virus in intestine epithelial cells by enhancing GPR43-mediated IFN-III production

The effects of taraxasterol on liver fibrosis revealed by RNA sequencing.

Fangchinoline inhibits the PEDV replication in intestinal epithelial cells via autophagic flux suppression