Suppression of ZEB1 by Ethyl caffeate attenuates renal fibrosis via switching glycolytic reprogramming.

Renal fibrosis (RF) is a common endpoint of various chronic kidney diseases, leading to functional impairment and ultimately progressing to end-stage renal failure. Glycolytic reprogramming plays a critical role in the pathogenesis of fibrosis, which maybe a potential therapeutic target for treating renal fibrosis. Here, we revealed the novel role of ZEB1 in renal fibrosis, and whether targeting ZEB1 is the underlying mechanism for the anti-fibrotic effects of ethyl caffeate (EC) to regulate the glycolytic process. Treatment of EC attenuated the renal fibrosis and inhibited ZEB1 expression in vivo and in vitro, reducing the upregulated expression of glycolytic enzymes (HK2, PKM2, PFKP) and key metabolites (lactic acid, pyruvate). ZEB1 overexpression promoted the renal fibrosis and glycolysis, whereas knockout of ZEB1 apparently attenuated renal fibrosis in vivo and in vitro. EC interacted with ZEB1 to modulate the glycolytic enzymes for suppressing the elevated glycolytic reprogramming during renal fibrosis. In summary, our study reveals that ZEB1 plays an important role in regulating glycolytic reprogramming during the renal tubular epithelial cell fibrosis, suggesting inhibition of ZEB1 may be a potential strategy for treating renal fibrosis. Additionally, EC is a potential new drug candidate for the treatment of renal fibrosis and CKD.

Liver X receptor inverse agonist SR9243 attenuates rheumatoid arthritis via modulating glycolytic metabolism of macrophages.

Liver X receptors (LXRs) which link lipid metabolism and inflammation, were overexpressed in experimental rheumatoid arthritis (RA) rats as observed in our previous studies, while suppression of LXRα by silybin ameliorates arthritis and abnormal lipid metabolism. However, the role of LXRs in RA remains undefined. In this study, we investigated the inhibition role of LXRs in the polarization and activation of M1 macrophage by using a special LXRs inverse agonist SR9243, which led to ameliorating the progression of adjuvant-induced arthritis (AIA) in rats. Mechanistically, SR9243 disrupted the LPS/IFN-γ-induced Warburg effect in M1 macrophages, while glycolysis inhibitor 2-DG attenuated the inhibition effect of SR9243 on M1 polarization and the cytokines expression of M1 macrophages including iNOS, TNF-α, and IL-6 in vitro. Furthermore, SR9243 downregulated key glycolytic enzymes, including LDH-A, HK2, G6PD, GLUT1, and HIF-1α in M1 macrophages, which is mediated by increased phosphorylation of AMPK (Thr172) and reduced downstream phosphorylation of mTOR (Ser2448). Importantly, gene silencing of LXRs compromises the inhibition effect of SR9243 on M1 macrophage polarization and activation. Collectively, for the first time, our findings suggest that the LXR inverse agonist SR9243 mitigates adjuvant-induced rheumatoid arthritis and protects against bone erosion by inhibiting M1 macrophage polarization and activation through modulation of glycolytic metabolism via the AMPK/mTOR/HIF-1α pathway.

Novel treatment for refractory rheumatoid arthritis with total glucosides of paeony and nobiletin codelivered in a self-nanoemulsifying drug delivery system.

Patients with refractory rheumatoid arthritis (RA) remain a substantial clinical problem, while the overexpression of P-glycoprotein (P-gp) on their lymphocytes may contribute to resistance to anti-rheumatic drugs. This study aims to develop a novel treatment for refractory RA consisting of the combination of total glucosides of paeony (TGPs) and the P-gp inhibitor nobiletin (N), which are codelivered in a self-nanoemulsifying drug delivery system (SNEDDS). Based on the solubility, compatibility, and pseudoternary phase diagram tests, a nano-SNEDDS formulation composed of capryol 90-cremophor EL35-tcranscutol HP (CET) to codeliver TGP and N was developed, and this formulation increased the bioavailability of TGP by 435.04% (indicated with paeoniflorin). A modified adjuvant-induced arthritis (AIA) rat model was verified for the overexpression of P-gp in lymphocytes and resistance to methotrexate (MTX) treatment at the reported anti-inflammatory dosage. CET formulation not only increased the solubility and permeability of TGP but also inhibited the function and expression of P-gp, leading to enhanced bioavailability and intracellular concentration in the lymphocytes of AIA rats and consequently boosting the anti-arthritic effects of TGP. Moreover, TGP and N coloaded CET reduced the expression of P-gp in AIA rats partly by inhibiting the phosphorylated AKT and HIF-1α pathways. In summary, TGP-N coloaded SNEDDS is a novel and effective treatment for refractory RA.

Liver X receptors conserve the therapeutic target potential for the treatment of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic multi-system autoimmune disease with extremely complex pathogenesis. Significantly altered lipid paradox related to the inflammatory burden is reported in RA patients, inducing 50% higher cardiovascular risks. Recent studies have also demonstrated that lipid metabolism can regulate many functions of immune cells in which metabolic pathways have altered. The nuclear liver X receptors (LXRs), including LXRα and LXRß, play a central role in regulating lipid homeostasis and inflammatory responses. Undoubtedly, LXRs have been considered as an attractive therapeutic target for the treatment of RA. However, there are some contradictory effects of LXRs agonists observed in previous animal studies where both pro-inflammatory role and anti-inflammatory role were revealed for LXRs activation in RA. Therefore, in addition to updating the knowledge of LXRs as the prominent regulators of lipid homeostasis, the purpose of this review is to summarize the effects of LXRs agonists in RA-associated immune cells, to explore the underlying reasons for the contradictory therapeutic effects of LXRs agonists observed in RA animal models, and to discuss future strategy for the treatment of RA with LXRs modulators.

Carboxylic acid reduction and sulfate-reducing bacteria stabilization combined remediation of Cr (VI)-contaminated soil.

For controlling heavy metal pollution, the utilization of carboxylic acids (CAs) combined with sulfate-reducing bacteria (SRB) for continuous and stable remediation of Cr (VI)-contaminated soil was comprehensively investigated. At pH 3, citrate and lactate had photocatalysis characteristics that enabled them to reduce high Cr (VI) concentrations. The reduction efficiencies of citrate and lactate were 99.16-100% and 80.78-87.00%, respectively. In the 40 mg L-1 Cr (VI) treatment, the total Cr adsorption rate of soil was 61.39-68.31%; as the pH increased, the Cr species adsorption capacity of the soil decreased. Following the addition of exogenous 100 mg L-1 Cr (VI), the Cr (VI) content of re-contaminated soil was reduced to 16.2734 ± 0.9505 mg L-1 or 15.8618 mg kg-1 by adding citrate or lactate. Then, using SRB via culture by mulching, addition of citrate or lactate markedly reduced the toxicity of Cr (VI). The respective citrate or lactate treatments had sulfur concentrations of sulfide from deep soil (high-sulfide layer) of 70.54 ± 17.59 and 98.85 ± 13.84 mg kg-1, respectively, and released Cr (VI) concentrations of 0.22 ± 0.25 and 3.64 ± 3.32 mg kg-1, respectively, due to oxidation upon air exposure. We used a two-stage remediation strategy for these treatments: First, CAs were used for photocatalytic reduction to reduce Cr (VI); next, CAs were utilized as carbon sources by SRB, which further reduced Cr (VI) and stabilized Cr species. In addition, citrate was more conducive than lactate to maintaining the stability of the soil microbial community. The results show that this method has potential in the remediation of Cr (VI)-contaminated soil.

Wutou decoction ameliorates experimental rheumatoid arthritis via regulating NF-kB and Nrf2: Integrating efficacy-oriented compatibility of traditional Chinese medicine.

BACKGROUND: Thousands of years of clinical application of Wutou decoction (WTD) support its reliable efficacy and safety in treating rheumatoid arthritis (RA). However, the underlying molecular mechanism remains unclear, and the synergistic involvement of assistant herbs in WTD in enhancing the sovereign herb in treating RA is unknown. PURPOSE: This study aimed to investigate the efficacy-oriented compatibility of five herbs in WTD and the underlying mechanisms. METHODS: The anti-arthritic effects of WTD and the compatibilities of the five herbs in WTD were studied in vivo with adjuvant-induced arthritis (AIA) rat model and in vitro with LPS-induced RAW264.7 macrophage. Network pharmacology analysis was conducted to identify the dominant pathways involved in the anti-arthritis mechanisms of WTD and how the five herbs work synergistically. The results were further verified by in vivo and in vitro experiments. RESULTS: Our data revealed that the five herbs in WTD exert synergistic anti-arthritic effects on RA. Moreover, Radix Aconite (AC) is the principal anti-inflammatory component in WTD according to the extent of therapeutic effects exerted on the AIA rats. In vivo and in vitro experiments demonstrated that WTD inhibited NF-κB phosphorylation and simultaneously increased the expression of Nrf2, which were the major pathways identified by the network pharmacology analysis. The major assistant component, Herba Ephedrae (EP), evidently inhibited NF-κB mediated inflammatory response. The other assistant component, Radix Astragali (AS), considerably enhanced the expression of Nrf2 when used alone or in combination with AC. These combinations improved the anti-arthritis effects on the AIA rats better than that of AC alone. Nevertheless, WTD always achieved the best effects than any combinations both in vivo and in vitro. CONCLUSION: The ministerial herbs EP and AS intensify the anti-arthritic effects of AC by regulating the NF-κB-mediated inflammatory pathway and the Nrf2-mediated anti-oxidation pathway which are the major pathways of WTD for alleviating the symptoms of RA.

Nobiletin ameliorates cardiac impairment and alleviates cardiac remodeling after acute myocardial infarction in rats via JNK regulation.

Nobiletin was found to protect against acute myocardial infarction (AMI)-induced cardiac function decline and myocardial remodeling, although the dose-effect relationship and underlying pathways remained unclear. In the current research, different doses of Nobiletin (7.5, 15 and 30 mg/kg/day) were administered to AMI rat model for 21 days. Survival rate, echocardiography, and histological analysis were assessed in vivo. In addition, MTT assay, flow cytometry, and Western blotting were conducted to explore Nobiletin's cytotoxicity and antiapoptotic effect on H9C2 cells. Mechanistically, the activation of MAPK effectors and p38 in vivo was studied. The results showed medium- and high-dose Nobiletin could significantly improve survival rate and cardiac function and reduce the area of infarction and cardiac fibrosis. Medium dose showed the best protection on cardiac functions, whereas high dose showed the best protective effect on cellular apoptosis and histological changes. JNK activation was significantly inhibited by Nobiletin in vivo, which could help to explain the partial contribution of autophagy to AMI-induced apoptosis and the discrepancy on dose-effect relationships. Together, our study suggested that JNK inhibition plays an important role in Nobiletin-induced antiapoptotic effect in myocardial infarction, and medium-dose Nobiletin demonstrated the strongest effect in vivo.

Nobiletin and its derivatives overcome multidrug resistance (MDR) in cancer: total synthesis and discovery of potent MDR reversal agents.

Our recent studies demonstrated that the natural product nobiletin (NOB) served as a promising multidrug resistance (MDR) reversal agent and improved the effectiveness of cancer chemotherapy in vitro. However, low aqueous solubility and difficulty in total synthesis limited its application as a therapeutic agent. To tackle these challenges, NOB was synthesized in a high yield by a concise route of six steps and fourteen derivatives were synthesized with remarkable solubility and efficacy. All the compounds showed improved sensitivity to paclitaxel (PTX) in P-glycoprotein (P-gp) overexpressing MDR cancer cells. Among them, compound 29d exhibited water solubility 280-fold higher than NOB. A drug-resistance A549/T xenograft model showed that 29d, at a dose of 50 mg/kg co-administered with PTX (15 mg/kg), inhibited tumor growth more effective than NOB and remarkably increased PTX concentration in the tumors via P-gp inhibition. Moreover, Western blot experiments revealed that 29d inhibited expression of NRF2, phosphorylated ERK and AKT in MDR cancer cells, thus implying 29d of multiple mechanisms to reverse MDR in lung cancer.

Urolithin B, a gut microbiota metabolite, protects against myocardial ischemia/reperfusion injury via p62/Keap1/Nrf2 signaling pathway.

Ischemia/reperfusion (IR) induces additional damage during the restoration of blood flow to ischemic myocardium. Urolithin B (UB) is one of the gut metabolites of ellagitannins, a class of antioxidant polyphenols, which was found to be protective against oxidative stress in multiple organs. However, the role of UB in cardiovascular disease remains elusive. Adult Sprague Dawley rats were subjected to left anterior descending artery ligation for 30 min followed by 120 min of reperfusion, with or without UB treatment. In vitro, the H9c2 cardiomyocytes were subjected to hypoxia (94 %N2/5 %CO2/1 %O2) for 3 h, followed by reoxygenation (74 %N2/5 %CO2/21 %O2) for 3 h (HR). UB was found to decrease myocardial infarct size and attenuate the cardiac dysfunction in the rats after IR, and protect against HR injury in H9c2 cardiomyocytes. Mechanistically, UB inhibited autophagy by activating Akt/mTOR/ULK1 pathway and protected against oxidative stress and caspase 3-dependent cell apoptosis. In particular, UB induced accumulation of p62 and its interaction with Keap1, which promoted Nrf2 nuclear translocation during HR insult. Of note, the protection of UB against superoxide production and apoptotic cell death was compromised with Nrf2 gene silencing. Taken together, our findings suggested that UB protected against myocardial IR injury at least partially via the p62/Keap1/Nrf2 signaling pathway, which highlights the potential of UB as a novel therapy for ischemic heart disease.

Increased expression of DRAM1 confers myocardial protection against ischemia via restoring autophagy flux.

BACKGROUND: DRAM1 (Damage-regulated autophagy modulator 1) was reported as one of the most important lysosome membrane protein that mediates the interaction between autophagosome and lysosome. Our aim was to investigate whether DRAM1 contributes to cardiac remodeling after acute myocardial infarction (AMI) and the underlying mechanisms. METHODS AND RESULTS: Adenovirus harboring DRAM1 was injected in the peri-infarct zone in a rat model of AMI experimentally produced by permanent ligation of left anterior descending (LAD) coronary artery. Increased DRAM1 expression protected the cardiomyocytes from ischemia stress-induced autophagy flux obstacle and improved cardiac prognosis after AMI. DRAM1 overexpression attenuated the accumulation of autophagy substrate protein, LC3IIand p62/SQSTM1 obviously both in vivo and in vitro. An adenovirus harboring mRFP-GFP-LC3 showed that DRAM1 overexpression restored the autophagic flux by enhancing autophagosome conversion to autophagolysosome. Although Atg12 mRNA was up-regulated with DRAM1 overexpression the free Atg12 protein was decreased accompanied by increased Atg12-Atg5 conjugate both in vitro and in vivo. Of interest, immunoprecipitation assay showed that DRAM1 interacted with Atg7, but without direct interaction with Atg5 or Atg12. Notably, the effect of DRAM1 on autophagy flux and cardiomyocyte protection could be mitigated by Atg7 siRNA. CONCLUSIONS: Our results indicated that DRAM1 protected cardiomyocytes from ischemia stress-induced autophagy flux obstacle and uncovered a novel DRAM1-Atg7-Atg12/Atg5 autophagy flux regulation pathway under conditions of myocardial ischemic stress.

Nobiletin attenuates adverse cardiac remodeling after acute myocardial infarction in rats via restoring autophagy flux.

BACKGROUND: Our previous study showed that autophagy flux was impaired with sustained heart ischemia, which exacerbated adverse cardiac remodeling after acute myocardial infarction (AMI). Here we investigated whether Nobiletin, a citrus polymethoxylated flavonoids, could restore the autophagy flux and improve cardiac prognosis after AMI. AMI was induced by ligating left anterior descending (LAD) coronary artery in rats. Nobiletin improved the post-infarct cardiac dysfunction significantly and attenuated adverse cardiac remodeling. Meanwhile, Nobiletin protected H9C2 cells against oxygen glucose deprivation (OGD) in vitro. The impaired autophagy flux due to ischemia was ameliorated after Nobiletin treatment by testing the autophagy substrate, LC3BⅡ and P62 protein level both in vivo and in vitro. GFP-mRFP-LC3 adenovirus transfection also supported that Nobiletin restored the impaired autophagy flux. Specifically, the autophagy flux inhibitor, chloroquine, but not 3 MA, alleviated Nobiletin-mediated protection against OGD. Notably, Nobiletin does not affect the activation of classical upstream autophagy signaling pathways. However, Nobiletin increased the lysosome acidation which also supported that Nobiletin accelerated autophagy flux. Taken together, our findings suggested that Nobiletin restored impaired autophagy flux and protected against acute myocardial infarction, suggesting a potential role of autophagy flux in Nobiletin-mediated myocardial protection.