GPR120/FFAR4 stimulation attenuates airway remodeling and suppresses IL-4- and IL-13-induced airway epithelial injury via inhibition of STAT6 and Akt.

BACKGROUND: Airway remodeling is associated with severity and treatment insensitivity in asthma. This study aimed to investigate the effects of G protein-coupled receptor 120 (GPR120) stimulation on alleviating allergic inflammation and remodeling of airway epithelium. RESEARCH DESIGN AND METHODS: Ovalbumin (OVA)-challenged BALB/c mice and type-2-cytokine (IL-4 and IL-13)-exposed 16HBE human bronchial epithelial cells were treated with GSK137647A, a selective GPR120 agonist. Markers of allergic inflammation and airway remodeling were determined. RESULTS: GSK137647A attenuated inflammation and mucus secretion in airway epithelium of OVA-challenged mice. Stimulation of GPR120 in 16HBE suppressed expression of asthma-associated cytokines and cytokine-induced expression of pathogenic mucin-MUC5AC. These effects were abolished by co-treatment with AH7614, a GPR120 antagonist. Moreover, GPR120 stimulation in 16HBE cells reduced expression of fibrotic markers including fibronectin protein and ACTA2 mRNA and inhibited epithelial barrier leakage induced by type-2 inflammation via rescuing expression of zonula occludens-1 protein. Furthermore, GPR120 stimulation prevented the cytokine-induced airway epithelial remodeling via suppression of STAT6 and Akt phosphorylation. CONCLUSIONS: Our findings suggest that GPR120 activation alleviates allergic inflammation and remodeling of airway epithelium partly through inhibition of STAT6 and Akt. GPR120 may represent a novel therapeutic target for diseases associated with remodeling of airway epithelium, including asthma.

Discovery of a chalcone derivative as an anti-fibrotic agent targeting transforming growth factor-ß1 signaling: Potential therapy of renal fibrosis.

As a final common pathway of renal injuries, renal fibrosis leads to chronic kidney disease (CKD). Currently, there is no safe and effective therapy to prevent the progression of renal fibrosis to CKD. Inhibition of transforming growth factor-ß1 (TGF-ß1) pathway is proposed as one of the most promising approaches for anti-renal fibrosis therapies. This study aimed to identify novel anti-fibrotic agents using the TGF-ß1-induced fibrosis in renal proximal tubule epithelial cells (RPTEC) and characterize their mechanism of action as well as in vivo efficacy. By screening 362 natural product-based compounds for their ability to reduce collagen accumulation assessed by picro-sirius red (PSR) staining in RPTEC cells, a chalcone derivative AD-021 was identified as an anti-fibrotic agent with IC50 of 14.93 µM. AD-021 suppressed TGF-ß1-induced collagen production, expression of pro-fibrotic proteins (fibronectin and α-smooth muscle actin (αSMA)), and Smad-dependent and Smad-independent signaling pathways via suppression of TGF-ß receptor II (TGFßRII) phosphorylation in RPTEC cells. Furthermore, TGF-ß1-induced mitochondrial fission in RPTEC cells was ameliorated by AD-021 via mechanisms involving inhibition of Drp1 phosphorylation. In a mouse model of unilateral ureteral obstruction (UUO)-induced renal fibrosis, AD-021 reduced plasma TGF-ß1, ameliorated renal fibrosis and improved renal function. Collectively, AD-021 represents a novel class of natural product-based anti-fibrotic agent that has therapeutic potential in the prevention of fibrosis-associated renal disorders including CKD.

Natural statin derivatives as potential therapy to reduce intestinal fluid loss in cholera.

As a leading cause of death in children under 5 years old, secretory diarrheas including cholera are characterized by excessive intestinal fluid secretion driven by enterotoxin-induced cAMP-dependent intestinal chloride transport. This study aimed to identify fungal bioactive metabolites possessing anti-secretory effects against cAMP-dependent chloride secretion in intestinal epithelial cells. Using electrophysiological analyses in human intestinal epithelial (T84) cells, five fungus-derived statin derivatives including α,ß-dehydrolovastatin (DHLV), α,ß-dehydrodihydromonacolin K, lovastatin, mevastatin and simvastatin were found to inhibit the cAMP-dependent chloride secretion with IC50 values of 1.8, 8.9, 11.9, 11.4 and 5 µM, respectively. Being the most potent statin derivatives, DHLV was evaluated for its pharmacological properties including cellular toxicity, mechanism of action, target specificity and in vivo efficacy. DHLV at concentrations up to 20 µM did not affect cell viability and barrier integrity of T84 cells. Electrophysiological analyses indicated that DHLV inhibited cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent apical chloride channel, via mechanisms not involving alteration of intracellular cAMP levels or its negative regulators including AMP-activated protein kinases and protein phosphatases. DHLV had no effect on Na+-K+ ATPase activities but inhibited Ca2+-dependent chloride secretion without affecting intracellular Ca2+ levels. Importantly, intraperitoneal (2 mg/kg) and intraluminal (20 µM) injections of DHLV reduced cholera toxin-induced intestinal fluid secretion in mice by 59% and 65%, respectively without affecting baseline intestinal fluid transport. This study identifies natural statin derivatives as novel natural product-derived CFTR inhibitors, which may be beneficial in the treatment of enterotoxin-induced secretory diarrheas including cholera.

Isolation of CFTR and TMEM16A inhibitors from Neorautanenia mitis (A. Rich) Verdcourt: Potential lead compounds for treatment of secretory diarrhea.

A phytochemical study on the root extracts of Neorautanenia mitis, a Nigerian medicinal plant used in the management of diarrhea, led to the isolation of one new and 19 known natural products. These compounds and crude extracts were evaluated for Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Cl- channel and calcium-activated Cl- channel (TMEM16A) inhibitory activities in T84 and Calu-3 cells, respectively. Four compounds namely dolineon, neodulin, pachyrrhizine, and neotenone inhibited cAMP-induced Cl- secretion across T84 cell monolayers with IC50 values of ~0.81 µM, ~2.42 µM, ~2.87 µM, and ~4.66 µM, respectively. Dolineon having the highest inhibitory activity also inhibited a Ca + activated Cl- channel (TMEM16A) with an IC50 value of ~4.38 µM. The in vitro antidiarrheal activity of dolineon was evaluated on cholera toxin (CT) induced chloride secretion in T84 cells, where it inhibited CT-induced chloride secretion by >70% at 100 µM. Dolineon also inhibited CT-induced fluid secretion by ~70% in an in vivo mouse closed loop model at a dose of 16.9 µg/loop. The cytotoxicity of the extracts and compounds was evaluated on KB, Vero and BHK21 cells, dolineon showed low cytotoxicity of >29.6 µM and 57.30 + 6.77 µM against Vero and BHK21 cells, respectively. Our study revealed that several compounds isolated from N. mitis showed antidiarrheal activity. The most active compound dolineon can potentially serve as a lead compound towards the development of CFTR and TMEM16A inhibitors as future therapeutics for secretory diarrhea.

Activation of constitutive androstane receptor inhibits intestinal CFTR-mediated chloride transport.

Constitutive androstane receptor (CAR) belonging to the nuclear receptor superfamily plays an important role in the xenobiotic metabolism and disposition. It has been reported that CAR regulates the expression of the ATP-binding cassette (ABC) transporters in the intestine, such as multidrug resistance protein 1 (MDR1) and multidrug resistance-associated protein 2/3 (MRP2 and MRP3). In this study, we investigated the role of CAR in the regulation of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride transport in T84 human colonic epithelial cells and mouse intestinal tissues. Treatments of T84 cell monolayers with specific CAR agonists (CITCO and phenytoin at concentrations of 1 µM and 5 µM, respectively) for 24 h decreased transepithelial Cl- secretion in response to cAMP-dependent agonist. This inhibition was abolished by coincubation of CITCO with a CAR antagonist, CINPA1. We confirmed that an inhibitory effect of CAR agonists was not due to their cytotoxicity. Basolateral membrane permeabilization experiments also revealed that activation of CAR decreased apical Cl- current stimulated by both CPT-cAMP and genistein (a direct CFTR activator). Such activation also reduced both mRNA and protein expression of CFTR. Furthermore, CITCO decreased cholera toxin (CT)-induced Cl- secretion across T84 cell monolayers. In ICR mice, administration of TCPOBOP (3 mg/kgBW), a murine-specific CAR agonist, for 7 days produced significant decreases in CFTR mRNA and protein expressions in intestinal tissues. Interestingly, TCPOBOP also inhibited CT-induced intestinal fluid accumulation in mice. This is the first evidence showing that CFTR was downregulated by CAR activation in the intestine. Our findings suggest that CAR has potential as a new drug target for treatment of condition with hyperactivity/ hyperfunction of CFTR especially secretory diarrheas.

Exhaustive exercise decreases renal organic anion transporter 3 function.

This study aimed to investigate the effects of various types of exercise on organic anion transporter 3 (Oat3) function, a major transporter that plays a role in the secretion of a variety of drugs and endogenous compounds. Male Wistar rats were randomly allocated to non-exercise, exhaustive, acute and training exercise groups. The function of Oat3 was assessed by the uptake of [3H]-estrone sulfate ([3H]-ES) into rat renal cortical slices. Acute and training exercises had no effect on [3H]-ES uptake whereas a marked reduction in [3H]-ES uptake occurred immediately after exhaustive exercise. However, the reduction in Oat3 function was gradually recovered at 6 and 24 h after the exercise session. Importantly, the impairment of Oat3 function was associated with a decrease in renal Oat3 protein expression. Our results indicate that exhaustive exercise produces a significant impact on renal organic anion transport function, which in turn could alter the plasma level of drugs and compounds in the body.

Steviol slows renal cyst growth by reducing AQP2 expression and promoting AQP2 degradation.

Overexpression of aquaporin 2 (AQP2) was observed and suggested to be involved in fluid secretion leading to cyst enlargement in polycystic kidney disease (PKD). The cyst expansion deteriorates the renal function and, therefore, therapies targeting cyst enlargement are of clinical interest. Of note, inhibition of vasopressin function using vasopressin 2 receptor (V2R) antagonist which decreased cAMP production along with AQP2 production and function can slow cyst growth in ADPKD. This finding supports the role of AQP2 in cyst enlargement. Steviol, a major metabolite of the sweetening compound stevioside, was reported to retard MDCK cyst growth and enlargement by inhibiting CFTR activity. Interestingly, its efficacy was found to be higher than that of CFTRinh-172. Since steviol was also found to produce diuresis in rodent, it is likely that steviol might have an additional effect in retarding cyst progression, such as inhibition of AQP2 expression and function. Here, we investigated the effect of steviol on AQP2 function and on cyst growth using an in vitro cyst model (MDCK and Pkd1-/- cells). We found that steviol could markedly inhibit cyst growth by reducing AQP2 expression in both Pkd1-/- and MDCK cells. Real-time PCR also revealed that steviol decreased AQP2 mRNA expression level as well. Moreover, a proteasome inhibitor, MG-132, and the lysosomotropic agent, hydroxychloroquine (HCQ) were found to abolish the inhibitory effect of steviol in Pkd1-/- cells. Increased lysosomal enzyme marker (LAMP2) expression following steviol treatment clearly confirmed the involvement of lysosomes in steviol action. In conclusion, our finding showed for the first time that steviol slowed cyst growth, in part, by reducing AQP2 transcription, promoted proteasome, and lysosome-mediated AQP2 degradation. Due to its multiple actions, steviol is a promising compound for further development in the treatment of PKD.