Solanum melongena L. Extract Promotes Intestinal Tight Junction Re-Assembly via SIRT-1-Dependent Mechanisms.

Tight junction disruption can lead to pathogenesis of various diseases without therapeutic strategy to recover intestinal barrier integrity. The main objective of this study is to demonstrate the effect of Solanum melongena L. extract (SMLE) on intestinal tight junction recovery and its underlying mechanism. Intestinal barrier function is attenuated by Ca2+ depletion. SMLE treatment increased TER value across T84 cell monolayers. Permeability assay reveals that Ca2+ depletion promotes 4-kDa FITC-dextran permeability, but not 70-kDa FITC-dextran. SMLE suppresses the rate of 4-kDa FITC-dextran permeability, indicating that SMLE inhibits paracellular leak pathway permeability. SMLE-mediated TER increase and leak pathway suppression are abolished by neither calcium/calmodulin-dependent protein kinase kinase ß (CaMKKß) inhibitor nor AMP-activated protein kinase (AMPK) inhibitor. Furthermore, mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinase (ERK) inhibitors have no effects on SMLE-mediated TER increase and leak pathway suppression. Interestingly, SMLE is unable to enhance TER value and diminish leak pathway permeability in T84 cell monolayers pre-treated with sirtuin-1 (SIRT-1) inhibitor. Immunofluorescence staining reveals that SMLE enhances re-assembly of tight junction proteins, including occludin and ZO-1 to intercellular space but this effect is abolished by SIRT-1 inhibitor. These data suggest that SMLE promotes intestinal tight junction re-assembly via SIRT-1-dependent manner.

A sulfonamide chalcone AMPK activator ameliorates hyperglycemia and diabetic nephropathy in db/db mice.

Diabetic nephropathy (DN) is a serious complication of diabetes mellitus (DM), which currently lacks effective treatments. AMP-activated protein kinase (AMPK) stimulation by chalcones, a class of polyphenols abundantly found in plants, is proposed as a promising therapeutic approach for DM. This study aimed to identify novel chalcone derivatives with improved AMPK-stimulating activity in human podocytes and evaluate their mechanisms of action as well as in vivo efficacy in a mouse model of DN. Among 133 chalcone derivatives tested, the sulfonamide chalcone derivative IP-004 was identified as the most potent AMPK activator in human podocytes. Western blot analyses, intracellular calcium measurements and molecular docking simulation indicated that IP-004 activated AMPK by mechanisms involving direct binding at allosteric site of calcium-dependent protein kinase kinase ß (CaMKKß) without affecting intracellular calcium levels. Interestingly, eight weeks of intraperitoneal administration of IP-004 (20 mg/kg/day) significantly decreased fasting blood glucose level, activated AMPK in the livers, muscles and glomeruli, and ameliorated albuminuria in db/db type II diabetic mice. Collectively, this study identifies a novel chalcone derivative capable of activating AMPK in vitro and in vivo and exhibiting efficacy against hyperglycemia and DN in mice. Further development of AMPK activators based on chalcone derivatives may provide an effective treatment of DN.

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.

Anti-inflammatory activity of soluble chito-oligosaccharides (CHOS) on VitD3-induced human THP-1 monocytes.

Chito-oligosaccharides (CHOS) are oligomers of D-glucosamine and N-acetyl-glucosamine. Anti-inflammatory activities of a wide variety of CHOS mixtures have previously been reported, mainly based on studies with mouse models and murine macrophages. Since the mouse and human immune systems are quite different, gaining insight into the activity of CHOS on human cell lines, using well-characterized CHOS mixtures, is of considerable interest. Bacillus subtilis chitosanase (BsCsn46A) can efficiently convert chitosan to mixtures of water soluble low molecular weight CHOS. Here, the anti-inflammatory activity of a properly characterized CHOS mixture was studied, using human THP-1 cells that were differentiated to mature monocytes using vitamin D3. Addition of CHOS reduced the production of multiple pro-inflammatory cytokines associated with bacterial lipopolyssacharide (LPS)-stimulated inflammation, in a dose-dependent manner and without affecting cell viability. Interestingly, only minimal effects of CHOS were observed in similar experiments with phorbol 12-myristate 13-acetate- (PMA-) differentiated, macrophage-like, THP-1 cells. Altogether, in addition to showing promising biological effects of well-characterized low molecular weight soluble CHOS in a human system, the present study also points at Vitamin D3-stimulated THP-1 cells as a favorable system for assessing the anti-inflammatory activity of bioactive compounds.

Suppressed de novo lipogenesis by plasma membrane citrate transporter inhibitor promotes apoptosis in HepG2 cells.

Suppression of the expression or activities of enzymes that are involved in the synthesis of de novo lipogenesis (DNL) in cancer cells triggers cell death via apoptosis. The plasma membrane citrate transporter (PMCT) is the initial step that translocates citrate from blood circulation into the cytoplasm for de novo long-chain fatty acids synthesis. This study investigated the antitumor effect of the PMCT inhibitor (PMCTi) in inducing apoptosis by inhibiting the DNL pathway in HepG2 cells. The present findings showed that PMCTi reduced cell viability and enhanced apoptosis through decreased intracellular citrate levels, which consequently caused inhibition of fatty acid and triacylglycerol productions. Thus, as a result of the reduction in fatty acid synthesis, the activity of carnitine palmitoyl transferase-1 (CPT-1) was suppressed. Decreased CPT-1 activity also facilitated the disruption of mitochondrial membrane potential (ΔΨm) leading to stimulation of apoptosis. Surprisingly, primary human hepatocytes were not affected by PMCTi. Increased caspase-8 activity as a consequence of reduction in fatty acid synthesis was also found to cause disruption of ΔΨm. In addition, apoptosis induction by PMCTi was associated with an enhanced reactive oxygen species generation. Taken together, we suggest that inhibition of the DNL pathway following reduction in citrate levels is an important regulator of apoptosis in HepG2 cells via suppression of CPT-1 activity. Thus, targeting the DNL pathway mediating CPT-1 activity by PMCTi may be a selective potential anticancer therapy.

Combination of Mitochondrial and Plasma Membrane Citrate Transporter Inhibitors Inhibits De Novo Lipogenesis Pathway and Triggers Apoptosis in Hepatocellular Carcinoma Cells.

Increased expression levels of both mitochondrial citrate transporter (CTP) and plasma membrane citrate transporter (PMCT) proteins have been found in various cancers. The transported citrates by these two transporter proteins provide acetyl-CoA precursors for the de novo lipogenesis (DNL) pathway to support a high rate of cancer cell viability and development. Inhibition of the DNL pathway promotes cancer cell apoptosis without apparent cytotoxic to normal cells, leading to the representation of selective and powerful targets for cancer therapy. The present study demonstrates that treatments with CTP inhibitor (CTPi), PMCT inhibitor (PMCTi), and the combination of CTPi and PMCTi resulted in decreased cell viability in two hepatocellular carcinoma cell lines (HepG2 and HuH-7). Treatment with citrate transporter inhibitors caused a greater cytotoxic effect in HepG2 cells than in HuH-7 cells. A lower concentration of combined CTPi and PMCTi promotes cytotoxic effect compared with either of a single compound. An increased cell apoptosis and an induced cell cycle arrest in both cell lines were reported after administration of the combined inhibitors. A combination treatment exhibits an enhanced apoptosis through decreased intracellular citrate levels, which consequently cause inhibition of fatty acid production in HepG2 cells. Apoptosis induction through the mitochondrial-dependent pathway was found as a consequence of suppressed carnitine palmitoyl transferase-1 (CPT-1) activity and enhanced ROS generation by combined CTPi and PMCTi treatment. We showed that accumulation of malonyl-CoA did not correlate with decreasing CPT-1 activity. The present study showed that elevated ROS levels served as an inhibition on Bcl-2 activity that is at least in part responsible for apoptosis. Moreover, inhibition of the citrate transporter is selectively cytotoxic to HepG2 cells but not in primary human hepatocytes, supporting citrate-mediating fatty acid synthesis as a promising cancer therapy.