Kesong Peng

Inhibition of JNK phosphorylation by a novel curcumin analog prevents high glucose-induced inflammation and apoptosis in cardiomyocytes and the development of diabetic cardiomyopathy

Hyperglycemia-induced inflammation and apoptosis have important roles in the pathogenesis of diabetic cardiomyopathy. We recently found that a novel curcumin derivative, C66, is able to reduce the high glucose (HG)-induced inflammatory response. This study was designed to investigate the protective effects on diabetic cardiomyopathy and its underlying mechanisms. Pretreatment with C66 significantly reduced HG-induced overexpression of inflammatory cytokines via inactivation of nuclear factor-κB in both H9c2 cells and neonatal cardiomyocytes. Furthermore, we showed that the inhibition of Jun NH2-terminal kinase (JNK) phosphorylation contributed to the protection of C66 from inflammation and cell apoptosis, which was validated by the use of SP600125 and dominant-negative JNK. The molecular docking and kinase activity assay confirmed direct binding of C66 to and inhibition of JNK. In mice with type 1 diabetes, the administration of C66 or SP600125 at 5 mg/kg significantly decreased the levels of plasma and cardiac tumor necrosis factor-α, accompanied by decreasing cardiac apoptosis, and, finally, improved histological abnormalities, fibrosis, and cardiac dysfunction without affecting hyperglycemia. Thus, this work demonstrated the therapeutic potential of the JNK-targeting compound C66 for the treatment of diabetic cardiomyopathy. Importantly, we indicated a critical role of JNK in diabetic heart injury, and suggested that JNK inhibition may be a feasible strategy for treating diabetic cardiomyopathy.

Inhibition of MAPK-mediated ACE expression by compound C66 prevents STZ-induced diabetic nephropathy.

A range of in vitro, experimental and clinical intervention studies have implicated an important role for hyperglycaemia‐induced activation of the renin‐angiotensin system (RAS) in the development and progression of diabetic nephropathy (DN). Blockade of RAS by angiotensin converting enzyme (ACE) inhibitors is an effective strategy in treating diabetic kidney diseases. However, few studies demonstrate the mechanism by which hyperglycaemia up‐regulates the expression of ACE gene. Our previous studies have identified a novel curcumin analogue, (2E,6E)‐2,6‐bis(2‐(trifluoromethyl)benzylidene)cyclohexanone (C66), which could inhibit the high glucose (HG)‐induced phosphorylation of mitogen‐activated protein kinases in mouse macrophages. In this study, we found that the renal protection of C66 in diabetic mice was associated with mitogen‐activated protein kinase (MAPK) inactivation and ACE/angiotensin II (Ang II) down‐regulation. Generally, MAPKs have been considered as a downstream signalling of Ang II and a mediator for Ang II‐induced pathophysiological actions. However, using C66 and specific inhibitors as small molecule probes, in vitro experiments demonstrate that the MAPK signalling pathway regulates ACE expression under HG stimulation, which contributes to renal Ang II activation and the development of DN. This study indicates that C66 is a potential candidate of DN therapeutic agents, and more importantly, that reduction in ACE expression by MAPKs inhibition seems to be an alternative strategy for the treatment of DN.

Novel EGFR inhibitors attenuate cardiac hypertrophy induced by angiotensin II.

Cardiac hypertrophy is an important risk factor for heart failure. Epidermal growth factor receptor (EGFR) has been found to play a role in the pathogenesis of various cardiovascular diseases. The aim of this current study was to examine the role of EGFR in angiotensin II (Ang II)‐induced cardiac hypertrophy and identify the underlying molecular mechanisms. In this study, we observed that both Ang II and EGF could increase the phospohorylation of EGFR and protein kinase B (AKT)/extracellular signal‐regulated kinase (ERK), and then induce cell hypertrophy in H9c2 cells. Both pharmacological inhibitors and genetic silencing significantly reduced Ang II‐induced EGFR signalling pathway activation, hypertrophic marker overexpression, and cell hypertrophy. In addition, our results showed that Ang II‐induced EGFR activation is mediated by c‐Src phosphorylation. In vivo, Ang II treatment significantly led to cardiac remodelling including cardiac hypertrophy, disorganization and fibrosis, accompanied by the activation of EGFR signalling pathway in the heart tissues, while all these molecular and pathological alterations were attenuated by the oral administration with EGFR inhibitors. In conclusion, the c‐Src‐dependent EGFR activation may play an important role in Ang II‐induced cardiac hypertrophy, and inhibition of EGFR by specific molecules may be an effective strategy for the treatment of Ang II‐associated cardiac diseases.

Novel Epidermal Growth Factor Receptor Inhibitor Attenuates Angiotensin II–Induced Kidney Fibrosis

Chronic activation of renin-angiotensin system (RAS) greatly contributes to renal fibrosis and accelerates the progression of chronic kidney disease; however, the underlying molecular mechanism is poorly understood. Angiotensin II (Ang II), the central component of RAS, is a key regulator of renal fibrogenic destruction. Here we show that epidermal growth factor receptor (EGFR) plays an important role in Ang II–induced renal fibrosis. Inhibition of EGFR activation by novel small molecules or by short hairpin RNA knockdown in Ang II–treated SV40 mesangial cells in vitro suppresses protein kinase B and extracellular signal-related kinase signaling pathways and transforming growth factor-β/Sma- and Mad-related protein activation, and abolishes the accumulation of fibrotic markers such as connective tissue growth factor, collagen IV. The transactivation of EGFR by Ang II in SV40 cells depends on the phosphorylation of proto-oncogene tyrosine-protein kinase Src (c-Src) kinase. Further validation in vivo demonstrates that EGFR small molecule inhibitor successfully attenuates renal fibrosis and kidney dysfunction in a mouse model induced by Ang II infusion. These findings indicate a crucial role of EGFR in Ang II–dependent renal deterioration, and reveal EGFR inhibition as a new therapeutic strategy for preventing progression of chronic renal diseases.

Discovery and evaluation of novel anti-inflammatory derivatives of natural bioactive curcumin

Curcumin is a natural active product that has various pharmacological activities such as anti-inflammatory effects. Here, we report the synthesis and evaluation of 34 monocarbonyl curcumin analogs as novel anti-inflammatory agents. Among the analogs, the symmetrical heterocyclic type displayed the strongest inhibition of lipopolysaccharide (LPS)-stimulated expression of pro-inflammatory cytokines in macrophages. Analogs S1–S5 and AS29 reduced tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) production in a dose-dependent manner and also displayed excellent stability and low cytotoxicity in vitro. In addition, analog S1 dose-dependently inhibited LPS-induced extracellular signal-regulated kinase (ERK) phosphorylation. Furthermore, analogs S1 and S4 displayed a significant protective effect on LPS-induced septic death in mouse models, with 40% and 50% survival rates, respectively. These data demonstrate that the heterocyclic monocarbonyl curcumin analogs have potential therapeutic effects in acute inflammatory diseases.

Inhibition of inflammation and oxidative stress by an imidazopyridine derivative X22 prevents heart injury from obesity

Inflammation and oxidative stress plays an important role in the development of obesity‐related complications and cardiovascular disease. Benzimidazole and imidazopyridine compounds are a class of compounds with a variety of activities, including anti‐inflammatory, antioxidant and anti‐cancer. X22 is an imidazopyridine derivative we synthesized and evaluated previously for anti‐inflammatory activity in lipopolysaccharide‐stimulated macrophages. However, its ability to alleviate obesity‐induced heart injury via its anti‐inflammatory actions was unclear. This study was designed to evaluate the cardioprotective effects of X22 using cell culture studies and a high‐fat diet rat model. We observed that palmitic acid treatment in cardiac‐derived H9c2 cells induced a significant increase in reactive oxygen species, inflammation, apoptosis, fibrosis and hypertrophy. All of these changes were inhibited by treatment with X22. Furthermore, oral administration of X22 suppressed high‐fat diet‐induced oxidative stress, inflammation, apoptosis, hypertrophy and fibrosis in rat heart tissues and decreased serum lipid concentration. We also found that the anti‐inflammatory and anti‐oxidative actions of X22 were associated with Nrf2 activation and nuclear factor‐kappaB (NF‐κB) inhibition, respectively, both in vitro and in vivo. The results of this study indicate that X22 may be a promising cardioprotective agent and that Nrf2 and NF‐κB may be important therapeutic targets for obesity‐related complications.

An Oxygen-Chelate Complex, Palladium Bis-acetylacetonate, Induces Apoptosis in H460 Cells via Endoplasmic Reticulum Stress Pathway Rather than Interacting with DNA

Current precious-metal-containing anticancer agents are mostly chelated with N-containing ligands and function by interacting with DNA. In the present study, Pd(acac)2, a Pd(II) complex containing four O-donor ligands, has been evaluated as an active anticancer agent. Pd(acac)2 showed no interaction with N-ligand-containing DNA and the S-ligand-containing DMSO, probably because of the two six-member chelate rings that limit the release of the central Pd nuclei to bind to other ligands. Importantly, we found that Pd(acac)2 exhibited better growth inhibitory effects than cisplatin in several cancer cells. Treatment with Pd(acac)2 significantly induced apoptosis in H460 cells. Mechanistically, Pd(acac)2 induced the activation of a series of key components in ER stress-mediated apoptotic pathway, followed by caspase cleavage and activation, while cisplatin showed no similar effects. CHOP knockdown by specific siRNA significantly attenuated Pd(acac)2-induced cell apoptosis. Finally, Pd(acac)2 significantly inhibits H460 cell growth in xenograft mouse models. Taken together, these mechanistic insights on Pd(acac)2 provide us with a novel mechanism and strategy for the development of precious-metal-based anticancer drugs.

New EGFR inhibitor, 453, prevents renal fibrosis in angiotensin II-stimulated mice

Chronic activation of renin-angiotensin system (RAS) greatly contributes to renal fibrosis through the over expression of angiotensin (Ang) II, ultimately leading to chronic kidney disease (CKD). As the main peptide in the RAS, Ang II is a key regulator of nephrotic inflammation, fibrogenic destruction and hypertensive nephropathy. Controlled by growth factors such as TGF-β, Ang II is thought to be affected by other such growth factors including epidermal growth factor (EGF) due to its ability to stimulate growth, regulate angiogenesis, and desensitize cells from apoptotic stimuli. Here we show that epidermal growth factor receptor (EGFR) plays a key role in Ang II induced renal fibrosis and its inhibition for the use as an effective treatment of CKD. 453, an AG1478 analog, was used to block the EGF-EGFR interaction in vivo in 4-week old mice treated with Ang II and 453. Along with the inhibition of EGFR and its downstream signaling pathways (AKT and ERK), 453 also prevented the activation of fibrotic (collagen, CFGF, TGF-β), inflammatory (COX2, IL-6, IL-1β, TNF-α), apoptosis and oxidative stress pathways. These findings suggest the use of 453 as a novel EGFR-inhibitor for therapeutic use in CKD kidney dysfunction.

Inhibition of 11β-HSD1 by LG13 improves glucose metabolism in type 2 diabetic mice

11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) controls the production of active glucocorticoid (GC) and has been proposed as a new target for the treatment of type 2 diabetes. We have previously reported that a natural product, curcumin, exhibited moderate inhibition and selectivity on 11β-HSD1. By analyzing the models of protein, microsome, cells and GCs-induced mice in vitro and in vivo, this study presented a novel curcumin analog, LG13, as a potent selective 11β-HSD1 inhibitor. In vivo, Type 2 diabetic mice were treated with LG13 for 42 days to assess the pharmacological benefits of 11β-HSD1 inhibitor on hepatic glucose metabolism. In vitro studies revealed that LG13 selectively inhibited 11β-HSD1 with IC50 values at nanomolar level and high selectivity over 11β-HSD2. Targeting 11β-HSD1, LG13 could inhibit prednisone-induced adverse changes in mice, but had no effects on dexamethasone-induced ones. Further, the 11β-HSD1 inhibitors also suppressed 11β-HSD1 and GR expression, indicating a possible positive feedback system in the 11β-HSD1/GR cycle. In type 2 diabetic mice induced by high fat diet plus low-dosage STZ injection, oral administration with LG13 for 6 weeks significantly decreased fasting blood glucose, hepatic glucose metabolism, structural disorders, and lipid deposits. LG13 exhibited better pharmacological effects in vivo than insulin sensitizer pioglitazone and potential 11β-HSD1 inhibitor PF-915275. These pharmacological and mechanistic insights on LG13 also provide us novel agents, leading structures, and strategy for the development of 11β-HSD1 inhibitors treating metabolic syndromes.

11β-Hydroxysteroid Dehydrogenase Type 1(11β-HSD1) mediates insulin resistance through JNK activation in adipocytes.

Glucocorticoids are used to treat a number of human diseases but often lead to insulin resistance and metabolic syndrome. 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is a key enzyme that catalyzes the intracellular conversion of cortisone to physiologically active cortisol. Despite the known role of 11β-HSD1 and active glucocorticoid in causing insulin resistance, the molecular mechanisms by which insulin resistance is induced remain elusive. The aim of this study is to identify these mechanisms in high fat diet (HFD) experimental models. Mice on a HFD were treated with 11β-HSD1 inhibitor as well as a JNK inhibitor. We then treated 3T3-L1-derived adipocytes with prednisone, a synthetic glucocorticoid, and cells with 11β-HSD1 overexpression to study insulin resistance. Our results show that 11β-HSD1 and JNK inhibition mitigated insulin resistance in HFD mice. Prednisone stimulation or overexpression of 11β-HSD1 also caused JNK activation in cultured adipocytes. Inhibition of 11β-HSD1 blocked the activation of JNK in adipose tissue of HFD mice as well as in cultured adipocytes. Furthermore, prednisone significantly impaired the insulin signaling pathway, and these effects were reversed by 11β-HSD1 and JNK inhibition. Our study demonstrates that glucocorticoid-induced insulin resistance was dependent on 11β-HSD1, resulting in the critical activation of JNK signaling in adipocytes.