Chelerythrine Chloride: A Potential Rumen Microbial Urease Inhibitor Screened by Targeting UreG

Inhibition of ruminal microbial urease is of particular interest due to its crucial role in regulating urea-N utilization efficiency and nitrogen pollution in the livestock industry. Acetohydroxamic acid (AHA) is currently the only commercially available urease inhibitor, but it has adverse side effects. The urease accessory protein UreG, which facilitates the functional incorporation of the urease nickel metallocentre, has been proposed in developing urease inhibitor through disrupting urease maturation. The objective of this study was to screen natural compounds as potential urease inhibitors by targeting UreG in a predominant ruminal microbial urease. In silico screening and in vitro tests for potential inhibitors were performed using molecular docking and an assay for the GTPase activity of UreG. Chelerythrine chloride was selected as a potential urease inhibitor of UreG with an inhibition concentration IC50 value of 18.13 μM. It exhibited mixed inhibition, with the Ki value being 26.28 μM. We further explored its inhibition mechanism using isothermal titration calorimetry (ITC) and circular dichroism (CD) spectroscopy, and we found that chelerythrine chloride inhibited the binding of nickel to UreG and induced changes in the secondary structure, especially the α-helix and β-sheet of UreG. Chelerythrine chloride formed a pi-anion interaction with the Asp41 residue of UreG, which is an important residue in initiating the conformational changes of UreG. In conclusion, chelerythrine chloride exhibited a potential inhibitory effect on urease, which provided new evidence for strategies to develop novel urease inhibitors targeting UreG to reduce nitrogen excretion from ruminants.

Ozone-oxidative-preconditioning alleviates hepatic ischemia-reperfusion injury in rats via PKC- ERK1/2 pathway

BACKGROUND: To explore the protective effect of ozone-oxidative-preconditioning (OzoneOP) through the protein kinase C (PKC), ERK1/2, and heat shock protein70 (HSP70) signal transduction in rats during hepatic ischemic-reperfusion (IR) injury. METHODS: We constructed an IR model by occluding all vessels of the rats’ left and median liver lobes for 45 min, followed by reperfusion for 3 h. Afterwards, we constructed the OzoneOP model via intraperitoneal injection of 1 mg · kg -1 · d -1 of 50 mg · L -1 ozone for 5 days to investigate the significance of PKC, ERK1/2 and HSP70 signal transduction in OzoneOP. The PKC inhibitor chelerythrine chloride (CHE), activator phorobol12-myristate13-acetate (PMA) and MEK inhibitor PD98059 were utilized to analyze the phosphorylation of PKC and the expression levels of ERK1/2 and HSP70. After ischemia and reperfusion, alanine aminotransferase (ALT) and aspartase aminotransferase (AST) were detected in the abdominal aorta blood. Meanwhile, the expression of HSP70 protein and the activities of PKC and ERK1/2 in the left hepatic lobe were analyzed, and the ultrastructure of the hepatic was observed. RESULTS: Compared with the control group, the phosphorylation of PKC and ERK1/2 and the expression of HSP70 were higher in the OzoneOP-treated model ( P <0.05). Conversely, inhibiting PKC and ERK1/2 abolished the protection conferred by OzoneOP ( P <0.05). CONCLUSION: OzoneOP significantly increased the expression of HSP70 by activating PKC and ERK1/2 signaling pathways, thus significantly alleviating hepatic IR injury in rats. KEYWORDS: ERK1/2 MAPKs; HSP70; Liver ischemia-reperfusion; O zone - oxidative - preconditioning; PKC

Chelerythrine Chloride Downregulates β-Catenin and Inhibits Stem Cell Properties of Non-Small Cell Lung Carcinoma

Plant secondary metabolites have been seen as alternatives to seeking new medicines for treating various diseases. Phytochemical scientists remain hopeful that compounds isolated from natural sources could help alleviate the leading problem in oncology—the lung malignancy that kills an estimated two million people annually. In the present study, we characterized a medicinal compound benzophenanthridine alkaloid, called chelerythrine chloride for its anti-tumorigenic activities. Cell viability assays confirmed its cytotoxicity and anti-proliferative activity in non-small cell lung carcinoma (NSCLC) cell lines. Immunofluorescence staining of β-catenin revealed that there was a reduction of nuclear content as well as overall cellular content of β-catenin after treating NCI-H1703 with chelerythrine chloride. In functional characterizations, we observed favorable inhibitory activities of chelerythrine chloride in cancer stem cell (CSC) properties, which include soft agar colony-forming, migration, invasion, and spheroid forming abilities. Interesting observations in chelerythrine chloride treatment noted that its action abides to certain concentration-specific-targeting behavior in modulating β-catenin expression and apoptotic cell death. The downregulation of β-catenin implicates the downregulation of CSC transcription factors like SOX2 and MYC. In conclusion, chelerythrine chloride has the potential to mitigate cancer growth due to inhibitory actions toward the tumorigenic activity of CSC in lung cancer and it can be flexibly adjusted according to concentration to modulate specific targeting in different cell lines.

Aβ1-42 increases the expression of neural KATP subunits Kir6.2/SUR1 via the NF-κB, p38 MAPK and PKC signal pathways in rat primary cholinergic neurons

ATP-sensitive potassium channels (KATP) may mediate a potential neuroprotective role in Alzheimer’s disease (AD). Given that exposure to Aβ1-42 in cultured primary cholinergic neurons for 72 h significantly upregulates the expression of KATP subunits Kir6.2/SUR1, we aim to study the underlying signal transduction mechanisms that are involved in Aβ1–42-induced upregulation of KATP subunits Kir6.2/SUR1. In the present study, we first identified the primary cultured rat cortical and hippocampal neurons using immunocytochemistry. 0.5 μM NF-κB inhibitor SN-50, 2 μM p38MAPK inhibitor SB203580 or 2 μM PKC inhibitor Chelerythrine chloride (CTC) were then added in three separate groups, followed by 2 μM Aβ1–42 30 min later in all 3 groups. Western Blot was performed 72 h later to detect the expression of KATP subunits Kir6.2/SUR1. We found that Aβ1–42 significantly increased the level of KATP subunits Kir6.2/SUR1 expression at 72 h when compared with the control group ( p < 0.05). However, when compared with the Aβ1–42 group, the level of KATP subunits Kir6.2/SUR1 expression at 72 h significantly decreased in the SN50 + Aβ1–42 group, SB203580 + Aβ1–42 group, and the CTC + Aβ1–42 group ( p < 0.05). Our findings suggest that the NF-κB, p38 MAPK, and PKC signal pathways are partially involved in the upregulation of KATP subunits Kir6.2/SUR1 expression induced by Aβ1–42 cytotoxicity in neurons, which supports a potential theoretical basis of targeting these signal pathways in the treatment of AD.

Human kidney-2 cells harbor functional dopamine D1 receptors that require Giα for Gq/11α signaling

A recent study demonstrated that the dopamine D1 receptor (D1R) is nonfunctional in human kidney cells, HK2 cells, in terms of their inability to couple to G s protein in response to the D1R agonist fenoldopam. Since D1R also couples to G q protein, we tested whether D1R is functional in HK2 cells in terms of their ability to couple to G q and produce downstream signaling. For comparison, we also studied another receptor, angiotensin II type 1 receptor (AT1R) known to couple to G q. Protein kinase C (PKC) and 86rubidium transport activities were determined as surrogate downstream signaling markers. Fenoldopam and angiotensin II increased PKC activity, which decreased in the presence of respective receptor antagonists (SCH23390 for D1R; candesartan for AT1R), PKC (chelerythrine chloride) and G i protein (pertussis toxin) inhibitors and G q/11α siRNA. Furthermore, fenoldopam and angiotensin II increased 35S-GTPγS binding, an index of receptor-G protein coupling, which decreased with pertussis toxin and in G q/11α-depleted cells. Also, fenoldopam-mediated inhibition of 86rubidium transport (an index of Na-K-ATPase activity) was attenuated with SCH23390, chelerythrine chloride, pertussis toxin, and G q/11α siRNA. Moreover, fenoldopam caused a decrease in cytosolic and increase in membranous abundance of G q/11α. The immunoprecipitated levels of G q/11α in the membranes were greater in fenoldopam-treated cells, and G iα coimmunoprecipitated with G q/11α. Our results suggest that both D1R and AT1R are functional in HK2 cells, enabling G q-mediated downstream signaling in a G i dependent manner.

Interaction between nitric oxide and superoxide in the macula densa in aldosterone-induced alterations of tubuloglomerular feedback

Tubuloglomerular feedback (TGF)-mediated constriction of the afferent arteriole is modulated by a balance between release of superoxide (O2−) and nitric oxide (NO) in macula densa (MD) cells. Aldosterone activates mineralocorticoid receptors that are expressed in the MD and induces both NO and O2− generation. We hypothesize that aldosterone enhances O2− production in the MD mediated by protein kinase C (PKC), which buffers the effect of NO in control of TGF response. Studies were performed in microdissected and perfused MD and in a MD cell line, MMDD1 cells. Aldosterone significantly enhanced O2− generation both in perfused MD and in MMDD1 cells. When aldosterone (10−7 mol/l) was added in the tubular perfusate, TGF response was reduced from 2.4 ± 0.3 μm to 1.4 ± 0.2 μm in isolated perfused MD. In the presence of tempol, a O2− scavenger, TGF response was 1.5 ± 0.2 μm. In the presence of both tempol and aldosterone in the tubular perfusate, TGF response was further reduced to 0.4 ± 0.2 μm. To determine if PKC is involved in aldosterone-induced O2− production, we exposed the O2− cells to a nonselective PKC inhibitor chelerythrine chloride, a specific PKCα inhibitor Go6976, or a PKCα siRNA, and the aldosterone-induced increase in O2− production was blocked. These data indicate that aldosterone-stimulated O2− production in the MD buffers the effect of NO in control of TGF response, an effect that was mediated by PKCα.