Molecular mechanism of the anti-inflammatory and skin protective effects of Syzygium formosum in human skin keratinocytes.

Irradiation injury, especially caused by UVB, of the skin is one of the critical reasons for skin inflammation and damage. The present study aimed to explore the protective effect of Syzygium formosum leafy extract (SFLE) and its mechanism of action against UVB-induced damages of human keratinocytes. In this study, SFLE was prepared from 100 kg dried leaves using industrial-scale processes. We found that SFLE markedly reduced markers of the skin inflammation in UVB-induced pro-inflammatory cytokines. Only 2 µg/mL of SFLE exhibited significantly stronger anti-inflammatory effects than the fivefold concentration of positive control. Intriguingly, an anti-inflammatory enzyme, heme oxygenase-1 expression was significantly induced by SFLE treatment. MMP-3 and -9 were, but not MMP-1, significantly reduced. SFLE inhibited the expression of the MAPK pathway, resulting in a decrease on UVB-induced reactive oxygen species. In conclusion, SFLE can potentially be used to treat skin inflammatory diseases. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-023-01380-4.

Preventive effects of inotodiol on polyinosinic-polycytidylic acid-induced inflammation in human dermal fibroblasts.

Double-strand RNA(dsRNA), which can induce inflammation, can be generated by necrotic keratinocytes in the skin environment. As an analog of dsRNA, polyinosinic-polycytidylic acid (poly(I:C)) is used to induce inflammation via the Toll-like Receptor 3 (TLR3) signaling pathway. Inotodiol, isolated from Inonotus obliquus, known as Chaga mushroom, is a natural lanostane-type triterpenoid with significant pharmacological activity and notable anti-inflammatory effects. However, the functions of inotodiol on dsRNA-induced inflammation in human dermal fibroblast (HDFs) remains unclear. In this study, we evaluated the anti-inflammatory effects of inotodiol inflammation induced on by poly(I:C) in HDFs. After pre-treatment with inotodiol, poly (I:C) was used to induce inflammation. Subsequently, mRNA expression and protein secretion of inflammatory cytokines, as well as TLR3 signaling protein levels were assessed. Inflammatory cytokines IL-1ß, IL-6, and TNF-α's increased mRNA expression by poly(I:C) in HDFs was significantly suppressed in the inotodiol pre-treatment group in a dose-dependent manner. A similar pattern was evaluated in the protein levels of these three cytokines. The inflammatory signals of TLR3 via p-IKK, p-p38, and NF-κB was reduced by inotodiol pre-treatment. Taken together, inotodiol possesses strong anti-inflammatory activity against poly(I:C)-induced inflammation in HDFs. Therefore, our findings support potential application of inotodiol as an effective anti-inflammatory agent in cosmetics.

Antiaging effect of inotodiol on oxidative stress in human dermal fibroblasts.

Oxidative damage is one of the major causes of human skin aging. Inotodiol is a lanostane triterpenoid that demonstrates antiviral, anticancer, and anti-inflammatory activities. Previous studies have reported that inotodiol also has antiallergic effects. However, whether inotodiol inhibits oxidative stress-induced human skin aging is not known. Stimulation of human dermal fibroblast cells with hydrogen peroxide is related to skin aging. Inotodiol inhibited the expression of mitogen-activated protein kinase (MAPK) and NADPH Oxidase 5 (NOX5). Moreover, inotodiol effectively decreased nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), as well as nitric oxide (NO), reactive oxygen species (ROS), cyclooxygenase-2 (COX-2), and cytokines such as IL-1ß, IL-6, and TNF-α. Based on our results, inotodiol protects human dermal fibroblast by preventing MAPK-NOX5 and NF-κB activation and attenuates the expression of aging genes. Inotodiol may therefore be considered a potential candidate for developing natural antiaging products, because it protects the human skin from oxidative stress-induced skin aging by inhibiting the MAPK-NOX5 and NF-κB signaling pathways.

High nuclear NADPH oxidase 4 expression levels are correlated with cancer development and poor prognosis in hepatocellular carcinoma.

NADPH oxidase (NOX) is a key source of reactive oxygen species (ROS). This study aimed to verify NOX2 and NOX4 expression levels in hepatocellular carcinoma (HCC). A total of 134 matched pairs of HCC cells and non-tumour hepatocytes from 134 patients were examined by immunohistochemical staining, and the association of NOX2 and NOX4 expression with clinicopathological parameters was analysed. Western blotting in four HCC cell lines and reverse transcription digital droplet polymerase chain reaction (RT-ddPCR) in 20 pairs of HCC and non-tumour tissue samples were also performed to detect NOX4. Cytoplasmic NOX2 and nuclear NOX4 expression levels were shown by immunohistochemistry to be higher in HCC cells than in non-tumour hepatocytes (p<0.001 each). The western blotting results for NOX4 in four HCC cell lines were consistent with the immunohistochemical results. Increased cytoplasmic expression of NOX2 and NOX4 in HCC cells was significantly correlated with liver cirrhosis (p<0.001 and p<0.031, respectively). However, decreased cytoplasmic expression of NOX2 and NOX4 was significantly correlated with advanced pathological TNM stage (p<0.029 and p<0.007, respectively). Multivariate analysis with clinicopathological parameters showed that high nuclear and low cytoplasmic NOX4 expression levels are correlated with short overall survival (p=0 .021). Our findings imply that cytoplasmic NOX2 and nuclear NOX4 expression is upregulated during HCC development. In particular, NOX4 translocation into the nucleus may affect the development and progression of HCC. NOX2 and NOX4 could be diagnostic markers and have therapeutic implications in HCC.

Cytochrome P450 4A11 expression in tumor cells: A favorable prognostic factor for hepatocellular carcinoma patients.

BACKGROUND AND AIM: Elevated cytochrome p450 (CYP) 4A gene expression has been linked to the aggravation of various cancers and affects various regulated metabolites. In hepatocellular carcinoma (HCC), the clinicopathological value of CYP4A has not yet been explored, although CYP4A is expressed at high levels in the liver. The goal of this study was to evaluate the clinicopathological value of CYP4A11 expression in HCC. METHODS: We performed immunohistochemical analysis of CYP4A11 and correlated the results with clinicopathological features of HCC (n = 155). Western blotting and reverse transcription-polymerase chain reaction against CYP4A11 and CYP4A22 were also performed for 15 and 20 pairs of fresh-frozen primary HCC and non-neoplastic liver tissue, respectively. Moreover, we analyzed the underlying mechanism by comparing the high and low CYP4A11 mRNA expression groups using gene set enrichment analysis. RESULTS: CYP4A11 expression level was higher in non-neoplastic hepatocytes than those in HCC cells (P < 0.001), and CYP4A11 expression positively correlated with favorable prognostic factors, including tumor size, histological grade, and pathological tumor stage (P = 0.007, P = 0.005, and P = 0.007). Multivariate analysis revealed that CYP4A11 expression was an independent prognostic factor of overall and disease-free survival (P = 0.002 and P = 0.033). Based on gene set enrichment analysis, high CYP4A11 mRNA expression negatively correlated with the expression of cell cycle-related genes. CONCLUSION: These findings support the notion that CYP4A11 expression is a favorable prognostic factor of HCC and suggest potential predictive diagnostic and prognostic roles of CYP4A11 expression in HCC.

PLD1 activation mediates Amb a 1-induced Th2-associated cytokine expression via the JNK/ATF-2 pathway in BEAS-2B cells.

The purpose of this study was to identify the role of phospholipase D1 (PLD1) in Amb a 1-induced IL-5 and IL-13 expression. When BEAS-2B cells were stimulated with Amb a 1, PLD activity increased, and knockdown of PLD1 decreased Amb a 1-induced IL-5 and IL-13 expression. Amb a 1 also activated the PLCγ/p70S6K/JNK pathway. Furthermore, Amb a 1-induced PLD activation was also attenuated by PLCγ inhibition, and knockdown of PLD1 decreased Amb a 1-induced activation of P70S6K and JNK. When ATF-2 activity was blocked with ATF-2 siRNA, Amb a 1-induced IL-5 and IL-13 expression was completely abolished, indicating that ATF-2 is a transcriptional factor required for the expression of IL-5 and IL-13 in response to Amb a 1. Taken together, we suggest that PLD1 acts as an important regulator in Amb a 1-induced expression of IL-5 and IL-13 via a PLCγ/p70S6K/JNK/ATF-2 pathway in BEAS-2B cells.

Leptin increases TNF-α expression and production through phospholipase D1 in Raw 264.7 cells.

Epidemiological evidence suggests that obesity is associated with inflammation of the respiratory tract and the pathogenesis of asthma. The purpose of this study was to examine the role of phospholipase D1 (PLD1) in leptin-induced expression of the proinflammatory cytokine, tumor necrosis factor (TNF)-α, and to suggest a molecular link between obesity and respiratory tract inflammation. We investigated whether leptin, a typical adipocytokine, plays a role in the expression of TNF-α through increased PLD1 activity in Raw 264.7. Leptin enhanced the activity of PLD1 through activation of PLCγ and Src, while PLD1 siRNA decreased the leptin-induced expression and production of TNF-α. Leptin-induced PLD activation was also inhibited by a PLCγ inhibitor (PAO) and Src kinase inhibitor (PP2), indicating that PLCγ and Src kinase are upstream activators of PLD1. Down-regulation of PLD1 also completely blocked activation of p70S6K, an activator of JNK. Leptin-induced expression of TNF-α was also prevented by inhibition of p70S6K and JNK. Taken together, these results indicate that PLD1 acts as an important regulator of leptin-induced expression of TNF-α by participating in the PLCγ/Src/PLD1/PA/p70S6K/JNK pathway.

Phospholipase D1 is required for lipopolysaccharide-induced tumor necrosis factor-α expression and production through S6K1/JNK/c-Jun pathway in Raw 264.7 cells.

The purpose of this study was to identify the role of phospholipase D1 (PLD1) in lipopolysaccharide (LPS)-induced tumor necrosis factor-α (TNF-α) expression and production. LPS-induced TNF-α expression and production were TLR4 (Toll-like receptor 4)/Myd88 dependent in Raw 264.7 cells. LPS enhanced PLD activation, which was attenuated by TLR4 inhibitor (Polymixin B) or knockdown of Myd88 with siRNA treatment. To investigate the role of PLD in LPS-induced TNF-α expression and production, we transfected PLD1 and PLD2 siRNAs to Raw 264.7 cells, respectively. Interestingly, only knockdown of PLD1 decreased TNF-α expression but not PLD2. Next, we investigated the S6K1-JNK-c-Jun signaling pathway in LPS-induced TNF-α expression mechanism. Knockdown of PLD1 also decreased phosphorylation of S6K1, JNK and c-Jun induced by LPS. Furthermore, we found that activated c-Jun63/73 bound to TNF-α promoter and turned on TNF-α expression. Taken together, our results demonstrate that PLD1 is activated by LPS/TLR4/Myd88 pathway and regulates TNF-α expression and production through S6K1/JNK/c-Jun in Raw 264.7 cells.