α-Mangostin suppresses ethanol-induced gastric ulceration by regulating the Nrf2/HO-1 and NF-κB/NLRP3/caspase-1 signaling pathways and gut microbiota.

α-Mangostin is a natural xanthone derivative isolated from Camellia atrophy (CA), commonly known as Lichuan black tea (LBT). The present study investigated the ameliorating effect and mechanism of α-mangostin on alcoholic gastric ulcers (GU) in rats. In vivo, α-mangostin relieved pathological symptoms. Moreover, α-mangostin regulated the activation of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/heme oxygenase 1 (HO-1) and nuclear factor κB (NF-κB)/NLR family pyrin domain containing 3 (NLRP3)/caspase-1 pathways. Reactive oxygen species (ROS), tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß) were significantly decreased and IL-10 were increased, the microtubule-associated protein light chain 3 (LC3)-II/LC3-I ratio was increased, p62 protein expression was decreased, and inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) protein expression was down-regulated. The relevant mechanisms were validated using GSE-1 and RAW264.7 cells in an in vitro model. Furthermore, α-mangostin increased Ligilactobacillus and Muribaculum abundance as well as propionic acid and butyric acid contents. Therefore, α-mangostin possesses antioxidant and anti-inflammatory properties, and remodels intestinal flora dysbiosis through mechanisms that may involve regulation of the Nrf2/HO-1 pathway and NF-κB/NLRP3/caspase-1 pathway. It also increases propionic acid and butyric acid contents. This study provides novel evidence regarding the use of α-mangostin for treating GU.

Hugan Qingzhi tablets attenuates endoplasmic reticulum stress in nonalcoholic fatty liver disease rats by regulating PERK and ATF6 pathways.

BACKGROUND: Endoplasmic reticulum (ER) stress, promoting lipid metabolism disorders and steatohepatitis, contributes significantly to the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Hugan Qingzhi tablets (HQT) has a definite effect in the clinical treatment of NAFLD patients, but its mechanism is still unclear. This study aims to investigate the effects of HQT on ER stress in the liver tissues of NAFLD rats and explore the underlying mechanism. METHODS: The NAFLD rat model was managed with high-fat diet (HFD) for 12weeks. HQT was administrated in a daily basis to the HFD groups. Biochemical markers, pro-inflammatory cytokines, liver histology were assayed to evaluate HQT effects in HFD-induced NAFLD rats. Furthermore, the expression of ER stress-related signal molecules including glucose regulating protein 78 (GRP78), protein kinase RNA-like endoplasmic reticulum kinase (PERK), p-PERK, eukaryotic translation initiation factor 2α (EIF2α), p-EIF2α, activating transcription factor 4 (ATF4), acetyl-coenzyme A-carboxylase (ACC), activating transcription factor (ATF6), and nuclear factor-kappa B-p65 (NF-κB-p65) were detected by western blot and/or qRT-PCR. RESULTS: The histopathological characteristics and biochemical data indicated that HQT exhibited protective effects on HFD-induced NAFLD rats. Furthermore, it caused significant reduction in the expression of ERS markers, such as GRP78, PERK, p-PERK, and ATF6, and subsequently downregulated the expression of EIF2α, p-EIF2α ATF4, ACC, and NF-κB-p65. CONCLUSIONS: The results suggested that HQT has protective effect against hepatic steatosis and inflammation in NAFLD rats by attenuating ER stress, and the potential mechanism is through inhibition of PERK and ATF6 pathways.

Isoliquiritigenin suppresses the progression of malignant melanoma via targeting H2A.Z.1-E2F1 pathway.

Cutaneous melanoma is one of the most prevalent tumors, and it is still a huge challenge in the current clinical treatment. Isoliquiritigenin (ISL), which is isolated from Glycyrrhiza uralensis Fisch., has been reported for its anti-tumor effect. However, the underlying mechanism and targets of ISL are still not be revealed clearly. In this study, differentiallyexpressedproteins were identified bylabel-free quantitative mass spectrometry. Two isoforms of the histone variant H2A.Z, including H2A.Z.1 and H2A.Z.2, were significantly down regulated after administration of ISL in melanoma. H2A.Z.1 was highly expressed in melanoma and correlated with poor prognosis of melanoma. The expression of H2A.Z was inhibited by ISL in a concentration-dependent manner. Overexpression of H2A.Z.1 in melanoma cell lines partly restored the repressed cell proliferation and cell cycle by ISL. Moreover, E2F1 was identified as one downstream target of H2A.Z.1, which was also highly expressed in melanoma and correlated with poor prognosis of melanoma. Furthermore, in vivo assays validated the inhibitory role of ISL in melanoma proliferation and the expression of H2A.Z.1 and E2F1.Aboveall,it is indicated that ISL inhibit melanoma proliferation via targeting H2A.Z.1-E2F1 pathway. These findings explain the anti-tumor mechanism of ISL and provide potential therapeutic targets for melanoma.

Alisol B 23-Acetate Ameliorates Lipopolysaccharide-Induced Intestinal Barrier Dysfunction by Inhibiting TLR4-NOX1/ROS Signaling Pathway in Caco-2 Cells.

Alisol B 23-Acetate (AB23A) is a naturally occurring triterpenoid, which can be indicated in the rhizome of medicinal and dietary plants from Alisma species. Previous studies have demonstrated that AB23A could inhibit intestinal permeability by regulating tight junction (TJ)-related proteins. Even so, the AB23A protective mechanism against intestinal barrier dysfunction remains poorly understood. This investigation seeks to evaluate the AB23A protective effects on intestinal barrier dysfunction and determine the mechanisms for restoring intestinal barrier dysfunction in LPS-stimulated Caco-2 monolayers. According to our findings, AB23A attenuated the inflammation by reducing pro-inflammatory cytokines production like IL-6, TNF-α, IL-1ß, and prevented the paracellular permeability by inhibiting the disruption of TJ in LPS-induced Caco-2 monolayers after treated with LPS. AB23A also inhibited LPS-induced TLR4, NOX1 overexpression and subsequent ROS generation in Caco-2 monolayers. Transfected with NOX1-specific shRNA diminished the up-regulating AB23A effect on ZO-1 and occludin expression. Moreover, transfected with shRNA of TLR4 not only enhanced ZO-1 and occludin expression but attenuated NOX1 expression and ROS generation. Therefore, AB23A ameliorates LPS-induced intestinal barrier dysfunction by inhibiting TLR4-NOX1/ROS signaling pathway in Caco-2 monolayers, suggesting that AB23A may have positive impact on maintaining the intestinal barrier's integrity.

Metabolomic Mechanisms of Radix Fici Hirtae against Carbon Tetrachloride-Induced Acute Liver Damage in Mice.

Background: Radix Fici Hirtae (RFH), known as Cantonese ginseng, is an alternative folk medicine that is widely used to treat various diseases in southern China. The aim of this study was to investigate the effect and metabolic mechanisms of pretreatment with RFH on the serum metabolic profiles of carbon tetrachloride (CCl4) induced acute liver injury in mice. Methods: Mice fed with the water extract of RFH at a dose of 1.5 g/kg and 0.75 g/kg for consecutive 7 days, and then serum samples were taken for the metabolomic analysis. Furthermore, the bioinformatics and pathways analysis were measured. Results: The UHPLC-Orbitrap/MS based-metabolomic analysis identified 20 differential metabolic markers in serum of CCl4-induced liver injury mice compared to that of the normal controls, which were mainly related to the metabolism of amino acids and fatty acids. Furthermore, most of these biomarkers contributing to CCl4 induction were ameliorated by RFH, and the bioinformatics and pathways analysis revealed that therapeutic actions of RFH were mainly involved in the regulation of the oxidative stress responses and energy homeostasis. Conclusion: These findings provide potential metabolic mechanism for future study and allow for hypothesis generation about the hepatoprotective effects of Radix Fici Hirtae.

Ficus hirta Vahl. Ameliorates Nonalcoholic Fatty Liver Disease through Regulating Lipid Metabolism and Gut Microbiota.

Nonalcoholic fatty liver disease (NAFLD) has gradually become one of the most serious liver diseases threatening human health in the world. Currently, Chinese herbal medicine is a potentially important treatment option for NAFLD, and the development of effective Chinese herbal medicine has a good prospect. Previous studies have suggested that Ficus hirta Vahl. (FV) has various protective effects on the liver. In this study, we investigated the therapeutic outcomes of FV treatment for the liver disease and its underlying mechanism using HepG2 cell lines induced by palmitate (PA) and mouse model fed with high-fat diet (HFD). FV mainly exerts pharmacological effects by mediating lipid metabolism and inflammation. During the lipid metabolism regulation process, CD36, SREBP-1, SCD1, PPAR γ, ACOX1, and CPT1α are the key factors related to the healing effects of FV on NAFLD. During the inflammation process, the downregulation of IL-6, IL-1ß, and TNF-α is involved in alleviation of NAFLD. Furthermore, CD36 overexpression promotes lipid abnormal metabolism and inflammation in PA-induced HepG2 cells, while CD36 knockdown and FV supplementation reverse these responses. In addition, FV also modulates gut microbiota composition, such as Allobaculum, Faecalibaculum, and Butyricicoccus in HFD-fed mice. In summary, our findings demonstrated that FV exerted a beneficial preventive and therapeutic effect on NAFLD by improving lipid metabolism and inflammation as well as regulating the structure of gut microbiota, and therefore, FV may be a candidate for the treatment of NAFLD.

New Prognostic Biomarkers and Drug Targets for Skin Cutaneous Melanoma via Comprehensive Bioinformatic Analysis and Validation.

Skin cutaneous melanoma (SKCM) is the most aggressive and fatal type of skin cancer. Its highly heterogeneous features make personalized treatments difficult, so there is an urgent need to identify markers for early diagnosis and therapy. Detailed profiles are useful for assessing malignancy potential and treatment in various cancers. In this study, we constructed a co-expression module using expression data for cutaneous melanoma. A weighted gene co-expression network analysis was used to discover a co-expression gene module for the pathogenesis of this disease, followed by a comprehensive bioinformatics analysis of selected hub genes. A connectivity map (CMap) was used to predict drugs for the treatment of SKCM based on hub genes, and immunohistochemical (IHC) staining was performed to validate the protein levels. After discovering a co-expression gene module for the pathogenesis of this disease, we combined GWAS validation and DEG analysis to identify 10 hub genes in the most relevant module. Survival curves indicated that eight hub genes were significantly and negatively associated with overall survival. A total of eight hub genes were positively correlated with SKCM tumor purity, and 10 hub genes were negatively correlated with the infiltration level of CD4+ T cells and B cells. Methylation levels of seven hub genes in stage 2 SKCM were significantly lower than those in stage 3. We also analyzed the isomer expression levels of 10 hub genes to explore the therapeutic target value of 10 hub genes in terms of alternative splicing (AS). All 10 hub genes had mutations in skin tissue. Furthermore, CMap analysis identified cefamandole, ursolic acid, podophyllotoxin, and Gly-His-Lys as four targeted therapy drugs that may be effective treatments for SKCM. Finally, IHC staining results showed that all 10 molecules were highly expressed in melanoma specimens compared to normal samples. These findings provide new insights into SKCM pathogenesis based on multi-omics profiles of key prognostic biomarkers and drug targets. GPR143 and SLC45A2 may serve as drug targets for immunotherapy and prognostic biomarkers for SKCM. This study identified four drugs with significant potential in treating SKCM patients.

The probiotic effects of AB23A on high-fat-diet-induced non-alcoholic fatty liver disease in mice may be associated with suppressing the serum levels of lipopolysaccharides and branched-chain amino acids.

Alisol B 23-acetate (AB23A) is a natural triterpenoid isolated from Rhizoma alisamatis that has been widely used as a traditional Chinese medicine (TCM). Previous studies have documented the beneficial effect of AB23A on non-alcoholic fatty liver disease (NAFLD), but the functional interactions between gut microbiota and the anti-NAFLD effect of AB23A remain unclear. In this study, we investigated the benefits of experimental treatment with AB23A on gut microbiota dysbiosis in NAFLD with an obesity model. C57BL/6J mice were administrated a high-fat diet (HFD) with or without AB23A for 12 weeks. AB23A significantly improved metabolic phenotype in the HFD-fed mice. Moreover, results of 16S rRNA gene-based amplicon sequencing in each group reveled that AB23A not only reduced the abundance of the Firmicutes/Bacteroidaeota ratio and Actinobacteriota/Bacteroidaeota ratio, but regulated the abundance of the top 10 genera, including norank_f__Muribaculaceae, Lactobacillus, Ileibacterium, Turicibacter, Faecalibaculum, the Lachnospiraceae_NK4A136_group, unclassified_f__Lachnospiraceae, and norank_f__Lachnospiraceae. AB23A significantly reduced the serum levels of lipopolysaccharide and branched-chain amino acids, which are positively correlated with the abundances of Ileibacterium and Turicibacter. Moreover, AB23A led to remarkable reductions in the activation of TLR4, NF-κB, and mTOR, and upregulated the expression of tight junction proteins, including ZO-1 and occludin. These results revealed that AB23A displayed a prebiotic capacity in HFD-fed NAFLD mice.

Identification of a novel tumour microenvironment-based prognostic biomarker in skin cutaneous melanoma.

Skin cutaneous melanoma (SKCM) is one of the most destructive skin malignancies and has attracted worldwide attention. However, there is a lack of prognostic biomarkers, especially tumour microenvironment (TME)-based prognostic biomarkers. Therefore, there is an urgent need to investigate the TME in SKCM, as well as to identify efficient biomarkers for the diagnosis and treatment of SKCM patients. A comprehensive analysis was performed using SKCM samples from The Cancer Genome Atlas and normal samples from Genotype-Tissue Expression. TME scores were calculated using the ESTIMATE algorithm, and differential TME scores and differentially expressed prognostic genes were successively identified. We further identified more reliable prognostic genes via least absolute shrinkage and selection operator regression analysis and constructed a prognostic prediction model to predict overall survival. Receiver operating characteristic analysis was used to evaluate the diagnostic efficacy, and Cox regression analysis was applied to explore the relationship with clinicopathological characteristics. Finally, we identified a novel prognostic biomarker and conducted a functional enrichment analysis. After considering ESTIMATEScore and tumour purity as differential TME scores, we identified 34 differentially expressed prognostic genes. Using least absolute shrinkage and selection operator regression, we identified seven potential prognostic biomarkers (SLC13A5, RBM24, IGHV3OR16-15, PRSS35, SLC7A10, IGHV1-69D and IGHV2-26). Combined with receiver operating characteristic and regression analyses, we determined PRSS35 as a novel TME-based prognostic biomarker in SKCM, and functional analysis enriched immune-related cells, functions and signalling pathways. Our study indicated that PRSS35 could act as a potential prognostic biomarker in SKCM by investigating the TME, so as to provide new ideas and insights for the clinical diagnosis and treatment of SKCM.

SIRT6 Promotes Osteogenic Differentiation of Adipose-Derived Mesenchymal Stem Cells Through Antagonizing DNMT1.

BACKGROUND: Adipose-derived stem cells (ADSCs) are increasingly used in regenerative medicine because of their potential to differentiate into multiple cell types, including osteogenic lineages. Sirtuin protein 6 (SIRT6) is a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase that plays important roles in cell differentiation. NOTCH signaling has also been reported to involve in osteogenic differentiation. However, the function of SIRT6 in osteogenic differentiation of ADSCs and its relation to the NOTCH signaling pathways are yet to be explored. METHODS: The in vitro study with human ADSCs (hADSCs) and in vivo experiments with nude mice have been performed. Alkaline phosphatase (ALP) assays and ALP staining were used to detect osteogenic activity. Alizarin Red staining was performed to detect calcium deposition induced by osteogenic differentiation of ADSCs. Western blot, RT-qPCR, luciferase reporter assay, and co-immunoprecipitation assay were applied to explore the relationship between of SIRT6, DNA methyltransferases (DNMTs) and NOTCHs. RESULTS: SIRT6 promoted ALP activity, enhanced mineralization and upregulated expression of osteogenic-related genes of hADSCs in vitro and in vivo. Further mechanistic studies showed that SIRT6 deacetylated DNMT1, leading to its unstability at protein level. The decreased expression of DNMT1 prevented the abnormal DNA methylation of NOTCH1 and NOTCH2, resulting in the upregulation of their transcription. SIRT6 overexpression partially suppressed the abnormal DNA methylation of NOTCH1 and NOTCH2 by antagonizing DNMT1, leading to an increased capacity of ADSCs for their osteogenic differentiation. CONCLUSION: This study demonstrates that SIRT6 physical interacts with the DNMT1 protein, deacetylating and destabilizing DNMT1 protein, leading to the activation of NOTCH1 and NOTCH2, Which in turn promotes the osteogenic differentiation of ADSCs.

Long non­coding RNA MIR4713HG aggravates malignant behaviors in oral tongue squamous cell carcinoma via binding with microRNA let­7c­5p.

Oral tongue squamous cell carcinoma (OTSCC) is one of the most aggressive pathological types of head and neck squamous cell carcinoma, and presents with rapid local invasion and metastasis. The present study confirmed that the long non­coding (lnc) RNA MIR4713HG was markedly upregulated in both OTSCC tissues and cell lines and associated with poor survival. The present study performed a series of experiments to investigate the impact of MIR4713HG on OTSCC and revealed that upregulation of MIR4713HG had a crucial role in promoting cell proliferation and metastasis of OTSCC cell lines both in vitro and in vivo. By applying bioinformatics analyses, micro RNA let­7c­5p was observed to physically bind with MIR4713HG, and the knockdown of let­7c­5p could counteract the influence of MIR4713HG on OTSCC. Furthermore, the present study demonstrated that let­7c­5p performed its regulating role in OTSCC via affecting the expression level of transmembrane channel like 7 (TMC7). In conclusion, the present study demonstrated that lncRNA MIR4713HG acted as a pro­tumor factor facilitating cell proliferation and metastasis of OTSCC via affecting the let­7c­5p/TMC7 signaling pathway, which presents as a promising therapeutic target in OTSCC.

LINC00958 promotes the proliferation of TSCC via miR-211-5p/CENPK axis and activating the JAK/STAT3 signaling pathway.

BACKGROUND: Tongue squamous cell carcinoma (TSCC) is one of the most common oral tumors. Recently, long intergenic noncoding RNA 00958 (LINC00958) has been identified as an oncogene in human cancers. Nevertheless, the role of LINC00958 and its downstream mechanisms in TSCC is still unknown. METHODS: The effect of LINC00958 on TSCC cells proliferation and growth were assessed by CCK-8, colony formation, 5-Ethynyl-2'-deoxyuridline (EdU) assay and flow cytometry assays in vitro and tumor xenograft model in vivo. Bioinformatics analysis was used to predict the target of LINC00958 in TSCC, which was verified by RNA immunoprecipitation and luciferase reporter assays. RESULTS: LINC00958 was increased in TSCC tissues, and patients with high LINC00958 expression had a shorter overall survival. LINC00958 knockdown significantly decreased the growth rate of TSCC cells both in vitro and in vivo. In mechanism, LINC00958 acted as a ceRNA by competitively sponging miR-211-5p. In addition, we identified CENPK as a direct target gene of miR-211-5p, which was higher in TSCC tissues than that in adjacent normal tissues. Up-regulated miR-211-5p or down-regulated CENPK could abolish LINC00958-induced proliferation promotion in TSCC cells. Furthermore, The overexpression of CENPK promoted the expression of oncogenic cell cycle regulators and activated the JAK/STAT3 signaling. CONCLUSIONS: Our findings suggested that LINC00958 is a potential prognostic biomarker in TSCC.

UTRN inhibits melanoma growth by suppressing p38 and JNK/c-Jun signaling pathways.

BACKGROUND: Utrophin (UTRN), as a tumor suppressor gene, is involved in various cancer progression. The function of UTRN in the melanoma process and the related molecular mechanisms are still unclear. Herein, we studied the function of UTRN in melanoma growth and the relevant molecular mechanisms. METHODS: Using the GEO database and UCSC Xena project, we compared the expression of UTRN in non-cancerous and melanoma tissues. Immunohistochemistry (IHC) staining, qRT-PCR and Western Blot (WB) were performed to evaluate UTRN expression in clinical samples. A total of 447 cases with UTRN expression data, patient characteristics and survival data were extracted from TCGA database and analyzed. After stable transduction and single cell cloning, the proliferation ability of A375 human melanoma cells was analyzed by Cell Counting Kit­8 (CCK) and 5­ethynyl­2'­deoxyuridine (EdU) incorporation assays. GSEA was performed to predict the mechanism by which UTRN regulated melanoma growth. Then WB analysis was used to assess the protein expression levels of pathway signaling in overexpression (EXP) melanoma cells. Epac activator 8-pCPT-2'-O-Me-cAMP was then used to evaluate the proliferation ability by activation of p38 and JNK/c-Jun signaling pathways. RESULTS: Data from GEO and UCSC Xena project indicated that UTRN expression was decreased in melanoma. Experiment on clinical samples further confirmed our finding. TCGA results showed that a reduced expression of UTRN in 447 melanoma samples was associated with advanced clinical characteristics (T stage, Clark level, ulceration), shorter survival time and poorer prognosis. In addition, up-regulated UTRN expression inhibited melanoma cell proliferation when compared to control group. MAPK signaling pathway was presented in both KEGG and BioCarta databases by using GSEA tool. WB results confirmed the down-regulated expression of p38, JNK1 and c-Jun in EXP group when compared to control group. Epac activator 8-pCPT-2'-O-Me-cAMP treatment could partially rescue proliferation of tumor cells. CONCLUSION: We have demonstrated that reduced UTRN predicted poorer prognosis and UTRN inhibited melanoma growth via p38 and JNK1/c-Jun pathways. Therefore, UTRN could serve as a tumor suppressor and novel prognostic biomarker for melanoma patients.

The dietary flavonoid isoliquiritigenin induced apoptosis and suppressed metastasis in melanoma cells: An in vitro and in vivo study.

AIMS: This study aimed to explore the role of Isoliquiritigenin (ISL) in the proliferation and invasion of melanoma cells and investigate the mechanism of action of this compound. MAIN METHODS: The functional roles of ISL in melanoma cells were determined by CCK8 assay, colony formation assay, flow cytometry and wound healing assay. The antitumor activity of ISL was assessed in vivo in a mouse xenograft model using A2058 cells. Quantitative real-time PCR analysis (RT-qPCR) and western blot assays were used to evaluate the gene and protein expression in cell lines or tumor tissue samples. Bioinformatic analysis, luciferase reporter assay, and gene set enrichment analysis (GSEA) were performed to confirm the mechanism of ISL effect on cell growth and metastasis of melanoma. KEY FINDINGS: ISL suppressed proliferation and migration of melanoma cells via downregulation of miR-27a expression. The inhibitory effect of ISL on growth and metastasis of melanoma cells was reversed by ectopic expression of miR-27a. Bioinformatic analysis showed that miR-27a targets POU class 2 homeobox 3 (POU2F3); this result was verified by the luciferase reporter assay and by a decrease in the expression of POU2F3 by miR-27a intervention. GSEA demonstrated that POU2F3 is associated with the c-MYC/p53 signaling pathway and metastasis. POU2F3 knockdown reversed the inhibitory effect of ISL on the growth and metastasis of melanoma. Additionally, POU2F3 was found to be downregulated in melanoma tissue samples and was negatively correlated with miR-27a. SIGNIFICANCE: ISL inhibits proliferation and metastasis of melanoma via the miR-27a/POU2F3/c-MYC/p53 axis; these results may provide a new thought for the treatment of melanoma.

Hugan Qingzhi medication ameliorates free fatty acid-induced L02 hepatocyte endoplasmic reticulum stress by regulating the activation of PKC-δ.

BACKGROUND: Previous studies have found that Hugan Qingzhi tablet (HQT) has significant lipid-lowering and antioxidant effects on non-alcoholic fatty liver disease (NAFLD). Moreover, the results of proteomic analysis confirmed that various proteins in endoplasmic reticulum stress (ERS) pathway were activated and recovered by HQT. However, its mechanism remains confused. The purpose of this study was to explore the effects of HQT-medicated serum on hepatic ERS and its relevant mechanisms. METHODS: L02 cells were induced by Free Fatty Acid (FFA) for 24 h to establish a model of hepatic ERS and pretreated with the drug-medicated rat serum for 24 h. Accumulation of intracellular lipid was evaluated using Oil Red O staining and Triglyceride detection kit. The morphological changes of ER were observed by TEM. PKC-δ was silenced by specific siRNA. Western blot and RT-qPCR were applied to detect the expression of markers related to ERS, calcium disorder, steatosis and insulin resistance. The fluorescence of Ca2+ influx was recorded using fluorescence spectrophotometer. RESULTS: HQT-medicated serum significantly decreased the intracellular TG content. Furthermore, it caused significant reduction in the expression of ERS markers and an improvement in ER structure of L02 cells. PKC-δ was activated into phosphorylated PKC-δ in FFA-induced L02 hepatocytes while these changes can be reversed by HQT-medicated serum. Silencing PKC-δ in L02 cells can restore the expression and activity of SERCA2 in ER and down-regulate the expression of IP3R protein to maintain intracellular calcium homeostasis, so as to relieve FFA-induced ERS and its lipid accumulation and insulin resistance. CONCLUSIONS: The results concluded that HQT-medicated serum exerts protective effects against hepatic ERS, steatosis and insulin resistance in FFA-induced L02 hepatocyte. And its potential mechanism might be down-regulating the activation of PKC-δ and stabilization of intracellular calcium.

Formulation and Characterization of a 3D-Printed Cryptotanshinone-Loaded Niosomal Hydrogel for Topical Therapy of Acne.

Cryptotanshinone (CPT) is an efficacious acne treatment, while niosomal hydrogel is a known effective topical drug delivery system that produces a minimal amount of irritation. Three-dimensional (3D) printing technologies have the potential to improve the field of personalized acne treatment. Therefore, this study endeavored to develop a 3D-printed niosomal hydrogel (3DP-NH) containing CPT as a topical delivery system for acne therapy. Specifically, CPT-loaded niosomes were prepared using a reverse phase evaporation method, and the formulation was optimized using a response surface methodology. In vitro characterization showed that optimized CPT-loaded niosomes were below 150 nm in size with an entrapment efficiency of between 67 and 71%. The CPT-loaded niosomes were added in a dropwise manner into the hydrogel to formulate CPT-loaded niosomal hydrogel (CPT-NH), which was then printed as 3DP-CPT-NH with specific drug dose, shape, and size using an extrusion-based 3D printer. The in vitro release behavior of 3DP-CPT-NH was found to follow the Korsmeyer-Peppas model. Permeation and deposition experiments showed significantly higher rates of transdermal flux, Q24, and CPT deposition (p < 0.05) compared with 3D-printed CPT-loaded conventional hydrogel (3DP-CPT-CH), which did not contain niosomes. In vivo anti-acne activity evaluated through an acne rat model revealed that 3DP-CPT-NH exhibited a greater anti-acne effect with no skin irritation. Enhanced skin hydration, wide inter-corneocyte gaps in the stratum corneum and a disturbed lipid arrangement may contribute towards the enhanced penetration properties of CPT. Collectively, this study demonstrated that 3DP-CPT-NH is a promising topical drug delivery system for personalized acne treatments.

Mechanisms of white mustard seed (Sinapis alba L.) volatile oils as transdermal penetration enhancers.

We investigated the transdermal drug permeation enhancement properties and associated mechanisms of white mustard (Sinapis alba L.) seed volatile oil (SVO). Using gas chromatography-mass spectrometry, we showed that SVO was composed primarily of allylisothiocyanate and isothiocyanatocyclopropane. Compared with azone, SVO had better penetration-enhancing effects on three model drugs (5-Fluorouracil, Osthole, and Paeonol), with each having different oil-water partition coefficients. Histopathology showed that SVO did not induce skin irritation when the concentration was lower than 2% (v/v), and it induced less irritation than azone. According to attenuated total reflection-Fourier transform infrared spectroscopy and transmission electron microscopy, SVO induced skin lipid structural disorder and increased the distance between the stratum corneum, which is beneficial to the penetration of drugs. Cellular experiments showed that SVO inhibited Ca2+-ATPase activity, increased intracellular Ca2+ concentration, and changed the membrane potential in HaCaT cells, which promoted drug transfer into the skin. Our findings reveal that SVO is a safe and efficient natural product that has great potential as skin penetration enhancer.

Altered metabolites in guinea pigs with allergic asthma after acupoint sticking therapy: New insights from a metabolomics approach.

BACKGROUND: Clinical evidence gathered in Chinese communities suggested that acupoint sticking therapy could be an alternative treatment for asthma-related diseases. However, its underlying mechanism is still poorly understood. AIM/HYPOTHESIS: In this study, we aimed to investigate the mechanism of the anti-inflammatory effect of acupoint sticking application with 'Treatment of Winter Disease in Summer' (TWDS) prescription by using metabolomics. METHODS: Allergic asthma in guinea pig was sensitized and challenged by ovalbumin (OVA). Histopathological evaluation of the lung tissue was performed by hematoxylin and eosin (H&E) staining and Masson's trichrome staining. The levels of Th2 cytokine and IgE level in serum were measured using enzyme-linked immunoassay (ELISA). The mRNA expression levels of IL-4, IL-5, IL-13 and orosomucoid-like 3 (ORMDL3) were measured using quantitative reverse transcription polymerase chain reaction (RT-qPCR). Proteins of NF-κB signaling pathway were measured using western blot. The serum metabolomics profiles were obtained by using ultra-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS). RESULTS: The overall results confirmed that AST with TWDS prescription had a significant protective effect against OVA-induced allergic asthma in guinea pig. This treatment not only attenuated airway inflammation and collagen deposition in the airway, but also decreased the levels of IL-4, IL-5, IL-13 and IgE in serum. In addition, metabolomics results indicated that metabolisms of phospholipid, sphingolipid, purine, amino acid and level of epinephrine were restored back to the normal control level. Moreover, results of the gene expression of ORMDL3 in lung tissues indicated that AST using TWDS could alter the sphingolipid metabolism. Further western blotting analysis also showed that its anti-inflammatory mechanism was by decreasing the phosphorylation of p65 and IκB. CONCLUSION: The study demonstrated that metabolomics provides a better understanding of the actions of TWDS acupoint sticking therapy on OVA-induced allergic asthma.

Isoliquiritigenin suppresses human melanoma growth by targeting miR-301b/LRIG1 signaling.

BACKGROUND: Isoliquiritigenin (ISL), a natural flavonoid isolated from the root of licorice (Glycyrrhiza uralensis), has shown various pharmacological properties including anti-oxidant, anti-inflammatory and anti-cancer activities. MicroRNAs (miRNAs), a class of small non-coding RNAs, have been reported as post-transcriptional regulators with altered expression levels in melanoma. This study aims to investigate the anti-melanoma effect of ISL and its potential mechanism. METHODS: We investigated the effect of ISL on the proliferation and apoptosis of melanoma cell lines with functional assays, such as CCK-8 assay, colony formation assay and flow cytometry. The protein level of apoptosis related genes were measured by western blotting. High-throughput genome sequencing was used for screening differentially expressed miRNAs of melanoma cell lines after the treatment of ISL. We performed functional assays to determine the oncogenic role of miR-301b, the most differentially expressed miRNA, and its target gene leucine rich repeats and immunoglobulin like domains 1 (LRIG1), confirmed by bioinformatic analysis, luciferase reporter assay, western blotting and immunohistochemical assay in melanoma. Immunocompromised mouse models were used to determine the role of miR-301b and its target gene in melanoma tumorigenesis in vivo. The relationship between miR-301b and LRIG1 was further verified in GEO data set and tissue specimens. RESULTS: Functional assays indicated that ISL exerted significant growth inhibition and apoptosis induction on melanoma cells. MiR-301b is the most differentially expressed miRNA after the treatment of ISL and significantly downregulated. The suppressive effect of ISL on cell growth is reversed by ectopic expression of miR-301b. Intratumorally administration of miR-301b angomir enhances the inhibitory effect of ISL on tumor growth in vivo. Bioinformatic analysis showed that miR-301b may target LRIG1, miR-301b suppresses the luciferase activity of reporter constructs containing 3'UTR of LRIG1 as well as the expression level of LRIG1. And the anti-cancer effect of ISL is mitigated when LRIG1 is silenced in vivo and in vitro. Analysis of the melanoma samples obtained from patients shows that LRIG1 is negatively correlated with miR-301b. CONCLUSIONS: ISL may inhibit the proliferation of melanoma cells by suppressing miR-301b and inducing its target LRIG1.