Expression Of Key Molecules Research Articles

Exosomes derived from mesenchymal stem cells attenuate NLRP3-related pyroptosis in autoimmune premature ovarian insufficiency via the NF-κB pathway

Research questionWhat is the effect of exosomes derived from bone marrow mesenchymal stem cells (MSC-Exos) on the pyroptosis and recovery of granulosa cells in autoimmune premature ovarian insufficiency (POI)? DesignIn vitro, KGN cells were exposed to interferon-gamma to simulate immune injury. Samples were collected after a 48 h incubation with MSC-Exos (30 μg/ml). The cell viability, secretion of oestrogen and expression of key molecules in pyroptosis and the nuclear factor kappa B (NF-κB) pathway were tested. In vivo, the BALB/c mouse model of autoimmune POI model induced by zona pellucida glycoprotein 3 was used. Fertility testing and sample collection were applied 4 weeks after the ovarian subcapsular injection of MSC-Exos (150 μg for each ovary). Hormone concentration measurements, follicle counting and pyroptotic pathway analyses were conducted for each group. ResultsIn vitro, MSC-Exos significantly promoted the proliferation rate and secretion of oestrogen, while at the same time suppressing apoptosis and pyroptosis. In vivo, exosomal treatment normalized the irregular oestrous cycles, rescued the follicular loss and increased the pregnancy rate and number of offspring in POI mice. Elevated serum concentrations of oestrogen and anti-Müllerian hormone, as well as decreased concentrations of FSH and interleukin-1β, were shown. Furthermore, MSC-Exos down-regulated the expression of the NLRP3/Casp1/GSDMD pathway and inhibited activation of the NF-κB pathway. ConclusionsThese findings demonstrate for the first time that MSC-Exos exert a significant effect on restoring ovarian function in autoimmune POI in vivo and in vitro by suppressing the NLRP3/Casp1/GSDMD pathway and pyroptosis. The NF-κB pathway may contribute to the regulation of NLRP3-related pyroptosis.

New insights into the intestinal barrier through "gut-organ" axes and a glimpse of the microgravity's effects on intestinal barrier.

Gut serves as the largest interface between humans and the environment, playing a crucial role in nutrient absorption and protection against harmful substances. The intestinal barrier acts as the initial defense mechanism against non-specific infections, with its integrity directly impacting the homeostasis and health of the human body. The primary factor attributed to the impairment of the intestinal barrier in previous studies has always centered on the gastrointestinal tract itself. In recent years, the concept of the "gut-organ" axis has gained significant popularity, revealing a profound interconnection between the gut and other organs. It speculates that disruption of these axes plays a crucial role in the pathogenesis and progression of intestinal barrier damage. The evaluation of intestinal barrier function and detection of enterogenic endotoxins can serve as "detecting agents" for identifying early functional alterations in the heart, kidney, and liver, thereby facilitating timely intervention in the disorders. Simultaneously, consolidating intestinal barrier integrity may also present a potential therapeutic approach to attenuate damage in other organs. Studies have demonstrated that diverse signaling pathways and their corresponding key molecules are extensively involved in the pathophysiological regulation of the intestinal barrier. Aberrant activation of these signaling pathways and dysregulated expression of key molecules play a pivotal role in the process of intestinal barrier impairment. Microgravity, being the predominant characteristic of space, can potentially exert a significant influence on diverse intestinal barriers. We will discuss the interaction between the "gut-organ" axes and intestinal barrier damage, further elucidate the signaling pathways underlying intestinal barrier damage, and summarize alterations in various components of the intestinal barrier under microgravity. This review aims to offer a novel perspective for comprehending the etiology and molecular mechanisms of intestinal barrier injury as well as the prevention and management of intestinal barrier injury under microgravity environment.

Role of Eukaryotic Translation Elongation Factor 1 Family Members in the Tumorigenesis and Progression of Lung Adenocarcinoma

Objective To investigate the role and mechanism of eukaryotic translation elongation factor 1(EEF1) family members (EEF1D,EEF1A1,and EEF1A2) in lung adenocarcinoma (LUAD) based on public databases.Methods We examined EEF1 member expression levels in human LUAD samples via The Cancer Genome Atlas in the UCSC Xena browser and the Clinical Proteomic Tumor Analysis Consortium.We analyzed the mRNA and protein levels of EEF1D,EEF1A1,and EEF1A2 and their correlations with pathological variables via the Mann-Whitney U test.The Kaplan-Meier curves were established to assess the prognostic values of EEF1D,EEF1A1,and EEF1A2.The single-sample gene set enrichment analysis algorithm was employed to explore the relationship between the expression levels of EEF1 members and tumor immune cell infiltration.Spearman and Pearson correlation analyses were performed to examine the relationship between the expression levels of EEF1 members and those of the genes in the phosphatidylinositol 3-kinase/protein kinase B signaling pathway.The immunohistochemical assay was employed to determine the expression levels of EEF1D,EEF1A1,and EEF1A2 in the LUAD tissue (n=75) and paracancer tissue (n=75) samples.Results The mRNA and protein levels of EEF1D,EEF1A1,and EEF1A2 showed significant differences between tumor and paracancer tissues (all P<0.001).The patients with high protein levels of EEF1A1 showed bad prognosis in terms of overall survival (P=0.039),and those with high protein levels of EEF1A2 showed good prognosis in terms of overall survival (P=0.012).The influence of the mRNA level of EEF1D on prognosis was associated with pathological characteristics.The expression levels of EEF1 members were significantly associated with the infiltration of various immune cells and the expression of key molecules in the phosphatidylinositol 3-kinase/protein kinase B signaling pathway.Conclusion EEF1D,EEF1A1,and EEF1A2 are associated with the progression of LUAD,serving as the candidate prognostic markers for LUAD.

Dopamine depletion alters neuroplasticity-related signaling in the rat hippocampus

ABSTRACT Dopamine (DA) plays a significant role in regulating hippocampal function, particularly in modulating synaptic plasticity. Despite this, a comprehensive understanding of the molecular mechanisms involved in neuroplasticity-related signaling influenced by DA remains incomplete. This study aimed to elucidate the changes in the expression of key molecules related to hippocampal neuroplasticity following DA depletion in rats. To induce DA depletion, unilateral striatal infusions of 6-hydroxydopamine (6-OHDA) were administered to adult Sprague-Dawley rats. The subsequent loss of nigrostriatal DAergic signaling in these 6-OHDA-lesioned rats was confirmed using an apomorphine-induced rotation test at 4 weeks post-infusion and by assessing the expression levels of tyrosine hydroxylase (TH) through immunohistochemistry and western blotting at 7 weeks post-infusion. A decrease in DAergic signaling, evidenced by reduced TH-positive immunoreactivity, was also noted in the ipsilateral hippocampus of the lesioned rats. Interestingly, 6-OHDA infusion led to increased phosphorylation of pivotal hippocampal plasticity-related proteins, including extracellular signal-regulated kinase (ERK), protein kinase B (Akt), glycogen synthase kinase 3β (GSK3β), and cAMP response element-binding protein (CREB), in the ipsilateral hippocampus 7 weeks following the infusion. To extend these findings, in vitro experiments were conducted on primary hippocampal neurons exposed to DA and/or the active D1/D2 DA receptor antagonist, flupentixol (Flux). DA inhibited the constitutive phosphorylation of ERK, Akt, GSK3, and CREB, while Flux restored these phosphorylation levels. Taken together, these findings indicate that DA depletion triggers an increase in plasticity-related signaling in the hippocampus, suggesting a possible compensatory mechanism that promotes activity-independent neuroplasticity following DA depletion.

Electroacupuncture ameliorates hepatic defects in a rat model of polycystic ovary syndrome induced by letrozole and a high-fat diet.

This study was designed to evaluate the effects of low-frequency electroacupuncture (EA) on glucose and lipid disturbances in a rat model of polycystic ovary syndrome (PCOS) characterized by insulin resistance (IR) and hepatic steatosis. The PCOS rat model was induced by continuous administration of letrozole (LET) combined with a high-fat diet (HFD). Female Sprague-Dawley rats were divided into the following four groups: control, control + EA, LET + HFD and LET + HFD + EA. EA was administered five or six times a week with a maximum of 20 treatment sessions. Body weight, estrous cyclicity, hormonal status, glucose and insulin tolerance, lipid profiles, liver inflammation factors, liver morphology and changes in the phosphatidylinositol 3-kinase (PI3-K)/Akt (protein kinase B) pathway were evaluated. The rat model presented anovulatory cycles, increased body weight, elevated testosterone, abnormal glucose and lipid metabolism, IR, liver inflammation, hepatic steatosis and dysregulation of the insulin-mediated PI3-K/Akt signaling axis. EA reduced fasting blood glucose, fasting insulin, area under the curve for glucose, homeostasis model assessment of IR indices, triglycerides and free fatty acids, and alleviated hepatic steatosis. Furthermore, low-frequency EA downregulated mRNA expression of tumor necrosis factor (TNF)-α and interleukin (IL)-6, upregulated mRNA expression of peroxisome proliferator-activated receptor (PPAR)-α, increased protein expression of phosphorylated (p)-Akt (Ser473), p-glycogen synthase kinase (GSK) 3β (Ser9) and glucose transporter 4 (GLUT4), increased the ratio of p-GSK3β to GSK3β and downregulated protein expression of GSK3β. An obese PCOS rat model with IR and hepatic steatosis was successfully established by the combination of LET and HFD. EA improved dysfunctional glucose and lipid metabolism in this PCOS-IR rat model, and the molecular mechanism appeared to involve regulation of the expression of key molecules of the PI3-K/Akt insulin signaling pathway in the liver.

Taurine promotes muscle fiber type transformation through CaN/NFATc1 signaling in porcine myoblasts.

This study investigated the effect of taurine (TAU) on the muscle fiber type transformation in porcine myoblasts and its molecular mechanisms. The findings revealed that TAU augmented the protein expression of slow MyHC and the enzyme activities of oxidative metabolism markers like malate dehydrogenase and succinic dehydrogenase. Conversely, it curtailed the expression of fast MyHC and glycolytic metabolism enzyme activity of lactate dehydrogenase. Moreover, TAU elevated the expression of genes associated with oxidative fiber while diminishing the expression of those linked to glycolytic fibers, suggesting that TAU promoted the muscle fiber type transformation from glycolytic fiber to oxidative fiber. Additionally, TAU notably enhanced the expression of key molecules of calcineurin (CaN)/nuclear factor of activated T cells c1 (NFATc1) signaling and the CaN activity in porcine myoblasts. However, CaN inhibitor cyclosporine A abolished these effects induced by TAU. Our results indicated that TAU regulated the muscle fiber type transformation from glycolytic to oxidative fiber by activation of CaN/NFATc1 signaling.

Preliminary Study on the Effect and Molecular Mechanism of Tetrandrine in Alleviating Pulmonary Inflammation and Fibrosis Induced by Silicon Dioxide.

This study aims to explore the molecular mechanism of tetrandrine (Tet) in alleviating pulmonary inflammation and fibrosis induced by silica (SiO2) from the perspective of autophagy. C57BL/6J mice were selected as experimental animals, and SiO2 was exposed by intranasal instillation. Tet was intervened by oral gavage. The mice were euthanized on the 7th and 42nd day of SiO2 exposure, and lung tissues were collected for histopathological, molecular biological, immunological, and transmission electron microscopy analysis. The results showed that SiO2 exposure could lead to significant lung inflammation and fibrosis, while Tet could significantly reduce SiO2 exposure-induced lung inflammation and fibrosis. Molecular mechanism research indicated that, compared with SiO2 expose group, Tet intervention could significantly reduce the expression levels of inflammatory cytokines and fibrosis markers (TNF-α, IL-1β, MCP-1, TGF-β1, HYP, Col-I, and Fn), and regulate the expression of key molecules ATG7, microtubule-associated protein 1 light chain 3B (LC3B), and P62 in the autophagy pathway to improve the blocking of autophagic flux, promote the recovery of autophagic lysosomal system function, and inhibit apoptosis. In summary, Tet can alleviate silica-induced lung inflammation and fibrosis, which may be achieved by regulating the expression of key molecules in the autophagy process and associated apoptotic pathway.

Mechanism of Zhenwu Decoction in improving renal inflammatory injury in mice with DN of spleen-kidney Yang deficiency syndrome by regulating ROCK/IKK/NF-κB pathway

To investigate the intervention effect and mechanism of Zhenwu Decoction on diabetic nephropathy(DN) mice of spleen-kidney Yang deficiency syndrome based on the Rho-associated coiled-coil kinase(ROCK)/IκB kinase(IKK)/nuclear factor-κB(NF-κB) pathway. Ninety-five 7-week-old db/db male mice and 25 7-week-old db/m male mice were fed adaptively for one week. The DN model of spleen-kidney Yang deficiency syndrome was induced by Dahuang Decoction combined with hydrocortisone by gavage, and then the model was evaluated. After modeling, they were randomly divided into a model group, high-dose, medium-dose, and low-dose Zhenwu Decoction groups(33.8, 16.9, and 8.45 g·kg~(-1)·d~(-1)), and an irbesartan group(25 mg·kg~(-1)·d~(-1)), with at least 15 animals in each group. The intervention lasted for eight weeks. After the intervention, body weight and food intake were measured. Serum crea-tinine(Scr), blood urea nitrogen(BUN), fasting blood glucose(FBG), urinary albumin(uALb), and urine creatinine(Ucr) were determined. The uALb/Ucr ratio(ACR) and 24 h urinary protein(UTP) were calculated. Renal pathological morphology was evaluated by HE staining and Masson staining. The levels of key molecular proteins in the ROCK/IKK/NF-κB pathway were detected by Western blot. Enzyme-linked immunosorbent assay(ELISA) was used to detect interleukin-1β(IL-1β), interleukin-6(IL-6), interleukin-8(IL-8), interleukin-10(IL-10), and tumor necrosis factor-α(TNF-α). Compared with the blank group, the model group showed increased content of BUN, uALb, and SCr, increased values of 24 h UTP and ACR, decreased content of Ucr(P&lt;0.05), enlarged glomeruli, thickened basement membrane, mesangial matrix proliferation, inflammatory cell infiltration, and collagen fiber deposition. The protein expression of ROCK1, ROCK2, IKK, NF-κB, phosphorylated IKK(p-IKK), phosphorylated NF-κB(p-NF-κB), and phosphorylated inhibitor of NF-κB(p-IκB) increased(P&lt;0.05), while the protein expression of inhibitor of NF-κB(IκB) decreased(P&lt;0.05). The levels of inflammatory factors IL-1β, IL-6, IL-8, and TNF-α increased(P&lt;0.05), while the level of IL-10 decreased(P&lt;0.05). Compared with the model group, the groups with drug treatment showed decreased levels of BUN, uALb, SCr, 24 h UTP, and ACR, increased level of Ucr(P&lt;0.05), and improved renal pathological status to varying degrees. The high-and medium-dose Zhenwu Decoction groups and the irbesartan group showed reduced protein expression of ROCK1, ROCK2, IKK, NF-κB, p-IKK, p-NF-κB, and p-IκB in the kidneys(P&lt;0.05), increased protein expression of IκB(P&lt;0.05), decreased levels of serum inflammatory factors IL-1β, IL-6, IL-8, and TNF-α(P&lt;0.05), and increased level of IL-10(P&lt;0.05). Zhenwu Decoction can significantly improve renal function and renal pathological damage in DN mice of spleen-kidney Yang deficiency syndrome, and its specific mechanism may be related to the inhibition of inflammatory response by down-regulating the expression of key molecules in the ROCK/IKK/NF-κB pathway in the kidney.

The dual functions of KDM7A in HBV replication and immune microenvironment.

KDM7A (lysine demethylase 7A, also known as JHDM1D) is a histone demethylase, it is mainly involved in the intracellular post-translational modifications process. Recently, it has been proved that the histone demethylase members can regulate the replication of hepatitis B virus (HBV) and the expression of key molecules in the Janus-activated kinase-signal transducer and activator of the transcription (JAK/STAT) signaling pathway by chromatin modifying mechanisms. In our study, we identify novel roles of KDM7A in HBV replication and immune microenvironment through two subjects: pathogen and host. On the one hand, KDM7A is highly expressed in HBV-infected cells and promotes HBV replication in vitro and in vivo. Moreover, KDM7A interacts with HBV covalently closed circular DNA and augments the activity of the HBV core promoter. On the other hand, KDM7A can remodel the immune microenvironment. It inhibits the expression of interferon-stimulated genes (ISGs) through the IFN-γ/JAK2/STAT1 signaling pathway in both hepatocytes and macrophages. Further study shows that KDM7A interacts with JAK2 and STAT1 and affects their methylation. In general, we demonstrate the dual functions of KDM7A in HBV replication and immune microenvironment, and then we propose a new therapeutic target for HBV infection and immunotherapy. IMPORTANCE Histone lysine demethylase KDM7A can interact with covalently closed circular DNA and promote the replication of hepatitis B virus (HBV). The IFN-γ/JAK2/STAT1 signaling pathway in macrophages and hepatocytes is also downregulated by KDM7A. This study provides new insights into the mechanism of HBV infection and the remodeling of the immune microenvironment.

MitoQ as an antenatal antioxidant treatment improves markers of lung maturation in healthy and hypoxic pregnancy.

Chronic fetal hypoxaemia is a common pregnancy complication that increases the risk of infants experiencing respiratory complications at birth. In turn, chronic fetal hypoxaemia promotes oxidative stress, and maternal antioxidant therapy in animal models of hypoxic pregnancy has proven to be protective with regards to fetal growth and cardiovascular development. However, whether antenatal antioxidant therapy confers any benefit on lung development in complicated pregnancies has not yet been investigated. Here, we tested the hypothesis that maternal antenatal treatment with MitoQ will protect the developing lung in hypoxic pregnancy in sheep, a species with similar fetal lung developmental milestones as humans. Maternal treatment with MitoQ during late gestation promoted fetal pulmonary surfactant maturation and an increase in the expression of lung mitochondrial complexes III and V independent of oxygenation. Maternal treatment with MitoQ in hypoxic pregnancy also increased the expression of genes regulating liquid reabsorption in the fetal lung. These data support the hypothesis tested and suggest that MitoQ as an antenatal targeted antioxidant treatment may improve lung maturation in the late gestation fetus. KEY POINTS: Chronic fetal hypoxaemia promotes oxidative stress, and maternal antioxidant therapy in hypoxic pregnancy has proven to be protective with regards to fetal growth and cardiovascular development. MitoQ is a targeted antioxidant that uses the cell and the mitochondrial membrane potential to accumulate within the mitochondria. Treatment of healthy or hypoxic pregnancy with MitoQ, increases the expression of key molecules involved in surfactant maturation, lung liquid reabsorption and in mitochondrial proteins driving ATP synthesis in the fetal sheep lung. There were no detrimental effects of MitoQ treatment alone on the molecular components measured in the present study, suggesting that maternal antioxidant treatment has no effect on other components of normal maturation of the surfactant system.

Mammary gland development in male rats perinatally exposed to propiconazole, glyphosate, or their mixture

This study aimed to assess whether perinatal exposure to propiconazole (PRO), glyphosate (GLY) or their mixture (PROGLY) alters key endocrine pathways and the development of the male rat mammary gland. To this end, pregnant rats were orally exposed to vehicle, PRO, GLY, or a mixture of PRO and GLY from gestation day 9 until weaning. Male offspring were euthanized on postnatal day (PND) 21 and PND60. On PND21, GLY-exposed rats showed reduced mammary epithelial cell proliferation, whereas PRO-exposed ones showed increased ductal p-Erk1/2 expression without histomorphological alterations. On PND60, GLY-exposed rats showed reduced mammary gland area and estrogen receptor alpha expression and increased aromatase expression, whereas PRO-exposed ones showed enhanced lobuloalveolar development and increased lobular hyperplasia. However, PROGLY did not modify any of the endpoints evaluated. In summary, PRO and GLY modified the expression of key molecules and the development of the male mammary gland individually but not together.

Characterization of a cuproptosis-related signature to evaluate immune features and predict prognosis in colorectal cancer.

Cuproptosis is a newly discovered type of cell death. Little is known about the roles that cuproptosis related genes (CRGs) play in colorectal cancer (CRC). The aim of this study is to evaluate the prognostic value of CRGs and their relationship with tumor immune microenvironment. TCGA-COAD dataset was used as the training cohort. Pearson correlation was employed to identify CRGs and paired tumor-normal samples were used to identify those CRGs with differential expression pattern. A risk score signature was constructed using LASSO regression and multivariate Cox stepwise regression methods. Two GEO datasets were used as validation cohorts for confirming predictive power and clinical significance of this model. Expression patterns of seven CRGs were evaluated in COAD tissues. In vitro experiments were conducted to validate the expression of the CRGs during cuproptosis. A total of 771 differentially expressed CRGs were identified in the training cohort. A predictive model termed riskScore was constructed consisting of 7 CRGs and two clinical parameters (age and stage). Survival analysis suggested that patients with higher riskScore showed shorter OS than those with lower (P<0.0001). ROC analysis revealed that AUC values of cases in the training cohort for 1-, 2-, and 3-year survival were 0.82, 0.80, 0.86 respectively, indicating its good predictive efficacy. Correlations with clinical features showed that higher riskScore was significantly associated with advanced TNM stages, which were further confirmed in two validation cohorts. Single sample gene set enrichment analysis (ssGSEA) showed that high-risk group presented with an immune-cold phenotype. Consistently, ESTIMATE algorithm analysis showed lower immune scores in riskScore-high group. Expressions of key molecules in riskScore model are strongly associated with TME infiltrating cells and immune checkpoint molecules. Patients with a lower riskScore exhibited a higher complete remission rate in CRCs. Finally, seven CRGs involved in riskScore were significantly altered between cancerous and paracancerous normal tissues. Elesclomol, a potent copper ionophore, significantly altered expressions of seven CRGs in CRCs, indicating their relationship with cuproptosis. The cuproptosis-related gene signature could serve as a potential prognostic predictor for colorectal cancer patients and may offer novel insights into clinical cancer therapeutics.

Preliminary study on the role of aryl hydrocarbon receptor in the neurotoxicity of three typical bisphenol compounds (BPA, BPS and TBBPA) at environmentally relevant concentrations to adult zebrafish (Danio rerio)

ObjectiveThis study was aimed to explore the role of AhR in the neurotoxicity of adult zebrafish induced by three typical bisphenol compounds (BPA, BPS, TBBPA) at environmentally relevant doses. MethodsThe adult zebrafish were randomly divided into solvent control group (DMSO) and AhR inhibitor CH223191 (CH) group (0.05 μmol/L), bisphenol exposure groups (10, 100, 1000 nmol/L) and combined exposure groups (0.05 μmol/L CH and 1000 nmol/L bisphenol compounds). Each tank contained 8 fish (4 male and 4 female), and two parallel tanks were set synchronously. After 30 days of exposure, zebrafish were put on ice plate for anesthesia, weighed and measured for body length, and dissected for brain tissue. The gene expression was detected by RT-qPCR, and the activities of antioxidant enzymes were detected by commercial kits. SPSS 26.0 was used to analyze the data. Additionally, GO, KEGG and principal component analysis (PCA) were carried out. ResultsCompared with the solvent control group, there were no significant differences in body weight and length among the exposed groups. In general, exposure to bisphenol compounds could affect the expression of Ahr2 and AhR target genes (cyp1a1, cyp1a2, and cyp1c1), key genes of neural function (elavl3, gfap, mbp, syn2a, gap43, Zn5, shha, and ache), oxidative stress related genes (nrf2, gpx1a, gstp1/gstp1.2, gstp2/gstp1.1, sod1, sod2, and cat), and the activities of antioxidant enzymes (SOD, CAT and GSH-Px/GPX) in zebrafish brain tissue to some extent. Compared with the groups exposed to bisphenols alone, CH could antagonize the above interference effects caused by bisphenols to some extent. Therefore, the toxic effects of BPA, BPS and TBBPA might be produced through similar mechanisms. ConclusionEnvironmentally related doses of bisphenols (BPA, BPS, TBBPA) could disturb the expression of key molecules of oxidative stress and neural function through activating the AhR signaling pathway, and ultimately lead to neurotoxicity.

Levistolide A ameliorates fibrosis in chronic kidney disease via modulating multitarget actions in vitro and in vivo.

The increasing incidence of chronic kidney disease (CKD) urgently calls for effective nephroprotective agents. Traditional Chinese Medicine Angelica sinensis and its formula are well known for CKD therapy, but the underlying mechanisms and effective substances of reno-protective effects remain unclear. To this end, we isolated eleven ligustilide dimers (1-11) from A. sinensis and examined the molecular mechanism of their nephroprotective effects. Because of internal RAS playing an important role in CKD, we used renin expression as a target and screened preliminarily for antifibrotic effects of ligustilide dimers (1-11) by constructing a dual luciferase reporter gene in vitro. Furthermore, the reno-protective effects of the ligustilides and their underlying mechanism were investigated in TGF-β1-stimulated HK-2 cells and 5/6 nephrectomy (Nx) mice. The ligustilide dimers exhibited anti-fibrotic effects by inhibiting human renin (hREN) promoter activity to decrease renin expression and down-regulate the expression of fibrosis-related factors, including α-SMA, collagen I, and fibronectin in vitro. Levistolide A (LA) and angeolide keto ester (AK) were screened out to identify their ability and underlying mechanism for treating CKD. Experimental validation further indicated that LA or AK treatment inhibited the expression of key molecules in RAS, TGF-β1/Smad, and MAPK pathways to downregulate ECM deposition. Furthermore, LA obviously meliorated renal injury in 5/6 Nx mice through ameliorating oxidant stress, inflammation, apoptosis and renal fibrosis. The experimental results demonstrated that ligustilide dimers were potential nephroprotective agents. LA might be an attractive drug candidate for renin-targeted CKD therapy.

Sex-specific effect of AQP9 deficiency on hepatic triglyceride metabolism in mice with diet-induced obesity.

Studies in obese rats and human cell models of non-alcoholic fatty liver disease have indicated that knockdown of the hepatic glycerol channel aquaporin 9 (AQP9) leads to decreased hepatic steatosis. However, a study in leptin receptor-deficient mice did not find that knockout (KO) of AQP9 alleviated hepatic steatosis. The aim of this study was to investigate the effect of high-fat diet (HFD) on hepatic glycerol and triglyceride metabolism in male and female AQP9 KO mice. Male and female AQP9 KO mice and wild-type (WT) littermates were fed a HFD for 12weeks. Weight, food intake and blood glucose were monitored throughout the study and tissue analysis included determination of hepatic triglyceride content and triglyceride secretion. The expression of key molecules for hepatic glycerol and triglyceride metabolism was evaluated using qPCR and western blotting. AQP9 KO and WT mice demonstrated a similar weight gain throughout the study period, and we found no evidence for AQP9 deficiency being associated with a reduced hepatic accumulation of triglyceride or a reduced blood glucose level. Instead, we show that the effect of AQP9 deficiency on hepatic lipid metabolism is sex-specific, with only male AQP9 KO mice having a reduced hepatic secretion of triglycerides and an elevated expression of peroxisome proliferator-activated receptor α. Male AQP9 KO mice had an elevated blood glucose level after 12weeks of HFD when compared to baseline levels. Thus, we found no evidence for AQP9 inhibition being a target for alleviating the development of hepatic steatosis in mice with diet-induced obesity. KEY POINTS: This study investigates the effect of AQP9 deficiency on hepatic triglyceride metabolism in both male and female mice fed a high-fat diet (HFD) for 12weeks. No evidence was found for AQP9 deficiency being associated with a reduced hepatic accumulation of triglyceride or a reduced blood glucose level. The effect of AQP9 deficiency on hepatic triglyceride metabolism is sex-specific. Male AQP9 KO mice had a reduced hepatic secretion of triglycerides and an elevated expression of peroxisome proliferator-activated receptor α, which likely promotes an increased hepatic fatty acid oxidation. Male AQP9 KO had an elevated blood glucose level after 12weeks of HFD when compared to baseline levels.

Barrier function-related genes and proteins have an altered expression in acne-involved skin.

Acne vulgaris provides a unique disease setting in which a prominent skin inflammation is coupled with the overproduction of lipid-rich sebum. Our goal was to evaluate the expression of barrier molecules in papular acne skin samples obtained from untreated patients and compare those to the results of healthy and of papulopustular rosacea-involved ones at the mRNA and protein levels. In addition, we aimed to assess the effects of various sebum composing lipids on the expression of proteins involved in barrier formation in keratinocytes. Available microarray data sets of papular acne and papulopustular rosacea-affected skin samples were re-analysed with a focus on epidermal barrier-related pathways. Immunohistochemistry was performed to detect barrier molecules in the interfollicular regions of human acne and healthy skin samples. Protein levels of barrier-related genes were measured by western blot in samples of HaCaT keratinocytes treated with selected lipids. Meta-analysis of whole transcriptome data sets revealed that barrier-related pathways are significantly affected in acne vulgaris skin samples. While an altered expression of key molecules in maintaining barrier functions such as filaggrin, keratin 1, involucrin, desmoglein 1, kallikrein 5 and 7, was also observed at the protein levels, our data demonstrated that sebum composing lipids may selectively modify the levels of epidermal barrier-related molecules. Our results suggest that although not as prominently as in the dry papulopustular rosacea skin, the epidermal barrier in the interfollicular region may be damaged also in the lipid-rich skin samples of papular acne. Furthermore, our findings indicating diverse regulatory effects of various sebum lipids on the expression of barrier molecules in keratinocytes suggest, that they may influence the moisturization of the skin as well. Altogether, our findings could have implications in the development of sebum-modulating anti-acne therapies and even in the care of symptom-free skin.

Anoikis resistance of small airway epithelium is involved in the progression of chronic obstructive pulmonary disease.

Anoikis resistance is recognized as a crucial step in the metastasis of cancer cells. Most epithelial tumors are distinguished by the ability of epithelial cells to abscond anoikis when detached from the extracellular matrix. However, no study has investigated the involvement of anoikis in the small airway epithelium (SAE) of chronic obstructive pulmonary disease (COPD). Anoikis-related genes (ANRGs) exhibiting differential expression in COPD were identified using microarray datasets obtained from the Gene Expression Omnibus (GEO) database. Unsupervised clustering was performed to classify COPD patients into anoikis-related subtypes. Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, gene set enrichment analysis (GSEA), and gene set variation analysis (GSVA) were used to annotate the functions between different subtypes. Differential expression analysis and weighted gene co-expression network analysis (WGCNA) were leveraged to identify key molecules. The relative proportion of infiltrating immune cells in the SAE was quantified using the CIBERSORT and ssGSEA computational algorithms, and the correlation between key molecules and immune cell abundance was analyzed. The expression of key molecules in BEAS-2B cells exposed to cigarette smoke extract (CSE) was validated using qRT-PCR. A total of 25 ANRGs exhibited differential expression in the SAE of COPD patients, based on which two subtypes of COPD patients with distinct anoikis patterns were identified. COPD patients with anoikis resistance had more advanced GOLD stages and cigarette consumption. Functional annotations revealed a different immune status between COPD patients with pro-anoikis and anoikis resistance. Tenomodulin (TNMD) and long intergenic non-protein coding RNA 656 (LINC00656) were subsequently identified as key molecules involved in this process, and a close correlation between TNMD and the infiltrating immune cells was observed, such as activated CD4+ memory T cells, M1 macrophages, and activated NK cells. Further enrichment analyses clarified the relationship between TNMD and the inflammatory and apoptotic signaling pathway as the potential mechanism for regulating anoikis. In vitro experiments showed a dramatic upregulation of TNMD and LINC00656 in BEAS-2B cells when exposed to 3% CSE for 48 hours. TNMD contributes to the progression of COPD by inducing anoikis resistance in SAE, which is intimately associated with the immune microenvironment.

WNT16b promotes the proliferation and self-renewal of human limbal epithelial stem/progenitor cells via activating the calcium/calcineurin A/NFATC2 pathway.

Our previous finding revealed that WNT16b promoted the proliferation of human limbal epithelial stem cells (hLESCs) through a β-catenin independent pathway. Here, we aimed to explore its underlying molecular mechanism and evaluate its potential in the treatment of limbal stem cell deficiency (LSCD). Based on the findings of mRNA-sequencing, the expression of key molecules in WNT/calcineurin A/NFATC2 signalling pathway was investigated in WNT16b-co-incubated hLESCs and control hLESCs. An epithelial wound healing model was established on Wnt16b-KO mice to confirm the regulatory effect of WNT16b in vivo. The therapeutic potential of WNT16b-co-incubated hLESCs was also evaluated in mice with LSCD. Our findings showed that WNT16b bound with Frizzled7, promoted the release of Ca2+ and activated calcineurin A and NFATC2. With the translocation of NFATC2 into cell nucleus and the activation of HDAC3, WDR5 and GCN5L2, the expression of H3K4me3, H3K14ac and H3K27ac in the promoter regions of FoxM1 and c-MYC increased, which led to hLESC proliferation. The effect of the WNT16b/calcium/calcineurin A/NFATC2 pathway on LESC homeostasis maintenance and corneal epithelial repair was confirmed in Wnt16b-KO mice. Moreover, WNT16b-coincubated hLESCs could reconstruct a stable ocular surface and inhibit corneal neovascularization in mice with LSCD. In conclusion, WNT16b enhances the proliferation and maintains the stemness of hLESCs by activating the non-canonical calcium/calcineurin A/NFATC2 pathway in vitro and in vivo, and accelerates corneal epithelial wound healing.

Pirfenidone Protects from UVB-Induced Photodamage in Hairless Mice.

Ultraviolet radiation (UV) is the main environmental factor that causes histological degenerative changes of the skin giving rise to a chronic process called photodamage. Non-melanoma skin cancer induced by UVB radiation is a result of a cascade of molecular events caused by DNA damage in epidermis cells, including persistent inflammation, oxidative stress, and suppression of T cell-mediated immunity. Retinoids such as tretinoin have been widely used in skin to treat photoaging and photodamage, though its secondary adverse effects have been recognized. Pirfenidone (PFD) has emerged as an antifibrogenic, anti-inflammatory and antioxidant agent, and in this work its efficacy was evaluated in a model of UVB-induced photodamage. Epidermal, dermal, and inflammatory changes were measured by histomorphometric parameters. In addition, gene, and protein expression of key molecules in these processes were evaluated. Our results revealed an anti-photodamage effect of topical PFD with absence of inflammatory skin lesions determined by dermoscopy. In addition, PFD reduced elastosis, improved organization, arrangement, and deposition of dermal collagens, downregulated several pro-inflammatory markers such as NF-kB, IL-1, IL-6 and TNFα, and decreased keratinocyte damage. Topical pirfenidone represents a promising agent for the treatment of cell photodamage in humans. Clinical trials need to be carried out to explore this premise.

Investigating the potential role of swertiamarin on insulin resistant and non-insulin resistant granulosa cells of poly cystic ovarian syndrome patients

Background and aimConventional drugs have limitations due to prevalence of contraindications in PCOS patients. To explore the potential effects of swertiamarin, on abrupted insulin and steroidogenic signaling in human luteinized granulosa cells from PCOS patients with or without insulin resistance.Experimental procedurehLGCs from 8 controls and 16 PCOS patients were classified for insulin resistance based on down regulation of protein expression of insulin receptor-β (INSR- β) as shown in our previous paper. Cells were grouped as control, PCOS-IR and PCOS-NIR, treated with swertiamarin (66 µM) and metformin (1 mM). Expression of key molecules involved in insulin signaling, fat metabolism, IGF system and steroidogenesis were compared between groups.ResultsSwertiamarin significantly (P < 0.05) reversed the expression of INSR-β, PI(3)K, p-Akt, PKC-ζ, PPARγ, (P < 0.01) IRS (Ser 307) and IGF system in PCOS-IR group and was equally potent to metformin. In the same group, candidate genes viz SREBP1c, FAS, ACC-1 and CPT-1 were down regulated by swertiamarin (P < 0.001) and metformin (P < 0.001). Significant upregulation was demonstrated in expression of StAR, CYP19A1, 17β-HSD and 3β-HSD when treated with swertiamarin (P < 0.01) and metformin (P < 0.01) in PCOS-IR followed by increase in 17β-HSD and 3β-HSD enzyme activity along with estradiol and progesterone secretions. However, swertiamarin did not reveal any effect on PCOS-NIR group as compared to metformin that significantly (P < 0.01) reversed all the parameters related to steroidogenesis and down regulated basal expression of insulin signaling genes.ConclusionSwertiamarin, presents itself as a potential fertility drug in hLGCs from PCOS-IR patients.