Thermo-sensitive PLGA-PEG-PLGA hydrogel for sustained release of EGF to inhibit cervical cancer recurrence.

Overexpression of epidermal growth factor receptor (EGFR) in cancer is a key cause of recurrence of cervical cancer (CC). Although the EGF-EGFR pathway has been studied for decades, preventing tumor growth and recurrence caused by peripheral EGF remains a great challenge. In this work, a strategy is proposed to reduce the stimulation of high concentration EGF on tumor growth by using a thermo-sensitive hydrogel. The hydrogel is a triblock copolymer composed of polyethylene glycol (PEG) and poly (lactide glycolide) (PLGA). Based on the excellent temperature sensitivity, carrier capacity, swelling property and biocompatibility, the hydrogel can absorb the liquid around the tumor by injection and release EGF continuously at low concentration. The inhibitory effect of hydrogel on tumor growth is fully confirmed by an implanted tumor mouse model with human cervical cancer cell lines (HeLa) using triple-immunodeficient NCG mice. Compared with free EGF, the EGF-loaded hydrogel can hardly induce surface plasmon resonance (SPR) response, which proves that hydrogel can effectively weaken cytoskeleton rearrangement and inhibit cell migration by continuously releasing low concentration EGF. In addition, the EGF-loaded hydrogel can reduce cell proliferation by delaying the progress of cell cycle progression. Taken together, the hydrogel can effectively protect tumor microenvironment from the stimulation of high concentration EGF, delay cancer cellular processes and tumor growth, and thus providing an approach for inhibiting tumor recurrence of CC.

Molecular and cellular pruritus mechanisms in the host skin.

Pruritus, also known as itching, is a complex sensation that involves the activation of specific physiological and cellular receptors. The skin is innervated with sensory nerves as well as some receptors for various sensations, and its immune system has prominent neurological connections. Sensory neurons have a considerable impact on the sensation of itching. However, immune cells also play a role in this process, as they release pruritogens. Disruption of the dermal barrier activates an immune response, initiating a series of chemical, physical, and cellular reactions. These reactions involve various cell types, including keratinocytes, as well as immune cells involved in innate and adaptive immunity. Collective activation of these immune responses confers protection against potential pathogens. Thus, understanding the molecular and cellular mechanisms that contribute to pruritus in host skin is crucial for the advancement of effective treatment approaches. This review provides a comprehensive analysis of the present knowledge concerning the molecular and cellular mechanisms underlying itching signaling in the skin. Additionally, this review explored the integration of these mechanisms with the broader context of itch mediators and the expression of their receptors in the skin.

DNA methylation and gene expression profiles to identify childhood atopic asthma associated genes.

BACKGROUND: Asthma is a chronic inflammatory disorder of the airways involving many different factors. This study aimed to screen for the critical genes using DNA methylation/CpGs and miRNAs involved in childhood atopic asthma. METHODS: DNA methylation and gene expression data (Access Numbers GSE40732 and GSE40576) were downloaded from the Gene Expression Omnibus database. Each set contains 194 peripheral blood mononuclear cell (PBMC) samples of 97 children with atopic asthma and 97 control children. Differentially expressed genes (DEGs) with DNA methylation changes were identified. Pearson correlation analysis was used to select genes with an opposite direction of expression and differences in methylation levels, and then Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed. Protein-protein interaction network and miRNA-target gene regulatory networks were then constructed. Finally, important genes related to asthma were screened. RESULTS: A total of 130 critical DEGs with DNA methylation changes were screened from children with atopic asthma and compared with control samples from healthy children. GO and KEGG pathway enrichment analysis found that critical genes were primarily related to 24 GO terms and 10 KEGG pathways. In the miRNA-target gene regulatory networks, 9 KEGG pathways were identified. Analysis of the miRNA-target gene network noted an overlapping KEGG signaling pathway, hsa04060: cytokine-cytokine receptor interaction, in which the gene CCL2, directly related to asthma, was involved. This gene is targeted by eight asthma related miRNAs (hsa-miR-206, hsa-miR-19a, hsa-miR-9,hsa-miR-22, hsa-miR-33b, hsa-miR-122, hsa-miR-1, and hsa-miR-23b). The genes IL2RG and CCl4 were also involved in this pathway. CONCLUSIONS: The present study provides a novel insight into the underlying molecular mechanism of childhood atopic asthma.

Role of the IL-33/ST2 receptor axis in ovarian cancer progression.

Ovarian cancer remains a significant health problem for women in the world due to its diagnosis at advanced stages of disease and the high mortality rate of patients. To date, ovarian cancer is frequently treated with tumor reduction surgery followed by platinum/paclitaxel-based chemotherapy; however, most patients eventually develop relapsed disease. The mRNA expression levels of interleukin-33 (IL-33) and the suppressor of tumorigenicity 2 (ST2) receptor are significantly upregulated in ovarian cancer tissues and metastatic tumor lesions. In addition, IL-33 and ST2 expression has been associated with a poor overall survival in patients with epithelial ovarian cancer. The IL-33 receptor ST2 is expressed as both a membrane-anchored receptor (ST2L) activated by IL-33, and as a soluble variant that exhibits anti-inflammatory properties. In the present review, the functions of the IL-33/ST2L axis in cells and their aberrant expression levels in ovarian cancer were discussed. In addition, targeting their expression as a novel strategy for the control of ovarian cancer progression was emphasized.

Immune response in fluid therapy with crystalloids of different ratios or colloid for rats in haemorrhagic shock.

This study investigated the association between different ratios of balanced salt based-crystalloid (PLASMA SOLUTION-A [CJ HealthCare, Seoul, Korea]) (the ratios of crystalloid for blood loss, 1:1, 1:2 and 1:3) or balanced salt-based colloid (VOLULYTE 6% [Fresenius Kabi, Germany]) (the ratio of colloid for blood loss, 1:1) to restore blood loss and immune response in rats with haemorrhagic shock. About 50% of total estimated blood volume was removed after anaesthesia. The fluid was administered for resuscitation after exsanguination, according to the type of fluid and the ratios of exsanguinated volume and fluid volume for resuscitation. After sacrifice, expression of immune cells in blood and tissues was evaluated. Histological analyses and syndecan-1 immunohistochemistry assays were performed on tissues. Endothelial damage according to syndecan-1 and cytokine levels in blood was also assessed. Fluid resuscitation with same, two-fold, or three-fold volumes of crystalloid, or same volume of colloid, to treat haemorrhagic shock in rats resulted in a similar increase in blood pressure. The expression of neutrophils in blood decreased significantly after colloid administration, compared to before exsanguination. Syndecan-1 expression increased after exsanguination and fluid resuscitation in all groups, without any significant difference. In conclusion, same volume of balanced salt-based crystalloid for blood loss was enough to restore BP at the choice of fluid for the management of haemorrhagic shock in the rats, compared with different ratios of crystalloid or same volume of colloid, on the aspect of immune response.

Mechanism of CNP-mediated DG-PKC and IP4 signaling pathway in diabetic rats with gastric motility disorder.

In the precedent research conducted by the same team, it concluded that the activities in C-type natriuretic peptide (CNP)/cyclic guanosine monophosphate (cGMP)/cyclic adenosine monophosphate (cAMP)/ß-type phospholipase C (PLCß) pathways of rat antral smooth muscle were changed due to diabetes, which was the key pathogenetic mechanism for diabetic gastric dysmotility. As the follow-on step, this study was designed to probe into the downstream signaling pathway of CNP/PLCß. The results showed that level of α-type protein kinase C (PKCα),cell membrane to cytoplasm ratio of PKCα, cell membrane to cytoplasmic ratio of ßI-type protein kinase C (PKCßI) and level of Phosphor-PKCα (P-PKCα) were significantly reduced in diabetes rat antral smooth muscle samples. The content of tetraphosphate inositol (IP4) in gastric antral smooth muscle of diabetic rats reduced, and the content of diacyl-glycerol (DG) was unchanged. CNP significantly decreased the content of IP4 and DG, this effect was more obvious in diabetic rats. Subsequent to the addition of protein kinase A (PKA) blocker N-[2- (p-Bromocin-namylamino)ethyl]-5 -isoquinolinesulfonamide dihydrochloride (H-89) before CNP treatment, the inhibitory effect of CNP was reduced; subsequent to the addition of protein kinase G (PKG) blocker KT5823 before CNP treatment, the inhibitory effect of CNP was also reduced. With the addition of the combination of H-89 and KT5823 before CNP treatment, the inhibition by CNP could be offset. These results were concluded that CNP inhibited the activity of PKC family in rat smooth muscle and reduced the levels of IP4 and DG through the PKG/PKA-PLCß pathways, causing inhibited muscular contractions, which may be a key pathogenetic factor for diabetic gastroparesis.

Downregulation of microRNA-124 prevents the development of acute liver failure through the upregulation of PIM-3.

NEW FINDINGS: • What is the central question of this study? Does miR-124 affect cell proliferation and apoptosis in acute liver failure (ALF) mice? • What is the main finding and its importance? Inhibiting miR-124 targets PIM-3 and thus upregulates its expression, consequently inhibiting liver cell apoptosis and promoting cell proliferation, ultimately preventing the progression of ALF. This highlights a promising competitive new target for ALF treatment. ABSTRACT: Acute liver failure (ALF) is a complicated syndrome frequently leading to dysfunction and failure of various organs. MicroRNAs (miRNAs) have played crucial roles in the development and progression of human diseases, including ALF. However, the potential role of miR-124 in ALF still remains elusive. Thus, we investigated the underlying mechanism by which miR-124 influences ALF in a mouse model of ALF. Initially, ALF mouse models were established using d-galactosamine and lipopolysaccharide. Then we detected the serum biochemical parameters of liver, and pathological characteristics and ultrastructure of liver tissues. Next, we determined miR-124 and PIM-3 expression in liver tissues and cells using RT-qPCR and western blot analysis. The interaction between miR-124 and PIM-3 was identified using the dual luciferase reporter gene assay. Subsequently, expression of miR-124 and PIM-3 in liver cells was altered to explore their effects on primary liver cell proliferation, the cell cycle and apoptosis. The results obtained showed that ALF mice exhibited a decreased cholinesterase level with increased levels of alanine aminotransferase, aspartate transaminase and total bilirubin as well as abundant liver cell apoptosis and necrosis. miR-124 was upregulated while PIM-3 was downregulated in ALF tissues and cells. Besides, the PIM-3 gene was a target of miR-124 and was inhibited by miR-124. Overexpression of miR-124 or silencing of PIM-3 reduced Bcl-2 expression but elevated tumour necrosis factor α expression, and resulted in a reduction in liver cell proliferation but an increase in cell apoptosis in ALF mice. Altogether, miR-124 functions as a disease-promoting miRNA with potential in stimulating ALF by targeting PIM-3.

microRNA-138 induces cell survival and reduces WNT/ß-catenin signaling of osteoarthritis chondrocytes through NEK2.

Osteoarthritis (OA) is a degenerative joint disease characterized by joint pain, stiffness, and function degeneration with high incidence. Recent studies have been inspired based on the association between microRNAs (miRs) and therapeutic research of OA. Hence, the present study evaluates the effects of miR-138 on chondrocyte proliferation, differentiation, and apoptosis through the WNT/ß-catenin signaling pathway in mice with OA by binding to NIMA-related kinase 2 (NEK2). Appropriate dataset was selected from the Gene Expression Omnibus database, and differentially expressed genes and potential miRNAs that could regulate NEK2 were explored. A mouse model of OA was established. The expressions of miR-138, NEK2, ß-catenin, GSK3ß, Bcl-2, Bcl-2-associated X protein (Bax), p53, MMP-13, Col2, and Aggrecan and the phosphorylation levels of ß-catenin were determined by the reverse transcription quantitative polymerase chain reaction and Western blot analysis. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and flow cytometry were employed to detect cell proliferation and apoptosis, respectively. The potential functional role of NEK2 was revealed to be related to the WNT/ß-catenin signaling pathway, and miR-138 was the putative regulator of NEK2. miR-138 expression was downregulated while expressions of NEK2 and ß-catenin as well as the phosphorylation levels of ß-catenin were upregulated in mice with OA. The chondrocytes treated with miR-138 mimic and siRNA-NEK2 exhibited reduced expressions of NEK2, ß-catenin, MMP-13, Bax, and p53 and elevated expressions of Col2, Aggrecan, and Bcl-2 as well as phosphorylation levels of ß-catenin along with enhanced chondrocytes' proliferation and suppressed cell apoptosis. Overexpression of miR-138 induces cell survival and reduces WNT/ß-catenin signaling of OA chondrocytes through NEK2. © 2019 IUBMB Life, 71(9):1355-1366, 2019.

The role of CNP-mediated PKG/PKA-PLCß pathway in diabetes-induced gastric motility disorder.

Our previous work demonstrated that the C-type natriuretic peptide (CNP)/cyclic guanosine monophosphate (cGMP)/cyclic adenosine monophosphate (cAMP) pathway in gastric antrum smooth muscle of rats with diabetes was upregulated and played an important role in the development of diabetic gastric dysmotility. Our goal for this study was to explore the downstream signaling pathways of CNP. We found that the expressions of protein kinase G (PKG) and protein kinase A (PKA) in gastric smooth muscle tissue of rats with diabetes were significantly upregulated. The expressions of ß-type phospholipase C 3(PLCß3) and ß-type phospholipase C 1(PLCß1) protein were reduced, whereas Phosphor-PLCß3Ser1105 (P-PLCß3Ser1105) was increased. The inhibitory effect of CNP on gastric antral smooth muscle in diabetic rats was significantly greater than in the normal group. The content of trisphosphate inositol (IP3) in the gastric antral smooth muscle of rats with diabetes was significantly lower than that of the normal group. After blocking PKA with N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89, a blockage PKA), the effect of CNP on the production of IP3 was decreased, while blocking PKG with KT5823 (a blockage PKG) simultaneously, and CNP can no longer reduce the IP3 production. CNP promoted the phosphorylation of PLCß3Ser1105, thereby inhibiting the activity of PLCß3 in gastric smooth muscle tissue of rats with diabetes; this effect can be abolished by blocking PKA and PKG. These results suggested that CNP can decrease IP3 level in gastric smooth muscle cells and thus inhibit gastric smooth muscle contraction through PKG/PKA-PLCß pathway, which may play an important role in the development of diabetic gastroparesis.

Simultaneous determination and pharmacokinetic study of three flavonoid glycosides in rat plasma by LC-MS/MS after oral administration of Rubus chingii Hu extract.

A simple and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of isoquercitrin, kaempferol-3-O-rutinoside and tiliroside in rat plasma. Plasma samples were deproteinized with methanol and separated on a Hypersil Gold C18 column (2.1 × 50 mm, i.d., 3.0 µm) using gradient elution with the mobile phase of water and methanol at a flow rate of 0.4 mL/min. Mass spectrometric detection was performed with negative ion electrospray ionization in selected reaction monitoring mode. All analytes showed good linearity over their investigated concentration ranges (r2 > 0.99). The lower limit of quantification was 1.0 ng/mL for isoquercitrin and 2.0 ng/mL for kaempferol-3-O-rutinoside and tiliroside, respectively. Intra- and inter-day precisions were <8.2% and accuracy ranged from -11.5 to 9.7%. The mean extraction recoveries of analytes and IS from rat plasma were >80.4%. The assay was successfully applied to investigate the pharmacokinetic study of the three ingredients after oral administration of Rubus chingii Hu to rats.

Significance of dynamic changes in gastric smooth muscle cell apoptosis, PI3K-AKT-mTOR and AMPK-mTOR signaling in a rat model of diabetic gastroparesis.

The aim of the present study was to investigate the significance of cell apoptosis, the phosphoinositide-3-kinase (PI3K)-protein kinase B (AKT)-mammalian target of rapamycin (mTOR) pathway, and the 5' adenosine monophosphate-activated protein kinase (AMPK)­mTOR pathways in the process of diabetic gastroparesis. Changes in gastric smooth muscle cells of diabetic rats with induced gastroparesis were examined. The diabetic rat model was established by dividing animals into a normal control group and diabetic model groups examined at 2, 4 and 6 weeks. Diabetic gastroparesis was evaluated by examining the rates of gastric residual pigment, whereas flow cytometry was used to detect the apoptosis of gastric smooth muscle cells. The expression levels of PI3K and phosphorylated (p­) AKT, AMPK, mTOR, tuberous sclerosis complex 2, p70 ribosomal S6 kinase, and eukaryotic translation initiation factor 4­binding protein 1 were determined in gastric muscles using western blot analysis. Diabetic gastroparesis was confirmed in models at 6 weeks. The apoptosis of gastric smooth muscle cells gradually increased in all diabetic groups, and significant changes were observed in key proteins involved in PI3K­AKT­mTOR and AMPK­mTOR signaling. The results indicated that apoptosis was important in the occurrence of diabetic gastroparesis, and the PI3K­AKT­mTOR and AMPK­mTOR pathways were activated during the apoptotic processes, but were incapable of regulating apoptosis.

Development and validation of a sensitive UHPLC-MS/MS method for quantitative analysis of farrerol in rat plasma: Application to pharmacokinetic and bioavailability studies.

Farrerol is a 2,3-dihydro-flavonoid isolated from rhododendron. In this study, a sensitive and selective ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for the determination of farrerol in rat plasma. Liquid-liquid extraction by ethyl ether was used for sample preparation. Chromatographic separation was achieved on an Agilent UHPLC XDB-C18 column (2.1 × 100 mm, 1.8 µm) with water and methanol (30:70, v/v) as the mobile phase. An electrospray source was applied and operated in negative ion mode; selection reaction monitoring was used for quantification using target fragment ions m/z 299 → 179 for farrerol and m/z 267 → 252 for internal standard. Calibration plots were linear in the range of 2.88-1440 ng/mL for farrerol in rat plasma. Intra- and inter-day precisions were <11.6%, and the accuracy ranged from -13.9 to 11.9%. The UHPLC-MS/MS method was successfully applied in pharmacokinetics and bioavailability studies of farrerol in rats.

CNP-pGC-cGMP-PDE3-cAMP Signal Pathway Upregulated in Gastric Smooth Muscle of Diabetic Rats.

Our previous studies have shown that CNP-NPR-B/pGC-cGMP is upregulated in the diabetic rats. The present study was designed to determine whether the upregulation of CNP-NPR-B/pGC-cGMP signal pathway affects cGMP-PDE3-cAMP signal pathway in diabetic gastric smooth muscle. The gastric smooth muscle motility was observed by using isometric measurement. PDEs expressions in diabetic gastric smooth muscle tissue were observed by using immunohistochemistry, Western blotting, and RT-PCR methods. The results demonstrated that the inhibitory effect of CNP on the spontaneous contraction of gastric antral circular smooth muscle was potentiated in STZ-induced diabetic rat. CNP-induced increase of cGMP and cAMP was much higher in diabetic gastric smooth muscle tissue than in controls. The expression of PDE3 is downregulated while the levels of gene expression of PDE1, PDE2, PDE4, and PDE5 were not altered in the diabetic gastric smooth muscle tissue. The results suggest that the sensitivity of gastric smooth muscle to CNP is potentiated via activation of CNP-pGC-cGMP-PDE3-cAMP signal pathway in STZ-induced diabetic rats, which may be associated with diabetes-induced gastric motility disorder.

[Forskolin inhibits spontaneous contraction of gastric antral smooth muscle in rats].

The aim of the present study was to investigate the effects of cyclic adenosine monophosphate (cAMP) on rat gastric antral circular smooth muscle function. Forskolin, a direct activator of adenylyl cyclase (AC), was used to observe the influences of cAMP. Multi-channel physiological recorder was used to record spontaneous contraction activity of gastric antral circular muscle from Wistar rats. And ELISA method was used to detect the change of cAMP production in perfusate. The results showed that forskolin concentration-dependently suppressed the amplitude and frequency of the spontaneous contraction of the gastric antral muscle, and lowered the baseline of contraction movement significantly. Forskolin concentration-dependently increased the production of cAMP in the perfusate, which showed a significant negative correlation with the contraction amplitude of gastric antral ring muscle. The inhibitory effect of forskolin on spontaneous contraction activity of rat gastric antral circular muscle could be blocked by cAMP-dependent protein kinase (PKA) inhibitor H-89. These results suggest forskolin increases cAMP production and then activates PKA pathway, resulting in the inhibition of the spontaneous contraction activity of rat gastric antral circular smooth muscle.

cGMP-PDE3-cAMP signal pathway involved in the inhibitory effect of CNP on gastric motility in rat.

In the present study, we investigated the mechanism of C-type natriuretic peptide (CNP)-induced inhibitory effect on spontaneous contraction of gastric antral smooth muscle to clarify CNP-NPR-B/pGC-cGMP downstream signal transduction pathway using organ bath and ELISA methods in rat. CNP significantly reduced the amplitude of the spontaneous contraction and increased the contents of cGMP and cAMP in the gastric antral smooth muscle tissue. In the presence of IBMX, a non-selective phosphodiesterase (PDE) inhibitor, the inhibitory effect of CNP on spontaneous contraction was significantly suppressed; however, the production of cGMP but not cAMP was still increased by CNP. EHNA, a PDE2 inhibitor, did not affect both CNP-induced inhibition of the contraction and CNP-induced increase of cGMP and cAMP generations in gastric smooth muscle tissue, while milrinone, a PDE3 inhibitor, similar to IBMX, attenuated the CNP-induced inhibitory effect on spontaneous contraction and increased the content of cGMP but not cAMP. The results suggest that cGMP-PDE3-cAMP signal pathway is also involved in the CNP-induced inhibition of gastric motility in rat.