Circular RNA METTL9 contributes to neuroinflammation following traumatic brain injury by complexing with astrocytic SND1.

BACKGROUND: Circular RNAs (circRNAs) are highly enriched in the central nervous system and have been implicated in neurodegenerative diseases. However, whether and how circRNAs contribute to the pathological processes induced by traumatic brain injury (TBI) has not been fully elucidated. METHODS: We conducted a high-throughput RNA sequencing screen for well-conserved, differentially expressed circRNAs in the cortex of rats subjected to experimental TBI. Circular RNA METTL9 (circMETTL9) was ultimately identified as upregulated post-TBI and further characterized by RT-PCR and agarose gel electrophoresis, Sanger sequencing, and RNase R treatment. To examine potential involvement of circMETTL9 in neurodegeneration and loss of function following TBI, circMETTL9 expression in cortex was knocked-down by microinjection of a shcircMETTL9 adeno-associated virus. Neurological functions were evaluated in control, TBI, and TBI-KD rats using a modified neurological severity score, cognitive function using the Morris water maze test, and nerve cell apoptosis rate by TUNEL staining. Pull-down assays and mass spectrometry were conducted to identify circMETTL9-binding proteins. Co-localization of circMETTL9 and SND1 in astrocytes was examined by fluorescence in situ hybridization and immunofluorescence double staining. Changes in the expression levels of chemokines and SND1 were estimated by quantitative PCR and western blotting. RESULTS: CircMETTL9 was significantly upregulated and peaked at 7 d in the cerebral cortex of TBI model rats, and it was abundantly expressed in astrocytes. We found that circMETTL9 knockdown significantly attenuated neurological dysfunction, cognitive impairment, and nerve cell apoptosis induced by TBI. CircMETTL9 directly bound to and increased the expression of SND1 in astrocytes, leading to the upregulation of CCL2, CXCL1, CCL3, CXCL3, and CXCL10, and ultimately to enhanced neuroinflammation. CONCLUSION: Altogether, we are the first to propose that circMETTL9 is a master regulator of neuroinflammation following TBI, and thus a major contributor to neurodegeneration and neurological dysfunction.

Thoracic perivascular adipose tissue inhibits VSMC apoptosis and aortic aneurysm formation in mice via the secretome of browning adipocytes.

Abdominal aortic aneurysm (AAA) is a dangerous vascular disease without any effective drug therapies so far. Emerging evidence suggests the phenotypic differences in perivascular adipose tissue (PVAT) between regions of the aorta are implicated in the development of atherosclerosis evidenced by the abdominal aorta more vulnerable to atherosclerosis than the thoracic aorta in large animals and humans. The prevalence of thoracic aortic aneurysms (TAA) is much less than that of abdominal aortic aneurysms (AAA). In this study we investigated the effect of thoracic PVAT (T-PVAT) transplantation on aortic aneurysm formation and the impact of T-PVAT on vascular smooth muscle cells. Calcium phosphate-induced mouse AAA model was established. T-PVAT (20 mg) was implanted around the abdominal aorta of recipient mice after removal of endogenous abdominal PVAT (A-PVAT) and calcium phosphate treatment. Mice were sacrificed two weeks after the surgery and the maximum external diameter of infrarenal aorta was measured. We found that T-PVAT displayed a more BAT-like phenotype than A-PVAT; transplantation of T-PVAT significantly attenuated calcium phosphate-induced abdominal aortic dilation and elastic degradation as compared to sham control or A-PVAT transplantation. In addition, T-PVAT transplantation largely preserved smooth muscle cell content in the abdominal aortic wall. Co-culture of T-PVAT with vascular smooth muscle cells (VSMCs) significantly inhibited H2O2- or TNFα plus cycloheximide-induced VSMC apoptosis. RNA sequencing analysis showed that T-PVAT was enriched by browning adipocytes and anti-apoptotic secretory proteins. We further verified that the secretome of mature adipocytes isolated from T-PVAT significantly inhibited H2O2- or TNFα plus cycloheximide-induced VSMC apoptosis. Using proteomic and bioinformatic analyses we identified cartilage oligomeric matrix protein (COMP) as a secreted protein significantly increased in T-PVAT. Recombinant COMP protein significantly inhibited VSMC apoptosis. We conclude that T-PVAT exerts anti-apoptosis effect on VSMCs and attenuates AAA formation, which is possibly attributed to the secretome of browning adipocytes.

Multiplex Assessment of Serum Chemokines CCL2, CCL5, CXCL1, CXCL10, and CXCL13 Following Traumatic Brain Injury.

Chemokines may promote neuroinflammation following traumatic brain injury (TBI), thereby exacerbating secondary injury. This study was designed to investigate the contributions of chemokines (CCL2, CCL5, CXCL1, CXCL10, and CXCL13) to TBI severity and clinical outcome. Peripheral blood was drawn from 92 TBI patients on admission, and 40 controls were recruited. Serum concentrations of CCL2, CCL5, CXCL1, CXCL10, and CXCL13 on admission were measured by ELISA. Preoperative clinical severity was evaluated using the Glasgow Coma Scale (GCS), and clinical outcome at 90 days post-TBI was evaluated using the Glasgow Outcome Scale (GOS). The associations were evaluated by calculating Spearman's correlation coefficients. A binary logistic regression model was used to identify clinicodemographic factors influencing outcome, and ROC curves were constructed. Serum concentrations of CCL2, CCL5, CXCL1, CXCL10, and CXCL13 were elevated significantly after TBI and negatively correlated with GCS and GOS scores except CCL5. CCL2 may be considered as an independent predictor to predict severity and outcome. Moreover, combination of GCS score, CCL2, and CXCL10 can be a better assessment prognosis of moderate and severe TBI.

Therapeutic Potential of Photobiomodulation for Chronic Kidney Disease.

Chronic kidney disease (CKD) is a growing global public health problem. The implementation of evidence-based clinical practices only defers the development of kidney failure. Death, transplantation, or dialysis are the consequences of kidney failure, resulting in a significant burden on the health system. Hence, innovative therapeutic strategies are urgently needed due to the limitations of current interventions. Photobiomodulation (PBM), a form of non-thermal light therapy, effectively mitigates mitochondrial dysfunction, reactive oxidative stress, inflammation, and gut microbiota dysbiosis, all of which are inherent in CKD. Preliminary studies suggest the benefits of PBM in multiple diseases, including CKD. Hence, this review will provide a concise summary of the underlying action mechanisms of PBM and its potential therapeutic effects on CKD. Based on the findings, PBM may represent a novel, non-invasive and non-pharmacological therapy for CKD, although more studies are necessary before PBM can be widely recommended.

[Effect of Rehmanniae Radix Praeparata on energy metabolism in prefrontal cortex of rats with attention deficit hyperactivity disorder based on "static Yin and dynamic Yang" theory].

The present study investigated the effect of Rehmanniae Radix Praeparata(RRP) on the energy metabolism of prefrontal cortex(PFC) of spontaneously hypertensive rats with attention deficit hyperactivity disorder(ADHD) based on the "static Yin and dynamic Yang" theory.Thirty spontaneously hypertensive male rats aged 3 weeks were randomly divided into a model group, a methylphenidate(MPH) group(2 mg·kg~(-1)), and an RRP group(2.4 g·kg~(-1)).Wistar-Kyoto(WKY) male rats of the same age were assigned to the normal group.Rats were treated with corresponding drugs twice per day, and those in the model group and the normal group received the same volume of 0.9% sodium carboxymethyl cellulose(CMC-Na) solution by gavage.The open-field test was performed to evaluate the spontaneous and impulsive behaviors of rats before treatment and on the 4~(th) week after treatment.Four weeks after treatment, PFC was isolated and mitochondria were prepared.The content of adenosine triphosphate(ATP), adenosine diphosphate(ADP), and adenosine monophosphate(AMP) in the PFC was determined by high-performance liquid chromatography(HPLC), and energy charge(EC) was calculated.The parameters related to mitochondrial respiratory function were measured by the Clark oxygen electrode, specifically, state 3 respiration(ST3), state 4 respiration(ST4), and respiratory control rate(RCR).Enzymatic activities of succinate dehydrogenase(SDH), cytochrome C oxidase(COX), Na~+-K~+-ATPase, and Ca~(2+)-Mg~(2+)-ATPase were measured by chemical colorimetry.Mitochondrial permeability transition pore(mPTP) opening was measured by spectrophotometry.Protein expression of glucose transporter 1(GLUT1) and GLUT3 in PFC was tested by Western blot.Compared with the results in the model group, RRP could significantly reduce the total distance of movement, vertical times, and distance in the central area in the open field test(P<0.05 or P<0.01), increase the content of ATP and EC, decrease the content of AMP(P<0.05), elevate ST3 and RCR(P<0.05), potentiate activities of SDH, COX, Na~+-K~+-ATPase, and Ca~(2+)-Mg~(2+)-ATPase(P<0.05 or P<0.01), inhibit the opening of mPTP, and increase the expression levels of GLUT1 and GLUT3 proteins(P<0.05).It was inferred that RRP could inhibit hyperacti-vity and impulsivity by improving the energy metabolism disorder in PFC of ADHD rats, and its mechanism may be related to the improvement of mitochondrial respiratory function, potentiation of Na~+-K~+-ATPase, Ca~(2+)-Mg~(2+)-ATPase, and mitochondrial respiratory enzymes, inhibition of the opening of mPTP, and up-regulation of the expression of glucose transporter proteins.This study initially reveals the biological connotation of the "static Yin and dynamic Yang" theory in ADHD.

Effect of surgical treatment on prognosis in preterm infants with obstructive hydrocephalus. / æ—©äº§å„¿æ¢—é˜»æ€§è„‘ç§¯æ°´å¤–ç§‘æ²»ç–—ä¸Žé¢„åŽçš„ä¸´åºŠç ”ç©¶.

OBJECTIVES: To study the effect of surgical treatment on prognosis in preterm infants with obstructive hydrocephalus. METHODS: A retrospective analysis was performed on the medical data of 49 preterm infants with obstructive hydrocephalus. According to the treatment regimen, they were divided into two groups: surgical treatment (n=12) and conservative treatment (n=37). The drainage methods, drainage complications, and eventual shunt outcome were analyzed in the surgical treatment group. The two groups were compared in terms of the etiology of hydrocephalus and prognosis. RESULTS: Among the 49 preterm infants with obstructive hydrocephalus, severe intracranial hemorrhage (37 cases; 76%) and central nervous system infection (10 cases, 20%) were the main causes of hydrocephalus. There was no significant difference in the composition of etiology between the two groups (P>0.05). In the surgical treatment group, 4 infants were treated with ventriculosubgaleal shunt and 8 were treated with Ommaya reservoir. One infant had secondary infection and 8 infants eventually underwent ventriculoperitoneal shunt. The surgical treatment group had a significantly higher survival rate than the conservative treatment group (P<0.05). As for the 37 preterm infants with severe intracranial hemorrhage, the surgical treatment group had a significantly higher proportion of infants with normal neurodevelopment than the conservative treatment group (P<0.05). As for the 10 preterm infants with central nervous system infection, neurodevelopmental abnormalities were observed in each of the two groups. CONCLUSIONS: Surgical treatment can improve the survival rate of preterm infants with obstructive hydrocephalus and the prognosis of preterm infants with severe intracranial hemorrhage.

Effect of Doxofylline on Reducing the Inflammatory Response in Mechanically Ventilated Rats with Chronic Obstructive Pulmonary Disease.

Objective: To evaluate the effect of doxofylline on reducing the inflammatory response in mechanically ventilated rats with chronic obstructive pulmonary disease (COPD). Methods: A total of 40 eight-week-old male Sprague Dawley rats were randomly divided into four groups of 10 rats each: a control group (group C), a model group (group M), a model + natural saline group (group N), and a doxofylline group (group D). Then mechanical ventilation, drug intervention, and the extraction of the experimental material were performed in each group. Pulmonary tissue samples were taken after 120 minutes of mechanical ventilation and the pulmonary histopathological changes and the wet/dry (W/D) weight ratio of the pulmonary tissue were identified. The levels of tumor necrosis factor α (TNF-α) and interleukin 10 (IL-10) were detected using an enzyme-linked immunosorbent assay, and the expression levels of c-Jun-N-terminal kinase (JNK) and phosphorylated c-Jun N-terminal kinase (p-JNK) were detected using immunohistochemistry. Results: Compared with group C, the pulmonary histopathology in groups M, N, and D showed typical changes associated with COPD. Furthermore, the W/D weight ratio and levels of TNF-α, JNK, and p-JNK in the pulmonary tissue increased in groups M, N, and D (P < 0.05), while the levels of IL-10 decreased (P < 0.05). Compared with group M, no statistically significant changes in the above indicators were detected in the pulmonary tissue of group N (P > 0.05). Compared with group N, the W/D weight ratio and levels of TNF-α, JNK, and p-JNK in the pulmonary tissue decreased in group D (P < 0.05), while the levels of IL-10 increased (P < 0.05). Conclusion: Doxofylline might attenuate pulmonary inflammatory responses in mechanically ventilated rats with COPD, and the JNK/stress-activated protein kinase signaling pathway is involved in doxofylline's inhibition of inflammatory responses in the pulmonary tissue of rats with COPD.

Exposure to ethephon compromises endometrial decidualization in mice during early pregnancy via GPR120.

Exposure to ethephon (ETH), a plant growth regulator commonly used for several purposes, can potentially decrease sperm numbers and viability. Occasional findings regarding ETH effects on female reproduction during early pregnancy have also been reported. During early pregnancy, endometrial decidualization is a critical event for embryo implantation and pregnancy maintenance. Thus, we aimed to explore the effect and mechanism of ETH on endometrial decidualization both in vivo and in vitro. Mice were gavaged with 0 and 285 mg/kg b.w. ETH from gestational days (GD)1 until sacrifice, whereas pseudopregnant mice from pseudopregnant day 1 (PPD-1) until PPD-8. Primary mouse endometrial stromal cells (mESCs) received 640 ug/ml ETH and added E2 and P4 to induce decidualization. Results indicated female albino CD1 mice exposed to high dose of ETH (285 mg/kg b.w.) by oral gavage, the number of embryo implantation sites on GD6 and GD8 were significantly decreased, the levels of serum E2 and P4 on GD8 were significantly decreased. Compared with the control group, the decidualization response artificially induced by corn oil in pseudopregnant mice and by E2 and P4 in primary mouse endometrial stromal cells (mESCs) was weakened in the high dose of ETH treated group. The high dose, 285 mg/kg b.w ETH treated group altered the expression of endometrial decidual markers on GD6 and GD8. The triglyceride and fatty acid metabolism-related genes were significantly increased after female albino CD1 mice exposed to high does, 285 mg/kg b.w ETH on GD6 and GD8. GPR120 was substantially reduced after ETH treatment. When overexpression of GPR120, the compromised decidualization induced by ETH treatment was rescued. Furthermore, molecular docking presented Thr234 and His251 of GPR120 as preferred binding sites for ETH. Mutation of these two sites rescued the compromised decidualization induced by ETH. In conclusion, we demonstrated that ETH exposure could impair decidualization during early pregnancy. GPR120 expression and binding between GPR120 and ETH are crucial for impaired decidualization mediated via ETH.

The Mechanism of Penehyclidine Hydrochloride and Its Effect on the Inflammatory Response of Lung Tissue in Rats with Chronic Obstructive Pulmonary Disease During Mechanical Ventilation.

BACKGROUND: Penehyclidine hydrochloride is a selective antagonist of M1 and M3 receptors. Clinical studies suggest that it is a potential drug for the treatment of chronic obstructive pulmonary disease (COPD). The purpose of this study was to evaluate the effect of penehyclidine hydrochloride on the inflammatory response of lung tissue during mechanical ventilation in rats with COPD and explore the role of the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) signaling pathway. METHODS: Eight-week-old male Sprague Dawley rats were exposed to cigarette smoke for 30 minutes every day for two months, and on the first and thirtieth days, 200 ug of lipopolysaccharide was injected into the trachea. Two months later, the rats were randomly divided into the control group (C), model group (M), model + normal saline group (N), and penehyclidine hydrochloride group (H) to undergo anesthesia and mechanical ventilation. In group H, 1 mg/kg of penehyclidine hydrochloride was injected intravenously. RESULTS: The results showed that: ① Compared with group C, the other groups all showed typical chronic obstructive pathological changes in the lung tissue; their wet/dry weight ratio (W/D), TNF-α, JNK, and p-JNK levels increased (P < 0.05), and their interleukin (IL)-10 levels decreased (P < 0.05). ② Compared with group M, there was no significant change in the lung tissue indexes in group N (P > 0.05). ③ Compared with group N, the W/D, TNF-α, JNK, and p-JNK levels in group H decreased (P < 0.05), while the levels of IL-10 increased (P < 0.05). CONCLUSION: Penehyclidine hydrochloride can alleviate the pulmonary inflammatory response in rats with COPD undergoing mechanical ventilation. The JNK/SAPK signaling pathway may be involved in this process.

The Mitochondrial Kinase PINK1 in Diabetic Kidney Disease.

Mitochondria are critical organelles that play a key role in cellular metabolism, survival, and homeostasis. Mitochondrial dysfunction has been implicated in the pathogenesis of diabetic kidney disease. The function of mitochondria is critically regulated by several mitochondrial protein kinases, including the phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1). The focus of PINK1 research has been centered on neuronal diseases. Recent studies have revealed a close link between PINK1 and many other diseases including kidney diseases. This review will provide a concise summary of PINK1 and its regulation of mitochondrial function in health and disease. The physiological role of PINK1 in the major cells involved in diabetic kidney disease including proximal tubular cells and podocytes will also be summarized. Collectively, these studies suggested that targeting PINK1 may offer a promising alternative for the treatment of diabetic kidney disease.

Bergaptol from blossoms of Citrus aurantium L. var. amara Engl inhibits LPS-induced inflammatory responses and ox-LDL-induced lipid deposition.

Aberrant activation of inflammation and excess accumulation of lipids play pivotal roles in atherosclerosis (AS) progression. Constituents from Citrus aurantium Linn variant amara Engl (CAVA) were effectively investigated for their various bioactivities, especially anti-inflammation. Bergaptol (BER) is particularly abundant in Citrus products. Accumulating studies have confirmed its predominant anti-cancer and antioxidant functions, whereas few studies focused on its antiatherogenic functions. In the current study, BER was isolated from CAVA for the first time. Macrophages were stimulated with lipopolysaccharides (LPSs) or oxidized low-density lipoproteins (ox-LDL) to mimic inflammatory responses and AS development. BER treatment significantly inhibited LPS-induced production of nitric oxide (NO), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), and gene expression of inducible nitric oxide synthase (iNOS), IL-6, TNF-α, interleukin-1 beta (IL-1ß) and cyclooxygenase-2 (COX-2). BER also potently blocked LPS-induced mitogen-activated protein kinase (MAPK) phosphorylation and nuclear factor-kappa B (NF-κB) activation, as evidenced by the inhibitory effects on c-Jun N-terminal kinase (JNK), P38, P65, IκBα and IκKα/ß phosphorylation, and NF-κB nuclear translocation. Furthermore, BER treatment markedly mitigated ox-LDL-induced foam cell formation by inhibiting scavenger receptor class A type I (SRA1) and cluster of differentiation 36 (CD36)-dependent cholesterol uptake. In conclusion, BER might be a novel therapeutic agent for AS prevention through inhibiting inflammatory responses and cholesterol uptake.

Characterization of TCF4-mediated oncogenic role in cutaneous squamous cell carcinoma.

The aberrant highly expressed T-cell factor 4 (TCF4) has been determined to be closely connected with carcinogenesis of cutaneous squamous cell carcinoma (cSCC) in previous studies. However, the underlying regulatory network and the potential therapeutic targets of TCF4 in SCC are still not fully understood. In this study, the highly expressed TCF4 was observed in human cSCC cancer compared to the paired adjacent tissues. A431 cell lines with TCF4 RNA silencing were found to be the repressive cell proliferation and invasion as well as the enhanced apoptosis. Furthermore, RNA-Seq was conducted and observed that 147 genes were up-regulated (including 113 coding genes and 34 lncRNA) while 172 genes were down-regulated (including 64 coding genes and 108 lncRNA) in TCF4 silencing compared to blank RNAi and untreated control A431 cells. 18 pathways including steroid, porphyrin, arachidonic acid, and retinol metabolism, as well as the functions associated with angiogenesis, inflammatory response, and cell adhesion were involved in the differentially expressed genes of A431 cells with TCF4 silencing. Finally, ChIP-qPCR of TCF4 and ß-catenin were performed and we found that the enrichments of ß-catenin were lost on the promoters on top ten down-regulated genes in A431 cells with TCF4 silencing compared to the untreated A431. Additionally, in untreated A431 cells, some genes such as ALDH8A1, DRICH1, and UGT1A5 were observed with high enrichment of TCF4, but without ß-catenin, which indicated a Wnt/ß-catenin independent way of TCF4 for gene transcriptional regulation. In conclusion, we declared that TCF4 played an important role in tumorigenesis of skin cancer via the aberrant activation of variety of signaling pathways, and could be considered as a potential therapeutic target for cSCC treatment.

Ratio of positive lymph nodes: The prognostic value in stage IV thyroid cancer.

To assess the prognostic value of lymph node ratio (LNR) in patients with stage IV thyroid cancer based on the Surveillance, Epidemiology, and End Results (SEER) database. A total of 4,940 eligible patients were included for the analysis. Kaplan-Meier survival analysis and Cox proportional hazard regression were used to reveal the effect of LNR on overall survival (OS) and disease specific survival (DSS). The optimal cut-off value of LNR for predicting OS and DSS was determined by the time-dependent Receiver Operating Characteristic analysis. By the univariate Cox proportional hazard regression, LNR was significantly associated with OS and DSS in patients with medullary thyroid cancer (MTC), papillary thyroid cancer and anaplastic thyroid cancer (all P < 0.05). With the optimal cut-off value, Kaplan-Meier analysis showed that MTC patients with LNR≥76.5% were significantly associated with poorer OS (log-rank test: P < 0.0001), and LNR≥40.7% were significantly associated with poorer DSS (log-rank test: P < 0.0001). LNR was an independent prognostic factor of poorer survival in MTC patients after adjusting for other variables by multivariable Cox analysis (OS: hazard ratio [HR] = 2.560, 95% confidence interval [CI] 1.690-3.879, P < 0.0001; DSS: HR=2.781, 95% CI 1.582-4.888, P = 0.0004). Our results demonstrated that LNR could predict clinical outcomes in patients with stage IV MTC, and 76.5% was the optimal cut-off value of LNR to predict OS. LNR, as a function of the nodes positive and the nodes examined, could provide suggestions on the postoperative prognosis of patients with stage IV MTC.

Polyphenols from Blossoms of Citrus aurantium L. var. amara Engl. Show Significant Anti-Complement and Anti-Inflammatory Effects.

Citrus aurantium L. var. amara Engl. (CAVA) was traditionally used as a digestant or expectorant in China. Crude polyphenols (CAVAP-W) extracted from blossoms of CAVA were mainly composed of eriocitrin/neoeriocitrin, eriocitrin/neoeriocitrin, rhoifolin, hesperidin, naringin, rutin, veronicastroside, neohesperidin, and hesperetin by LC-MS analysis. CAVAP-W showed significant anticomplement and anti-inflammatory effects. Due to the close relationship between anticomplement and anti-inflammatory activity, the anti-inflammatory effect was further investigated and the results showed that CAVAP-W significantly suppressed production of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1ß), and mRNA expression of inducible nitric oxide synthase (iNOS), IL-6, TNF-α, IL-1ß, and cyclooxygenase-2 (COX-2) in lipopolysaccharides-stimulated RAW264.7 cells. Furthermore, CAVAP-W inhibited mitogen-activated protein kinase (MAPK) phosphorylation and NF-κB activation through suppressing nuclear translocation of nuclear factor-kappa B (NF-κB) P65, degradation and phosphorylation of IκBα, phosphorylation of IκKα/ß, c-Jun N-terminal kinase (JNK), and P38, and activation of COX-2, thereby exerting the anti-inflammatory effects.

Deanxit relieves symptoms in a patient with jackhammer esophagus: A case report.

Jackhammer (hypercontractile) esophagus presents with dysphagia and chest pain. Current treatments are limited. We describe a 60-year-old man who presented with dysphagia, chest pain and heartburn for a period of 1 year. His workup showed Barrett's esophagus on endoscopy and high-resolution manometry demonstrated jackhammer esophagus with esophagogastric junction outflow obstruction. The patient was treated with proton pump inhibitor and nifedipine but without resolution of his symptoms. He was followed up to assess the efficacy of treatment with deanxit (flupentixol + melitracen). Dysphagia and chest pain resolved during the therapeutic trial and efficacy was maintained on maintenance treatment without troublesome side effects.

KCa3.1 mediates dysfunction of tubular autophagy in diabetic kidneys via PI3k/Akt/mTOR signaling pathways.

Autophagy is emerging as an important pathway in many diseases including diabetic nephropathy. It is acknowledged that oxidative stress plays a critical role in autophagy dysfunction and diabetic nephropathy, and KCa3.1 blockade ameliorates diabetic renal fibrosis through inhibiting TGF-ß1 signaling pathway. To identify the role of KCa3.1 in dysfunctional tubular autophagy in diabetic nephropathy, human proximal tubular cells (HK2) transfected with scrambled or KCa3.1 siRNAs were exposed to TGF-ß1 for 48 h, then autophagosome formation, the autophagy marker LC3, signaling molecules PI3K, Akt and mTOR, and oxidative stress marker nitrotyrosine were examined respectively. In vivo, LC3, nitrotyrosine and phosphorylated mTOR were examined in kidneys of diabetic KCa3.1+/+ and KCa3.1-/- mice. The results demonstrated that TGF-ß1 increased the formation of autophagic vacuoles, LC3 expression, and phosphorylation of PI3K, Akt and mTOR in scrambled siRNA transfected HK2 cells compared to control cells, which was reversed in KCa3.1 siRNA transfected HK2 cells. In vivo, expression of LC3 and nitrotyrosine, and phosphorylation of mTOR were significantly increased in kidneys of diabetic KCa3.1+/+ mice compared to non-diabetic mice, which were attenuated in kidneys of diabetic KCa3.1-/- mice. These results suggest that KCa3.1 activation contributes to dysfunctional tubular autophagy in diabetic nephropathy through PI3K/Akt/mTOR signaling pathways.

High glucose induces CCL20 in proximal tubular cells via activation of the KCa3.1 channel.

BACKGROUND: Inflammation plays a key role in the development and progression of diabetic nephropathy (DN). KCa3.1, a calcium activated potassium channel protein, is associated with vascular inflammation, atherogenesis, and proliferation of endothelial cells, macrophages, and fibroblasts. We have previously demonstrated that the KCa3.1 channel is activated by TGF-ß1 and blockade of KCa3.1 ameliorates renal fibrotic responses in DN through inhibition of the TGF-ß1 pathway. The present study aimed to identify the role of KCa3.1 in the inflammatory responses inherent in DN. METHODS: Human proximal tubular cells (HK2 cells) were exposed to high glucose (HG) in the presence or absence of the KCa3.1 inhibitor TRAM34 for 6 days. The proinflammatory cytokine chemokine (C-C motif) ligand 20 (CCL20) expression was examined by real-time PCR and enzyme-linked immunosorbent assay (ELISA). The activity of nuclear factor-κB (NF-κB) was measured by nuclear extraction and electrophoretic mobility shift assay (EMSA). In vivo, the expression of CCL20, the activity of NF-κB and macrophage infiltration (CD68 positive cells) were examined by real-time PCR and/or immunohistochemistry staining in kidneys from diabetic or KCa3.1-/- mice, and in eNOS-/- diabetic mice treated with the KCa3.1 channel inhibitor TRAM34. RESULTS: In vitro data showed that TRAM34 inhibited CCL20 expression and NF-κB activation induced by HG in HK2 cells. Both mRNA and protein levels of CCL20 significantly decreased in kidneys of diabetic KCa3.1-/- mice compared to diabetic wild type mice. Similarly, TRAM34 reduced CCL20 expression and NF-κB activation in diabetic eNOS-/- mice compared to diabetic controls. Blocking the KCa3.1 channel in both animal models led to a reduction in phosphorylated NF-κB. CONCLUSIONS: Overexpression of CCL20 in human proximal tubular cells is inhibited by blockade of KCa3.1 under diabetic conditions through inhibition of the NF-κB pathway.

KCa3.1 mediates activation of fibroblasts in diabetic renal interstitial fibrosis.

BACKGROUND: Fibroblast activation plays a critical role in diabetic nephropathy (DN). The Ca2+-activated K+ channel KCa3.1 mediates cellular proliferation of many cell types including fibroblasts. KCa3.1 has been reported to be a potential molecular target for pharmacological intervention in a diverse array of clinical conditions. However, the role of KCa3.1 in the activation of myofibroblasts in DN is unknown. These studies assessed the effect of KCa3.1 blockade on renal injury in experimental diabetes. METHODS: As TGF-ß1 plays a central role in the activation of fibroblasts to myofibroblasts in renal interstitial fibrosis, human primary renal interstitial fibroblasts were incubated with TGF-ß1+/- the selective inhibitor of KCa3.1, TRAM34, for 48 h. Two streptozotocin-induced diabetic mouse models were used in this study: wild-type KCa3.1+/+ and KCa3.1-/- mice, and secondly eNOS-/- mice treated with or without a selective inhibitor of KCa3.1 (TRAM34). Then, markers of fibroblast activation and fibrosis were determined. RESULTS: Blockade of KCa3.1 inhibited the upregulation of type I collagen, fibronectin, α-smooth muscle actin, vimentin and fibroblast-specific protein-1 in renal fibroblasts exposed to TGF-ß1 and in kidneys from diabetic mice. TRAM34 reduced TGF-ß1-induced phosphorylation of Smad2/3 and ERK1/2 but not P38 and JNK MAPK in interstitial fibroblasts. CONCLUSIONS: These results suggest that blockade of KCa3.1 attenuates diabetic renal interstitial fibrogenesis through inhibiting activation of fibroblasts and phosphorylation of Smad2/3 and ERK1/2. Therefore, therapeutic interventions to prevent or ameliorate DN through targeted inhibition of KCa3.1 deserve further consideration.

Clinical significance and expression of cyclin kinase subunits 1 and 2 in hepatocellular carcinoma.

BACKGROUND: The mammalian cyclin kinase subunit (Cks) family has two members, Cks1 and Cks2, which were identified based on the protein sequence homology to yeast Cks. Overexpression of Cks1 and Cks2 has been reported to be associated with high aggressiveness and a poor prognosis in various malignancies, including gastric, breast and prostate carcinomas. Yet, whether Cks1 and Cks2 are overexpressed in hepatocellular carcinoma (HCC) remains uncharacterized. AIMS: To investigate whether overexpression of the Cks family is clinically relevant to HCC, and whether expression patterns of Cks1 and Cks2 in HCC have diagnostic and prognostic value. METHODS: Real-time quantitative reverse transcriptase polymerase chain reaction, immunostaining and Western blot analyses were used to detect the expression of Cks1 and Cks2 at the mRNA and protein levels respectively. The associations between Cks1 and Cks2 expressions and clinical features, as well as the association between Cks1 or Cks2 and p27(kip1) expressions in HCC, were analysed. RESULTS: Expressions of Cks1 and Cks2 at both mRNA and protein levels were significantly higher in HCC than those in the adjacent noncancerous tissues (including chronic hepatitis and cirrhosis) and normal liver tissues. Overexpressions of Cks1 and Cks2 in HCC were closely associated with poor differentiation features. The expressions of both Cks1 and Cks2 were negatively associated with p27(kip1) at the protein level. CONCLUSIONS: Overexpression of Cks1 and Cks2 is associated with the aggressive tumour behaviours of HCC, and thus has diagnostic and prognostic value. Further efforts are needed to develop novel biomarkers for HCC based on CKs1 and Cks2 expressions.

Collagen type II enhances chondrogenesis in adipose tissue-derived stem cells by affecting cell shape.

Ideally, biomaterials have inductive properties, favoring specific lineage differentiation. For chondrogenic induction, these properties have been attributed to collagen type II. However, the underlying mechanisms are largely unknown. This study aimed to investigate whether collagen type II favors chondrogenic induction by affecting cell shape through beta1 integrins and Rho A/Rock signaling. For this purpose, adipose tissue-derived stem cells (ASCs) were encapsulated in collagen type I or II gels and cultured in plain and chondrogenic medium. It was demonstrated that (i) ASCs showed more efficient chondrogenic induction (higher collagen X, aggrecan, sox6, sox9, and collagen II gene expression) in both plain and chondrogenic media in collagen type II versus collagen type I gels; (ii) ASCs showed lower Rock 2 gene expression and a more rounded cell shape in collagen type II versus type I gels when grown in plain medium; (iii) Rock inhibitor (Y27632) more effectively enhanced chondrogenic gene expression of ASCs in collagen type I than in collagen type II gels, and diminished differences in chondrogenic gene expression and cell shape of ASCs between the two gel types; and (iv) beta1 integrins blocking not only reduced the differences of chondrogenic gene expression but also eliminated the differences of Rock 1 and Rock 2 gene expressions and cell shape when comparing ASCs embedded in collagen type I and II gels. We conclude that collagen type II provides the inductive signaling for chondrogenic differentiation in ASCs by evoking a round cell shape through beta1 integrin-mediated Rho A/Rock signaling pathway.