Glucosamine inhibits myoblast proliferation and differentiation, and stimulates myotube atrophy through distinct signal pathways.

Glucosamine (GlcN) is one of the dietary supplements used in the treatment of osteoarthritis. Endogenously, GlcN is synthesized from glucose through the hexosamine pathway. In addition to ameliorating arthritis, several biological functions of GlcN have been reported, including insulin resistance in skeletal muscle. However, the regulatory role of GlcN in skeletal muscle development is not clear. We therefore investigated the effect of GlcN on myoblast proliferation, differentiation, and myotube development and their underlying mechanisms in C2C12 cells. Myoblast proliferation was measured by MTT assay. The expressions of MyoD, myogenin (MyoG), and myosin heavy chain (MyHC) were identified as determinants of myoblast differentiation. Expressions of atrogin-1 and muscle RING-finger protein-1 (MuRF-1) were identified as markers of myotube atrophy. The results show that treatment with GlcN significantly reduced myoblast proliferation and phosphorylation of Stat3 and S6K. These findings suggest that GlcN can inhibit growth of myoblasts through inhibiting phosphorylation of Stat3 and S6K. In addition, GlcN significantly suppressed the expression of MyoD, MyoG, and MyHC, as well as myotube formation. Pretreatment of C2C12 myoblast cells with ER stress inhibitors significantly blocked GlcN-inhibited MyHC expression and myotube formation. It can be concluded that GlcN suppressed myogenic differentiation via a pathway that involved ER stress. Moreover, GlcN decreased myotube diameter and expression of MyHC, as well as increased MuRF-1 in C2C12 myotubes. Meanwhile, GlcN also reduced the expressions of phosphorylated Akt and mTOR were stimulated after GlcN treatment in C2C12 myotubes. Thus, GlcN induced skeletal muscle atrophy by inhibiting the protein synthesis pathway. Chronic GlcN infusion also caused skeletal muscle atrophy in mice. In conclusion, GlcN regulated important stages of skeletal muscle development through different signaling pathways.

Endothelin-1 impairs skeletal muscle myogenesis and development via ETB receptors and p38 MAPK signaling pathway.

Myopenia is a condition marked by progressive decline of muscle mass and strength and is associated with aging or obesity. It poses the risk of falling, with potential bone fractures, thereby also increasing the burden on family and society. Skeletal muscle wasting is characterized by a reduced number of myoblasts, impaired muscle regeneration and increased muscle atrophy markers (Atrogin-1, MuRF-1). Endothelin-1 (ET-1) is a potent vasoconstrictor peptide. Increased circulating levels of ET-1 is noted with aging and is associated with muscular fibrosis and decline of strength. However, the regulatory mechanism controlling its effect on myogenesis and atrophy remains unknown. In the present study, the effects of ET-1 on myoblast proliferation, differentiation and development were investigated in C2C12 cells and in ET-1-infused mice. The results show that ET-1, acting via ETB receptors, reduced insulin-stimulated cell proliferation, and also reduced MyoD, MyoG and MyHC expression in the differentiation processes of C2C12 myoblasts. ET-1 inhibited myoblast differentiation through ETB receptors and the p38 mitogen-activated protein kinase (MAPK)-dependent pathway. Additionally, ET-1 decreased MyHC expression in differentiated myotubes. Inhibition of proteasome activity by MG132 ameliorated the ET-1-stimulated protein degradation in differentiated C2C12 myotubes. Furthermore, chronic ET-1 infusion caused skeletal muscle atrophy and impaired exercise performance in mice. In conclusion, ET-1 inhibits insulin-induced cell proliferation, impairs myogenesis and induces muscle atrophy via ETB receptors and the p38 MAPK-dependent pathway.

Intratesticular Testosterone and Its Precursors among Azoospermic Men: A Pilot Study.

PURPOSE: The study aimed to comprehensively analyze testosterone and precursor concentrations in the testicular interstitial fluid (TIF) of men with azoospermia, exploring their significance in the testicular microenvironment and their correlation with testicular sperm retrieval outcomes. MATERIALS AND METHODS: We analyzed 37 TIF samples, including 5 from men with obstructive azoospermia (OA) and 32 from men with non-obstructive azoospermia (NOA). Liquid chromatography with tandem mass spectrometry quantified testosterone and precursor levels. Comparative assessments of the outcomes of testicular sperm retrieval were performed between the OA and NOA groups as well as among men with NOA. RESULTS: Men with NOA who had not undergone hormone treatment exhibited significantly higher intratesticular concentrations of testosterone (median 1,528.1 vs. 207.5 ng/mL), androstenedione (median 10.6 vs. 1.9 ng/mL), and 17-OH progesterone (median 13.0 vs. 1.8 ng/mL) than men diagnosed with OA. Notably, in the subgroup of patients with NOA subjected to medical treatment, men with successful sperm retrieval had significantly reduced levels of androstenedione (median androstenedione 5.7 vs. 18.5 ng/mL, p=0.004). Upon a more detailed analysis of these men who underwent hormone manipulation treatment, the testosterone/androstenedione ratio (indicative of HSD17B3 enzyme activity) was markedly increased in men with successful sperm retrieval (median: 365.8 vs. 165.0, p=0.008) compared with individuals with NOA who had unsuccessful sperm recovery. Furthermore, within the subset of men with NOA who did not undergo medical treatment before microdissection testicular sperm extraction but achieved successful sperm retrieval, the ratio of 17-OH progesterone/progesterone (indicative of CYP17A1 activity) was substantially higher. CONCLUSIONS: The study suggests distinct testosterone biosynthesis pathways in men with compromised spermatogenesis and those with normal spermatogenesis. Among NOA men with successful retrieval after hormone optimization therapy, there was decreased androstenedione and increased HSD17B3 enzyme activity. These findings have diagnostic and therapeutic implications for the future.

Proteomic Analysis of Testicular Interstitial Fluid in Men with Azoospermia.

Background: The primary microenvironment of the testis comprises testicular interstitial fluid (TIF) surrounding the seminiferous tubules and testicular interstitial tissue. The pathological alterations of germ and Sertoli cells could affect the TIF composition and might contain putative biomarkers for monitoring active spermatogenesis. Objective: We identified differentially expressed proteins in the TIF of patients with obstructive (OA) or nonobstructive (NOA) azoospermia to elucidate the underlying etiology of defective spermatogenesis. Design setting and participants: We prospectively enrolled nine patients, including three men with OA and six with NOA with (n = 3) and without (n = 3) successful sperm retrieval. Their TIF was collected during the testicular sperm extraction procedure. Outcome measurements and statistical analysis: TIF was analyzed using liquid chromatography-tandem mass spectrometry to identify differentially expressed proteins specific to OA and NOA with or without successful sperm retrieval. The dysregulated protein was further validated using Western blotting. Results and limitations: Among the 555 TIF proteins identified in NOA patients, 14 were downregulated relative to OA patients. These proteins participate in biological processes such as proteolysis, complement activation, and immune responses; complement and coagulation cascade pathways were also enriched. Furthermore, 68 proteins with significantly higher levels were identified in the TIF of NOA patients with successful sperm retrieval than in those with failed sperm retrieval; these are mainly implicated in oxidation-reduction processes. The expression of calreticulin, which can distinguish successful and failed testicular sperm retrieval in the NOA group, was validated by Western blotting. Conclusions: We provide the first scientific evaluation of TIF protein composition in men with azoospermia. These findings will help identify the physiological and pathological roles of each protein in regulating sperm production. Thus, our study underscores the potential of TIF in sperm retrieval biomarker discovery and would serve as a foundation for further studies to improve treatment strategies against azoospermia. Patient summary: Using a proteomic approach, we identified and analyzed the total protein content of testicular interstitial fluid in humans with defective spermatogenesis for the first time and discovered altered protein expression patterns in patients with nonobstructive azoospermia (NOA). Proteins related to oxidation-reduction processes were upregulated in NOA patients with successful sperm retrieval compared with those with failed sperm retrieval. This can aid the development of novel diagnostic tools for successful testicular sperm retrieval.

Cysteine-cysteine Chemokine Receptor Type 5 Plays a Critical Role in Exercise Performance by Regulating Mitochondrial Content in Skeletal Muscle.

Cysteine-cysteine chemokine receptor type 5 (CCR5) is thought to play an important role in the trafficking of lymphoid cells but has recently also been associated with AMPK signaling pathways that are implicated in energy metabolism in skeletal muscle. We hypothesized that genetic deletions of CCR5 would alter mitochondria content and exercise performance in mice. CCR5-/- and wild-type mice with the same genetic background were subjected to endurance exercise and grip strength tests. The soleus muscle was stained with immunofluorescence for myosin heavy chain 7 (MYH7) and succinate dehydrogenase (SDH) analysis as well as the expression of genes associated with muscle atrophy and mitochondrial oxidative phosphorylation were measured using qPCR. Although there were no differences in the weight of the soleus muscle between the CCR5-/- group and the wild-type mice, the CCR5-/- mice showed the following muscular dysfunctions: (i) decreased MYH7 percentage and cross-section area, (ii) higher myostatin and atrogin-1 mRNA levels, (iii) dropped expression of mitochondrial DNA-encoded electron respiratory chain genes (cytochrome B, cytochrome c oxidase subunit III, and ATP synthase subunit 6) as well as mitochondrial generation genes (PPARγ and PGC-1α), and (iv) lower SDH activity and exercise performance when compared with wild-type mice. In addition, genes associated with mitochondrial biogenesis (PGC-1α, PPARγ, and MFN2) and mitochondrial complex (ND4 and Cytb) were upregulated when the skeletal muscle cell line C2C12 was exposed to cysteine-cysteine chemokine ligand 4 (a ligand of CCR5) in vitro. These findings suggested that attenuation of endurance exercise performance is related to the loss of mitochondrial content and lower SDH activity of soleus muscle in CCR5 knockout mice. The present study provides evidence indicating that the chemokine receptor CCR5 might modulate the skeletal muscle metabolic energy system during exercise.

The impact of low-intensity extracorporeal shock waves on testicular spermatogenesis demonstrated in a rat model.

BACKGROUND: In rodent models, low-intensity extracorporeal shock wave therapy has been shown to negatively impact semen concentration after treatment on the penis, implying that the reproductive system in close proximity may be indirectly affected by this modality. We hypothesized that shock waves are detrimental to spermatogenesis, and the aim of this study was to evaluate the effect of shock waves on spermatogenesis after direct shockwave treatment on testes using different energy settings. METHODS: Twenty-five male Sprague Dawley rats, 8 weeks old, were divided into five groups, including one control group and four treatment groups each treated using shock waves of different intensities. All rats in the treatment groups received 2000 shocks on the left testis twice a week for 4 weeks, with shock wave intensity and frequency varied by treatment group: 0.1 mJ/mm 2 at 4 Hz for Group A, 0.15 mJ/mm 2 at 4 Hz for Group B, 0.35 mJ/mm 2 at 4 Hz for Group C, and 0.55mJ/mm 2 at 3 Hz for Group D. At the end of the experiment, sperm collected from the epididymis was evaluated for concentration and motility. Testicular spermatogenesis, the apoptotic index of germ cells, and the expression of a meiotic-specific gene were also analyzed. RESULTS: The treatment group receiving shock wave intensity at 0.55 mJ/mm 2 showed a significant decrease in sperm concentration, motility, and Johnsen score as compared to other groups. The apoptotic index of spermatogenic cells increased as the intensity of the shock wave treatment escalated, and reach a statistically significant difference at 4 weeks posttreatment. Treating testes with intensity levels of 0.55 mJ/mm 2 at 3 Hz interfere with the quality or quantity of spermatogenesis and also increases in spermatogenic cell apoptosis, whereas the expression of the SYCP3 gene significantly decreased after treatment with intensity levels of 0.10 mJ/mm 2 , 0.15 mJ/mm 2 , and 0.35 mJ/mm 2 at 4 Hz. CONCLUSION: Treating testes with intensity levels of 0.55 mJ/mm 2 at 3 Hz interfere with the quality or quantity of spermatogenesis and also increases spermatogenic cell apoptosis, whereas the expression of the SYCP3 gene significantly decreased after treatment with intensity levels of 0.10 mJ/mm 2 , 0.15 mJ/mm 2 , and 0.35 mJ/mm 2 at 4 Hz.

Effect of ultramarathon running on iron metabolism.

BACKGROUND: Iron is a vital trace element for energy production and oxygen transportation; importantly, it is essential to athletic performance. Maintaining iron balance is tightly controlled at systemic and cellular levels. This study aimed to determine serum iron tests, hepcidin levels, and cellular iron import and export activities in peripheral blood mononuclear cells (PBMCs) in ultramarathon runners to elucidate the association of systemic inflammation response and iron metabolism. METHODS: Sixteen amateur runners were enrolled. Blood samples were taken 1 week before, immediately, and 24 h after the run. Plasma hepcidin levels were measured by enzyme-linked immunosorbent assay. The expression levels of divalent metal iron transporter 1 (DMT1), ZRT/IRT-like protein 14 (ZIP14), transferrin receptor 1 (TfR1), and ferroportin (FPN) in PBMCs were measured using real-time quantitative reverse transcription-polymerase chain reaction. RESULTS: Serum iron concentrations and transferrin saturation significantly decreased immediately after the race and dramatically recovered 24 h post-race. Serum ferritin levels had a statistically significant rise immediately after the race and remained high 24 h after the completion of the race. Ultramarathons were associated with increased plasma interleukin-6 concentrations corresponding to the state of severe systemic inflammation and therefore boosted plasma hepcidin levels. The expression levels of DMT1 and FPN mRNA were markedly decreased immediately and 24 h after the race. The ZIP14 and TfR1 mRNA expression in PBMCs significantly decreased immediately after the race and returned to the baseline level at 24 h post-race. Positive significant correlations were observed between plasma hepcidin and ferritin levels. CONCLUSION: Iron homeostasis and systemic inflammatory response are closely interconnected. Cellular iron import and export mRNA activities in PBMCs were acutely inhibited during an ultramarathon.

Granulocytic MDSC with Deficient CCR5 Alleviates Lipogenesis and Inflammation in Nonalcoholic Fatty Liver Disease.

C-C chemokine receptor type 5 (CCR5) positively contributes to the pathogenesis of nonalcoholic fatty liver disease (NAFLD), a common metabolic liver disease associated with chronic inflammation. CCR5 signaling also facilitates the immunosuppressive activity of a group of immature myeloid cells known as granulocytic myeloid-derived suppressor cells (g-MDSCs). While both hepatocyte and g-MDSC express CCR5, how CCR5 coordinates these two distinct cell types in the hepatic microenvironment remains largely unknown. Here, we used in vivo and ex vivo approaches to define the molecular details of how CCR5 mediates the crosstalk between hepatocytes and g-MDSCs in a mouse model of NAFLD. Global CCR5-deficient mice exhibited more severe steatosis, increased hepatic gene expression of lipogenesis, and exacerbated liver damage in diet-induced obesity. Either NAFLD or CCR5-deficiency per se is causative for the increase of g-MDSCs. Purified g-MDSCs have a higher survival rate in the fatty liver microenvironment, and blockade of CCR5 significantly decreases g-MDSCs' expression of anti-inflammatory factors. On the other hand, the null of CCR5 signaling increases hepatocytes' expression of lipogenic genes in the NAFLD microenvironment. Most importantly, inhibiting g-MDSCs' CCR5 signaling in the fatty liver microenvironment dramatically reduces STAT3 signaling, lipogenic, and pro-inflammatory gene expression in primary hepatocytes. Adoptive cell transfer experiments further demonstrate that CCR5-deficient g-MDSCs mitigate hepatic lipogenic gene expression without facilitating pro-inflammatory cytokine production and liver damage in NAFLD mice. These results suggest that targeting g-MDSCs' CCR5 signaling might serve as a potential therapeutic strategy for NAFLD.

Nitric Oxide Mobilizes Intracellular Zn2+ via the GC/cGMP/PKG Signaling Pathway and Stimulates Adipocyte Differentiation.

Plasma and tissue zinc ion levels are associated with the development of obesity. Previous studies have suggested that zinc ions may regulate adipocyte metabolism and that nitric oxide (NO) plays a pivotal role in the regulation of adipocyte physiology. Our previous study showed that chronic NO deficiency causes a significant decrease in adipose tissue mass in rats. Studies also suggested that zinc ions play an important modulatory role in regulating NO function. This study aims to explore the role of zinc ions in NO-regulated adipocyte differentiation. We hypothesized that NO could increase intracellular Zn2+ level and then stimulate adipocyte differentiation. ZnCl2 and the NO donor, NONOate, were used to explore the effects of Zn2+ and NO on adipocyte differentiation. Regulatory mechanisms of NO on intracellular Zn2+ mobilization were determined by detection. Then, Zn2+-selective chelator TPEN was used to clarify the role of intracellular Zn2+ on NO-regulated adipocyte differentiation. Furthermore, the relationship between adipocyte size, Zn2+ level, and NOS expression in human subcutaneous fat tissue was elucidated. Results showed that both ZnCl2 and NO stimulated adipocyte differentiation in a dose-dependent manner. NO stimulated intracellular Zn2+ mobilization in adipocytes through the guanylate cyclase (GC)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) pathway, and NO-stimulated adipocyte differentiation was Zn2+-dependent. In human subcutaneous adipose tissue, adipocyte size was negatively correlated with expression of eNOS. In conclusion, NO treatment stimulates intracellular Zn2+ mobilization through the GC/cGMP/PKG pathway, subsequently stimulating adipocyte differentiation.

Endothelin-1 induces lipolysis through activation of the GC/cGMP/Ca2+/ERK/CaMKIII pathway in 3T3-L1 adipocytes.

Endothelin-1 (ET-1) is a potent vasoconstrictive peptide produced and secreted mainly by endothelial cells. Recent studies indicate that ET-1 can regulate lipid metabolism, which may increase the risk of insulin resistance. Our previous studies revealed that ET-1 induced lipolysis in adipocytes, but the underlying mechanisms were unclear. 3T3-L1 adipocytes were used to investigate the effect of ET-1 on lipolysis and the underlying mechanisms. Glycerol levels in the incubation medium and hormone-sensitive lipase (HSL) phosphorylation were used as indices for lipolysis. ET-1 significantly increased HSL phosphorylation and lipolysis, which were completely inhibited by ERK inhibitor (PD98059) and guanylyl cyclase (GC) inhibitor (LY83583). LY83583 reduced ET-1-induced ERK phosphorylation. A Ca2+-free medium and PLC inhibitor caused significant decreases in ET-1-induced lipolysis as well as ERK and HSL phosphorylation, and IP3 receptor activator (D-IP3) increased lipolysis. ET-1 increased cGMP production, which was not affected by depletion of extracellular Ca2+. On the other hand, LY83583 diminished the ET-1-induced Ca2+ influx. Transient receptor potential vanilloid-1 (TRPV-1) antagonist and shRNA partially inhibited ET-1-induced lipolysis. ET-1-induced lipolysis was completely suppressed by CaMKIII inhibitor (NH-125). These results indicate that ET-1 stimulates extracellular Ca2+ entry and activates the intracellular PLC/IP3/Ca2+ pathway through a cGMP-dependent pathway. The increased cytosolic Ca2+ that results from ET-1 treatment stimulates ERK and HSL phosphorylation, which subsequently induces lipolysis. ET-1 induces HSL phosphorylation and lipolysis via the GC/cGMP/Ca2+/ERK/CaMKIII signaling pathway in 3T3-L1 adipocytes.

Glucosamine Interferes With Myelopoiesis and Enhances the Immunosuppressive Activity of Myeloid-Derived Suppressor Cells.

Glucosamine (GlcN) is the most widely consumed dietary supplement and exhibits anti-inflammatory effects. However, the influence of GlcN on immune cell generation and function is largely unclear. In this study, GlcN was delivered into mice to examine its biological function in hematopoiesis. We found that GlcN promoted the production of immature myeloid cells, known as myeloid-derived suppressor cells (MDSCs), both in vivo and in vitro. Additionally, GlcN upregulated the expression of glucose transporter 1 in hematopoietic stem and progenitor cells (HSPCs), influenced HSPC functions, and downregulated key genes involved in myelopoiesis. Furthermore, GlcN increased the expression of arginase 1 and inducible nitric oxide synthase to produce high levels of reactive oxygen species, which was regulated by the STAT3 and ERK1/2 pathways, to increase the immunosuppressive ability of MDSCs. We revealed a novel role for GlcN in myelopoiesis and MDSC activity involving a potential link between GlcN and immune system, as well as the new therapeutic benefit.

Increased regulated on activation, normal T-cell expressed and secreted levels and cysteine-cysteine chemokine receptor 5 upregulation in omental adipose tissue and peripheral blood mononuclear cells are associated with testosterone level and insulin resistance in polycystic ovary syndrome.

OBJECTIVE: To study the relationship between circulating chemokine cysteine-cysteine motif ligand (CCL) 5 levels and cysteine-cysteine chemokine receptor type 5 (CCR5) expression in peripheral blood mononuclear cells (PBMCs) and adipose tissue with hyperandrogenism and insulin resistance in patients with polycystic ovary syndrome (PCOS). DESIGN: Case-control study. SETTING: University teaching hospital. PATIENT(S): Fifteen women with PCOS and 15 controls matched for body mass index and age were enrolled in this study. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Plasma levels of CCL3, CCL4, and CCL5 were determined using enzyme-linked immunosorbent assay kits, and omental adipose tissue and PBMCs were analyzed using real-time polymerase chain reaction to determine the expression level of CCR5 in participants. RESULT(S): Levels of CCL5 were significantly higher in women with PCOS. Expression of CCR5 in adipose tissue and PBMCs was significantly higher in women with PCOS compared with that in women in the control group. Cysteine-cysteine chemokine receptor type 5 expression also was upregulated in THP-1 cells after chronic exposure to testosterone. Levels of CCL5 had a significant positive correlation with testosterone levels in women with PCOS. Moreover, CCR5 showed a positive correlation with fasting glucose levels, homeostasis model insulin resistance index, and C-reactive protein. CONCLUSION(S): Increased levels of CCL5 and overexpression of CCR5 in PBMCs and adipose tissue are associated with hyperandrogenism and insulin resistance in women with PCOS. Additionally, CCR5 and CCL5 may be used as biomarkers in the pathogenesis of PCOS.

METTL3-dependent N6-methyladenosine RNA modification mediates the atherogenic inflammatory cascades in vascular endothelium.

Atherosclerosis is characterized by the plaque formation that restricts intraarterial blood flow. The disturbed blood flow with the associated oscillatory stress (OS) at the arterial curvatures and branch points can trigger endothelial activation and is one of the risk factors of atherosclerosis. Many studies reported the mechanotransduction related to OS and atherogenesis; however, the transcriptional and posttranscriptional regulatory mechanisms of atherosclerosis remain unclear. Herein, we investigated the role of N6-methyladenosine (m6A) RNA methylation in mechanotransduction in endothelial cells (ECs) because of its important role in epitranscriptome regulation. We have identified m6A methyltransferase METTL3 as a responsive hub to hemodynamic forces and atherogenic stimuli in ECs. OS led to an up-regulation of METTL3 expression, accompanied by m6A RNA hypermethylation, increased NF-κB p65 Ser536 phosphorylation, and enhanced monocyte adhesion. Knockdown of METTL3 abrogated this OS-induced m6A RNA hypermethylation and other manifestations, while METTL3 overexpression led to changes resembling the OS effects. RNA-sequencing and m6A-enhanced cross-linking and immunoprecipitation (eCLIP) experiments revealed NLRP1 and KLF4 as two hemodynamics-related downstream targets of METTL3-mediated hypermethylation. The METTL3-mediated RNA hypermethylation up-regulated NLRP1 transcript and down-regulated KLF4 transcript through YTHDF1 and YTHDF2 m6A reader proteins, respectively. In the in vivo atherosclerosis model, partial ligation of the carotid artery led to plaque formation and up-regulation of METTL3 and NLRP1, with down-regulation of KLF4; knockdown of METTL3 via repetitive shRNA administration prevented the atherogenic process, NLRP3 up-regulation, and KLF4 down-regulation. Collectively, we have demonstrated that METTL3 serves a central role in the atherogenesis induced by OS and disturbed blood flow.

Circulating microRNAs Associated With Reversible Cerebral Vasoconstriction Syndrome.

OBJECTIVE: The purpose of this study was to investigate the significance of circulating micro RNAs (miRNAs) in the pathogenesis of reversible cerebral vasoconstriction syndrome (RCVS). METHODS: We prospectively recruited 3 independent cohorts of patients with RCVS and age-matched and sex-matched controls in a single medical center. Next-generation small RNA sequencing followed by quantitative polymerase chain reaction (PCR) was used to identify and validate differentially expressed miRNAs, which was cross-validated in migraine patients in ictal stage or interictal stage. Computational analysis was used to predict the target genes of miRNAs, followed by in vitro functional analysis. RESULTS: We identified a panel of miRNAs including miR-130a-3p, miR-130b-3p, let-7a-5p, let-7b-5p, and let-7f-5p that well differentiated patients with RCVS from controls (area under the receiver operating characteristics curve [AUC] was 0.906, 0.890, and 0.867 in the 3 cohorts, respectively). The abundance of let-7a-5p, let-7b-5p, and let-7f-5p, but not miR-130a-3p nor miR-130b-3p, was significantly higher in patients with ictal migraine compared with that of controls and patients with interictal migraine. Target prediction and pathway enrichment analysis suggested that the transforming growth factor-ß signaling pathway and endothelin-1 responsible for vasomotor control might link these miRNAs to RCVS pathogenesis, which was confirmed in vitro by transfecting miRNAs mimics or incubating the patients' cerebrospinal fluid (CSF) in 3 different vascular endothelial cells. Moreover, miR-130a-3p was associated with imaging-proven disruption of the blood-brain barrier (BBB) in patients with RCVS and its overexpression led to reduced transendothelial electrical resistance (ie, increased permeability) in in vitro human BBB model. INTERPRETATION: We identified the circulating miRNA signatures associated with RCVS, which may be functionally linked to its headache, BBB integrity, and vasomotor function. ANN NEUROL 2021;89:459-473.

Cysteine-Cysteine Motif Chemokine Receptor 5 Expression in Letrozole-Induced Polycystic Ovary Syndrome Mice.

Polycystic ovary syndrome (PCOS), which affects 5-10% of women of reproductive age, is associated with reproductive and metabolic disorders, such as chronic anovulation, infertility, insulin resistance, and type 2 diabetes. However, the mechanism of PCOS is still unknown. Therefore, this study used a letrozole-exposed mouse model in which mice were orally fed letrozole for 20 weeks to investigate the effects of letrozole on the severity of reproductive and metabolic consequences and the expression of cysteine-cysteine motif chemokine receptor 5 (CCR5) in letrozole-induced PCOS mice. The letrozole-treated mice showed a disrupted estrous cycle and were arrested in the diestrus phase. Letrozole treatment also increased plasma testosterone levels, decreased estradiol levels, and caused multicystic follicle formation. Furthermore, histological analysis of the perigonadal white adipose tissue (pgWAT) showed no significant difference in the size and number of adipocytes between the letrozole-treated mice and the control group. Further, the letrozole-treated mice demonstrated glucose intolerance and insulin resistance during oral glucose and insulin tolerance testing. Additionally, the expression of CCR5 and cysteine-cysteine motif ligand 5 (CCL5) were significantly higher in the pgWAT of the letrozole-treated mice compared with the control group. CCR5 and CCL5 were also significantly correlated with the homeostasis model assessment of insulin resistance (HOMA-IR). Finally, the mechanisms of insulin resistance in PCOS may be caused by an increase in serine phosphorylation and a decrease in Akt phosphorylation.

Expression of ABC transporter and scavenger receptor mRNAs in PBMCs in 100-km ultramarathon runners.

BACKGROUND: Cholesterol metabolism is tightly regulated at the cellular level. This study was to measure the expression levels of ATP-binding cassette transporter A1 (ABCA1) and G1 (ABCG1), scavenger receptor class B type I (SR-BI) and class A (SRA), and CD36 mRNAs in peripheral blood mononuclear cells (PBMCs) in response to 100-km ultramarathon event and determine any correlation between these ABC transporters/scavenger receptor expression levels and plasma cholesterol homeostasis. MATERIALS AND METHODS: Twenty-six participants were enrolled. Blood was drawn from each individual 1 week prior, immediately after, and 24 hours after the race. The expression levels of ABCA1, ABCG1, SR-BI, SRA and CD36 in PBMCs were measured by using real-time quantitative reverse transcription polymerase chain reaction. RESULTS: Plasma triglyceride levels were significantly increased immediately after the race and dropped at 24-hour post-race compared with pre-race values. The 100-km ultramarathon boosted high-density lipoprotein cholesterol (HDL-C) levels and decreased low-density lipoprotein cholesterol (LDL-C) levels 24-hour post-race. The expression levels of ABCA1, ABCG1 and SR-BI were markedly decreased, whereas that of CD36 was slightly but significantly upregulated in runners' PBMCs immediately after the race. Ultramarathon resulted in immediate large-scale stimulation of inflammatory cytokines with increased plasma interleukin-6 and tumour necrosis factor-alpha levels. Moreover, by using in vitro models with human monocytic cell lines, incubation of runners' plasma immediately after the race significantly downregulated ABCA1 and ABCG1, and upregulated CD36 expression in these cells. CONCLUSIONS: ABCA1, ABCG1 and CD36 gene expressions in PBMCS might be associated with endurance exercise-induced plasma cholesterol homeostasis and systemic inflammatory response.

Long-term aerobic exercise training-induced anti-inflammatory response and mechanisms: Focusing on the toll-like receptor 4 signaling pathway.

Toll-like receptor 4 (TLR-4), which regulate inflammatory reactions, has become a popular research topic in recent years. This article reviews the latest scientific evidence on the regulation of TLR-4 by regular aerobic exercise training. The literature shows that long-term regular aerobic exercise training can effectively attenuate the expression of TLR-4 in immune cells and regulate its downstream intracellular cascade, including the p38 and PI3K/Akt signaling pathways. This further reduces cytokines secretion by inflammatory cells, which enhances immune system. We consider that the scientific evidence that long-term aerobic exercise training improves the inflammatory response provides a reasonable basis for using aerobic exercise training as a treatment for patients.

Molecular Mechanisms of Laparoscopic Ovarian Drilling and Its Therapeutic Effects in Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is a common endocrinopathy, characterized by chronic anovulation, hyperandrogenism, and multiple small subcapsular cystic follicles in the ovary during ultrasonography, and affects 5-10% of women of reproductive age. PCOS is frequently associated with insulin resistance (IR) accompanied by compensatory hyperinsulinemia and, therefore, presents an increased risk of type 2 diabetes mellitus (DM). The pathophysiology of PCOS is unclear, and many hypotheses have been proposed. Among these hypotheses, IR and hyperandrogenism may be the two key factors. The first line of treatment in PCOS includes lifestyle changes and body weight reduction. Achieving a 5-15% body weight reduction may improve IR and PCOS-associated hormonal abnormalities. For women who desire pregnancy, clomiphene citrate (CC) is the front-line treatment for ovulation induction. Twenty five percent of women may fail to ovulate spontaneously after three cycles of CC treatment, which is called CC-resistant PCOS. For CC-resistant PCOS women, there are many strategies to improve ovulation rate, including medical treatment and surgical approaches. Among the various surgical approaches, one particular surgical method, called laparoscopic ovarian drilling (LOD), has been proposed as an alternative treatment. LOD results in an overall spontaneous ovulation rate of 30-90% and final pregnancy rates of 13-88%. These benefits are more significant for women with CC-resistant PCOS. Although the intra- and post-operative complications and sequelae are always important, we believe that a better understanding of the pathophysiological changes and/or molecular mechanisms after LOD may provide a rationale for this procedure. LOD, mediated mainly by thermal effects, produces a series of morphological and biochemical changes. These changes include the formation of artificial holes in the very thick cortical wall, loosening of the dense and hard cortical wall, destruction of ovarian follicles with a subsequently decreased amount of theca and/or granulosa cells, destruction of ovarian stromal tissue with the subsequent development of transient but purulent and acute inflammatory reactions to initiate the immune response, and the continuing leakage or drainage of "toxic" follicular fluid in these immature and growth-ceased pre-antral follicles. All these factors contribute to decreasing local and systemic androgen levels, the following apoptosis process with these pre-antral follicles to atresia; the re-starting of normal follicular recruitment, development, and maturation, and finally, the normalization of the "hypothalamus-pituitary-ovary" axis and subsequent spontaneous ovulation. The detailed local and systematic changes in PCOS women after LOD are comprehensively reviewed in the current article.

The Mechanism of the Zika Virus Crossing the Placental Barrier and the Blood-Brain Barrier.

Zika virus (ZIKV) infection causes severe neurological symptoms in adults and fetal microcephaly and the virus is detected in the brain of microcephaly and meningoencephalitis patient. However, the mechanism of ZIKV crossing the physiological barrier to the central nervous systems (CNS) remains elusive. The placental barrier and the blood brain barrier (BBB) protect the fetus from pathogens and ensure healthy brain development during pregnancy. In this study, we used human placenta trophoblasts cells (JEG-3) and human brain-derived endothelial cells (hCMEC/D3) as in vitro models of the physiological barriers. Results showed that ZIKV could infect JEG-3 cells effectively and reduce the amounts of ZO-1 and occludin between adjacent cells by the proteasomal degradation pathway, suggesting that the permeability of the barrier differentially changed in response to ZIKV infection, allowing the virus particle to cross the host barrier. In contrast, ZIKV could infect hCMEC/D3 cells without disrupting the BBB barrier permeability and tight junction protein expression. Although no disruption to the BBB was observed during ZIKV infection, ZIKV particles were released on the basal side of the BBB model and infected underlying cells. In addition, we observed that fluorescence-labeled ZIKV particles could cross the in vitro placenta barrier and BBB model by transcytosis and the action of transcytosis could be blocked by either low temperature or pharmacological inhibitors of endocytosis. In summary, the ZIKV uses a cell-type specific paracellular pathway to cross the placenta monolayer barrier by disrupting cellular tight junction. In addition, the ZIKV can also cross both the placenta barrier and the BBB by transcytosis. Our study provided new insights into on the mechanism of the cellular barrier penetration of ZIKV particles.

Curcumin Attenuates Adipogenesis by Inducing Preadipocyte Apoptosis and Inhibiting Adipocyte Differentiation.

Patients with metabolic syndrome are at an increased risk of developing type 2 diabetes and cardiovascular diseases. The principal risk factor for development of metabolic syndrome is obesity, defined as a state of pathological hyperplasia or/and hypertrophy of adipose tissue. The number of mature adipocytes is determined by adipocyte differentiation from preadipocytes. The purpose of the present study is to investigate the effects of curcumin on adipogenesis and the underlying mechanism. To examine cell toxicity of curcumin, 3T3-L1 preadipocytes were treated with 0-50 µM curcumin for 24, 48, or 72 h, then cell viability was measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The effect of curcumin on the cell cycle was determined by flow cytometry. Curcumin-induced cell apoptosis was determined by the TUNEL assay and curcumin-induced caspase activation was measured by immunoblotting. The effect of curcumin on adipocyte differentiation was determined by measuring mitotic clonal expansion (MCE), expression of adipogenic transcription factors, and lipid accumulation. Results showed the viability of preadipocytes was significantly decreased by treatment with 30 µM curcumin, a concentration that caused apoptosis in preadipocytes, as assessed by the TUNEL assay, and caused activation of caspases 8, 9, and 3. A non-cytotoxic dose of curcumin (15 µM) inhibited MCE, downregulated the expression of PPARγ and C/EBPα, prevented differentiation medium-induced ß-catenin downregulation, and decreased the lipid accumulation in 3T3-L1 adipocytes. In conclusion, our data show that curcumin can induce preadipocyte apoptosis and inhibit adipocyte differentiation, leading to suppression of adipogenesis.