Association Between Telomere Length and Risk of Lung Cancer in an Asian Population: A Mendelian Randomization Study.

Background: Several traditional observational studies and Mendelian randomization (MR) studies have indicated an association between leukocyte telomere length (LTL) and the risk of lung cancer in the European population. However, the results in the Asian population are still unclear. The objective was to reveal the genetic causal association between LTL and the risk of lung cancer in the Asian population. Methods: We conducted a two-sample MR analysis using summary statistics. Instrumental variables (IVs) were obtained from the genome-wide association studies (GWAS) of LTL (n = 23,096) and lung cancer (n = 212,453) of Asian ancestry. We applied the random-effects inverse-variance weighted (IVW) model as the main method. As well, several other models were performed as complementary methods to assess the impact of potential MR assumption violations, including MR-Egger regression, weighted median, and weighted mode models. Results: We included eight single-nucleotide polymorphisms (SNPs) as IVs for LTL and found that LTL was significantly associated with the risk of lung cancer in the IVW model (odds ratio (OR): 1.60; 95% confidence interval (CI): 1.31 - 1.97; P = 5.96 × 10-6), which was in line with the results in the weighted median and weighted mode models. However, the relationship was not statistically significant in the MR-Egger regression model (OR: 1.44; 95% CI: 0.92 - 2.26; P = 0.160). Sensitivity analyses indicated the robustness of the results. Conclusions: This two-sample MR study confirmed that longer telomere length significantly increased the risk of lung cancer in the Asian population, which was in accord with findings in the Western population.

Icaritin Attenuates Lipid Accumulation by Increasing Energy Expenditure and Autophagy Regulated by Phosphorylating AMPK.

BACKGROUND AND AIMS: Lipid accumulation is the major characteristic of non-alcoholic fatty liver disease, the prevalence of which continues to rise. We aimed to investigate the effects and mechanisms of icaritin on lipid accumulation. METHODS: Cells were treated with icaritin at 0.7, 2.2, 6.7, or 20 µM for 24 h. The effects on lipid accumulation in L02 and Huh-7 cells were detected by Bodipy and oil red O staining, respectively. Mitochondria biogenesis of L02 cells was detected by MitoTracker Orange staining. Glucose uptake and adenosine triphosphate content of 3T3-L1 adipocytes and C2C12 myotubes were detected. The expression levels of proteins in the adenosine 5'-monophosphate-activated protein kinase (AMPK) signaling pathway, biomarkers of autophagy, and mitochondria biogenesis were measured by western blotting. LC3 puncta were detected by immunofluorescence. RESULTS: Icaritin significantly attenuated lipid accumulation in L02 and Huh-7 cells and boosted the mitochondria biogenesis of L02 cells. Icaritin enhanced glucose uptake, decreased adenosine triphosphate content, and activated the AMPK signaling pathway in 3T3-L1 adipocytes and C2C12 myotubes. Icaritin boosted autophagy and also enhanced the initiation of autophagic flux in 3T3-L1 preadipocytes and C2C12 myoblasts. However, icaritin decreased autophagy and promoted mitochondria biogenesis in 3T3-L1 adipocytes and C2C12 myotubes. CONCLUSIONS: Icaritin attenuates lipid accumulation by increasing energy expenditure and regulating autophagy by activating the AMPK pathway.

Decreased expression of ApoF associates with poor prognosis in human hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is frequently associated with metabolism dysfunction. Increasing evidence has demonstrated the crucial role of lipid metabolism in HCC progression. The function of apolipoprotein F (ApoF), a lipid transfer inhibitor protein, in HCC is incompletely understood. We aimed to evaluate the functional role of ApoF in HCC in this study. METHODS: We used quantitative reverse-transcription polymerase chain reaction (qRT-PCR) to detect ApoF mRNA expression in HCC tissues and hepatoma cell lines (SMMC-7721, HepG2, and Huh7). Immunohistochemistry was performed to detect the expression of ApoF in HCC tissues. The associations between ApoF expression and clinicopathological features as well as HCC prognosis were analyzed. The effect of ApoF on cellular proliferation and growth of SMMC-7721 and Huh7 cells was examined in vitro and in vivo. RESULTS: ApoF expression was significantly down-regulated at both mRNA and protein levels in HCC tissues as compared with adjacent tissues. In SMMC-7721 and Huh7 HCC cells, ApoF overexpression inhibited cell proliferation and migration. In a xenograft nude mouse model, ApoF overexpression effectively controlled HCC growth. Kaplan-Meier analysis results showed that the recurrence-free survival rate of HCC patients with low ApoF expression was significantly lower than that of other HCC patients. Low ApoF expression was associated with several clinicopathological features such as liver cirrhosis, Barcelona Clinic Liver Cancer stage and tumor-node-metastasis stage. CONCLUSIONS: ApoF expression was down-regulated in HCC, which was associated with low recurrence-free survival rate. ApoF may serve as a tumor suppressor in HCC and be a potential application for the treatment of this disease.

Whole-Brain Monosynaptic Afferent Projections to the Cholecystokinin Neurons of the Suprachiasmatic Nucleus.

The suprachiasmatic nucleus (SCN) is the principal pacemaker driving the circadian rhythms of physiological behaviors. The SCN consists of distinct neurons expressing neuropeptides, including arginine vasopressin (AVP), vasoactive intestinal polypeptide (VIP), gastrin-releasing peptide (GRP), cholecystokinin (CCK), and so on. AVP, VIP, and GRP neurons receive light stimulation from the retina to synchronize endogenous circadian clocks with the solar day, whereas CCK neurons are not directly innervated by retinal ganglion cells and may be involved in the non-photic regulation of the circadian clock. To better understand the function of CCK neurons in non-photic circadian rhythm, it is vital to clarify the direct afferent inputs to CCK neurons in the SCN. Here, we utilized a recently developed rabies virus- and Cre/loxP-based, cell type-specific, retrograde tracing system to map and quantitatively analyze the whole-brain monosynaptic inputs to SCN CCK neurons. We found that SCN CCK neurons received direct inputs from 29 brain nuclei. Among these nuclei, paraventricular nucleus of the hypothalamus (PVH), paraventricular nucleus of the thalamus (PVT), supraoptic nucleus (SON), ventromedial nucleus of the hypothalamus, and seven other nuclei sent numerous inputs to CCK neurons. Moderate inputs originated from the zona incerta, periventricular hypothalamic nucleus, and five other nuclei. A few inputs to CCK neurons originated from the orbital frontal cortex, prelimbic cortex, cingulate cortex, claustrum, and seven other nuclei. In addition, SCN CCK neurons were preferentially innervated by AVP neurons of the ipsilateral PVH and SON rather than their contralateral counterpart, whereas the contralateral PVT sent more projections to CCK neurons than to its ipsilateral counterpart. Taken together, these results expand our knowledge of the specific innervation to mouse SCN CCK neurons and provide an important indication for further investigations on the function of CCK neurons.

Increased B cell activating factor is associated with B cell class switching in patients with tuberculous pleural effusion.

B cell activating factor (BAFF), a member of the tumor necrosis factor family, is a key cytokine for B cell survival, a function that is essential for B cell maturation and memory. The expression levels of BAFF and its potential contribution to B cell maturation remain elusive in patients with tuberculous pleural effusion (TPE). The present study enrolled 40 healthy controls (HC) and 45 TPE patients, and investigated the levels of BAFF in the plasma and pleural effusion. Concomitantly, B cell subsets including naïve B cell (CD19+IgD+CD27­), unswitched B cell (CD19+IgD+CD27+), switched B cell (CD19+IgD­CD27+), total memory B cell (CD19+CD27+), plasma B cell (CD19+IgD­CD38+CD27+) and transitional B cell (CD19+IgDdim CD38+) in peripheral blood mononuclear cells (PBMCs) and pleural fluid mononuclear cells (PFMCs) were assessed using multicolor flow cytometry. Finally, the associations between BAFF and each sub­group of B cells in TPE patients were analyzed. Compared with HC cases, an increased BAFF level and elevated frequency of switched B cell were observed in the blood and pleural effusion from patients with TPE. The proportions of naïve B cell, plasma B cell and transitional B cell were lower in the PFMCs of TPE patients. Furthermore, a significant correlation was observed between the level of BAFF, and the proportion of switched B cell in the peripheral blood and pleural effusion of TPE patients. These findings indicated that the B cell profile may be different in the pleural effusion, and BAFF may activate switched B cells to enhance the humoral immune responses in patients with TPE. Further studies are required to elucidate the underlying mechanisms and determine the potential immunotherapy of the BAFF­switched B cell axis.

Striatal adenosine A2A receptor neurons control active-period sleep via parvalbumin neurons in external globus pallidus.

Dysfunction of the striatum is frequently associated with sleep disturbances. However, its role in sleep-wake regulation has been paid little attention even though the striatum densely expresses adenosine A2A receptors (A2ARs), which are essential for adenosine-induced sleep. Here we showed that chemogenetic activation of A2AR neurons in specific subregions of the striatum induced a remarkable increase in non-rapid eye movement (NREM) sleep. Anatomical mapping and immunoelectron microscopy revealed that striatal A2AR neurons innervated the external globus pallidus (GPe) in a topographically organized manner and preferentially formed inhibitory synapses with GPe parvalbumin (PV) neurons. Moreover, lesions of GPe PV neurons abolished the sleep-promoting effect of striatal A2AR neurons. In addition, chemogenetic inhibition of striatal A2AR neurons led to a significant decrease of NREM sleep at active period, but not inactive period of mice. These findings reveal a prominent contribution of striatal A2AR neuron/GPe PV neuron circuit in sleep control.

The circular RNA of peripheral blood mononuclear cells: Hsa_circ_0005836 as a new diagnostic biomarker and therapeutic target of active pulmonary tuberculosis.

It has been reported that circular RNA (circRNA) is associated with human cancer. However, few studies have been reported in active pulmonary tuberculosis (APTB). The global circRNA expression was detected in the peripheral blood mononuclear cells (PBMCs) of APTB patients (n=5) and health controls (HC) (n=5) by using high-throughput sequencing. According to the systematical bioinformatics analysis, the basic content of circRNAs and their fold changes in the two groups were calculated. We selected 6 significant differentially expressed circRNAs, hsa_circ_0005836, hsa_circ_0009128, hsa_circ_0003519, hsa_circ_0023956, hsa_circ_0078768, and hsa_circ_0088452 and validated the expression in PBMCs from APTB (n=10) and HC (n=10) by real-time quantitative reverse transcription-polymerase chain reactions (qRT-PCRs). Further, the verification of these specific circRNAs (hsa_circ_0005836 and hsa_circ_0009128) between APTB (n=34) and HC (n=30) in PBMCs was also conducted by qRT-PCRs. The RNA-seq data showed the significant differential expression of the 523 circRNAs between the APTB and HC groups (199 circRNAs were significantly up-regulated and 324 circRNAs were down-regulated). Hsa_circ_0005836 and hsa_circ_0009128 expression was significantly down-regulated in the PBMCs of APTB (P<0.05) in the samples of APTB compared to HC in our study. The gene ontology based enrichment analysis of the circRNA-miRNA-mRNAs network showed that cellular catabolic process (P=7.10E-08), regulation of metabolic process (P=2.10E-06), catalytic activity (P=3.67E-08), protein binding (P=1.71E-07), cell part (P=3.46E-06), intracellular part (P=1.71E-07), and intracellular (P=3.67E-08) were recognized in the comparisons between APTB and HC. Based on KEGG analysis, HTLV-I infection, regulation of actin cytoskeleton, neurotrophin signaling pathway and mTOR signaling pathway were relevant during tuberculosis bacillus infection. We found for the first time that hsa_circ_0005836 and hsa_circ_0009128 were significantly down-regulated in the PBMCs of APTB compared with HC. Our findings indicate hsa_circ_0005836 might serve as a novel potential biomarker for TB infection.

Kallistatin protects against diabetic nephropathy in db/db mice by suppressing AGE-RAGE-induced oxidative stress.

Kallistatin is a serine protease inhibitor with anti-inflammatory, anti-angiogenic, and anti-oxidative properties. Since oxidative stress plays a critical role in the pathogenesis of diabetic nephropathy, we studied the effect and mechanisms of action of kallistatin superinduction. Using ultrasound-microbubble-mediated gene transfer, kallistatin overexpression was induced in kidney tubules. In db/db mice, kallistatin overexpression reduced serum creatinine and BUN levels, ameliorated glomerulosclerosis and tubulointerstitial injury, and attenuated renal fibrosis by inhibiting TGF-ß signaling. Additionally, downstream PAI-1 and collagens I and IV expression were reduced and kallistatin partially suppressed renal inflammation by inhibiting NF-κB signaling and decreasing tissue kallikrein activity. Kallistatin lowered blood pressure and attenuated oxidative stress as evidenced by suppressed levels of NADPH oxidase 4, and oxidative markers (nitrotyrosine, 8-hydroxydeoxyguanosine, and malondialdehyde) in diabetic renal tissue. Kallistatin also inhibited RAGE expression in the diabetic kidney and AGE-stimulated cultured proximal tubular cells. Reduced AGE-induced reactive oxygen species generation reflected an anti-oxidative mechanism via the AGE-RAGE-reactive oxygen species axis. These results indicate a renoprotective role of kallistatin against diabetic nephropathy by multiple mechanisms including suppression of oxidative stress, anti-fibrotic and anti-inflammatory actions, and blood pressure lowering.

BMP7 reduces inflammation and oxidative stress in diabetic tubulopathy.

Bone morphogenetic protein 7 (BMP7) has been reported to confer renoprotective effects in acute and chronic kidney disease models, but its potential role in Type 2 diabetic nephropathy remains unknown. In cultured human proximal tubular epithelial cells (PTECs), exposure to advanced glycation end-products (AGEs) induced overexpression of intercellular adhesion molecule 1 (ICAM1), monocyte chemoattractant protein 1 (MCP1), interleukin 8 (IL-8) and interleukin 6 (IL-6), involving activation of p44/42 and p38 mitogen-activated protein kinase (MAPK) signalling. BMP7 dose-dependently attenuated AGE-induced up-regulation of ICAM1, MCP1, IL-8 and IL-6 at both mRNA and protein levels. Moreover, BMP7 suppressed AGE-induced p38 and p44/42 MAPK phosphorylation and reactive oxygen species production in PTECs. Compared with vehicle control, uninephrectomized db/db mice treated with BMP7 for 8 weeks had significantly lower urinary albumin-to-creatinine ratio (3549±816.2 µg/mg compared with 8612±2037 µg/mg, P=0.036), blood urea nitrogen (33.26±1.09 mg/dl compared with 37.49±0.89 mg/dl, P=0.006), and renal cortical expression of ICAM1 and MCP1 at both gene and protein levels. In addition, BMP7-treated animals had significantly less severe tubular damage, interstitial inflammatory cell infiltration, renal cortical p38 and p44/42 phosphorylation and lipid peroxidation. Our results demonstrate that BMP7 attenuates tubular pro-inflammatory responses in diabetic kidney disease by suppressing oxidative stress and multiple inflammatory signalling pathways including p38 and p44/42 MAPK. Its potential application as a therapeutic molecule in diabetic nephropathy warrants further investigation.

Mesenchymal stem cells modulate albumin-induced renal tubular inflammation and fibrosis.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) have recently shown promise as a therapeutic tool in various types of chronic kidney disease (CKD) models. However, the mechanism of action is incompletely understood. As renal prognosis in CKD is largely determined by the degree of renal tubular injury that correlates with residual proteinuria, we hypothesized that BM-MSCs may exert modulatory effects on renal tubular inflammation and epithelial-to-mesenchymal transition (EMT) under a protein-overloaded milieu. Using a co-culture model of human proximal tubular epithelial cells (PTECs) and BM-MSCs, we showed that concomitant stimulation of BM-MSCs by albumin excess was a prerequisite for them to attenuate albumin-induced IL-6, IL-8, TNF-α, CCL-2, CCL-5 overexpression in PTECs, which was partly mediated via deactivation of tubular NF-κB signaling. In addition, albumin induced tubular EMT, as shown by E-cadherin loss and α-SMA, FN and collagen IV overexpression, was also prevented by BM-MSC co-culture. Albumin-overloaded BM-MSCs per se retained their tri-lineage differentiation capacity and overexpressed hepatocyte growth factor (HGF) and TNFα-stimulating gene (TSG)-6 via P38 and NF-κB signaling. Albumin-induced tubular CCL-2, CCL-5 and TNF-α overexpression were suppressed by recombinant HGF treatment, while the upregulation of α-SMA, FN and collagen IV was attenuated by recombinant TSG-6. Neutralizing HGF and TSG-6 abolished the anti-inflammatory and anti-EMT effects of BM-MSC co-culture in albumin-induced PTECs, respectively. In vivo, albumin-overloaded mice treated with mouse BM-MSCs had markedly reduced BUN, tubular CCL-2 and CCL-5 expression, α-SMA and collagen IV accumulation independent of changes in proteinuria. These data suggest anti-inflammatory and anti-fibrotic roles of BM-MSCs on renal tubular cells under a protein overloaded condition, probably mediated via the paracrine action of HGF and TSG-6.

Transplantation of oligodendrocyte precursor cells improves locomotion deficits in rats with spinal cord irradiation injury.

Demyelination contributes to the functional impairment of irradiation injured spinal cord. One potential therapeutic strategy involves replacing the myelin-forming cells. Here, we asked whether transplantation of Olig2(+)-GFP(+)-oligodendrocyte precursor cells (OPCs), which are derived from Olig2-GFP-mouse embryonic stem cells (mESCs), could enhance remyelination and functional recovery after spinal cord irradiation injury. We differentiated Olig2-GFP-mESCs into purified Olig2(+)-GFP(+)-OPCs and transplanted them into the rats' cervical 4-5 dorsal spinal cord level at 4 months after irradiation injury. Eight weeks after transplantation, the Olig2(+)-GFP(+)-OPCs survived and integrated into the injured spinal cord. Immunofluorescence analysis showed that the grafted Olig2(+)-GFP(+)-OPCs primarily differentiated into adenomatous polyposis coli (APC(+)) oligodendrocytes (54.6±10.5%). The staining with luxol fast blue, hematoxylin & eosin (LFB/H&E) and electron microscopy demonstrated that the engrafted Olig2(+)-GFP(+)-OPCs attenuated the demyelination resulted from the irradiation. More importantly, the recovery of forelimb locomotor function was enhanced in animals receiving grafts of Olig2(+)-GFP(+)-OPCs. We concluded that OPC transplantation is a feasible therapy to repair the irradiated lesions in the central nervous system (CNS).

The TLR4 antagonist CRX-526 protects against advanced diabetic nephropathy.

We recently showed that Toll-like receptor (TLR) TLR4 was overexpressed in the human diabetic kidney, which could promote tubular inflammation. Here we explored whether the TLR4 antagonist, CRX-526, has therapeutic potential to attenuate renal injuries and slow the progression of advanced diabetic nephropathy in wild-type and endothelial nitric oxide synthase (eNOS) knockout mice. In the latter, the endogenous TLR4 ligand, high-mobility group box 1, was upregulated more than in wild-type animals. Four weeks after streptozotocin induction of diabetes, mice were injected with either CRX-526 or vehicle for 8 weeks. CRX-526 significantly reduced albuminuria and blood urea nitrogen without altering blood glucose and systolic blood pressure in diabetic mice. Glomerular hypertrophy, glomerulosclerosis, and tubulointerstitial injury were attenuated by CRX-526, which was associated with decreased chemokine (C-C motif) ligand (CCL)-2, osteopontin, CCL-5 overexpression, subsequent macrophage infiltration, and collagen deposition. These effects were associated with inhibition of TGF-ß overexpression and NF-κB activation. In vitro, CRX-526 inhibited high glucose-induced osteopontin upregulation and NF-κB nuclear translocation in cultured human proximal tubular epithelial cells. Thus, we provided evidence that inhibition of TLR4 with the synthetic antagonist CRX-526 conferred renoprotective effects in eNOS knockout diabetic mice with advanced diabetic nephropathy.

Projections of nucleus accumbens adenosine A2A receptor neurons in the mouse brain and their implications in mediating sleep-wake regulation.

Adenosine A2A receptors (A2ARs) in the nucleus accumbens (Acb) have been demonstrated to play an important role in the arousal effect of adenosine receptor antagonist caffeine, and may be involved in physiological sleep. To better understand the functions of these receptors in sleep, projections of A2AR neurons were mapped utilizing adeno-associated virus (AAV) encoding humanized Renilla green fluorescent protein (hrGFP) as a tracer for long axonal pathways. The Cre-dependent AAV was injected into the core (AcbC) and shell (AcbSh) of the Acb in A2AR-Cre mice. Immunohistochemistry was then used to visualize hrGFP, highlighting the perikarya of the A2AR neurons in the injection sites, and their axons in projection regions. The data revealed that A2AR neurons exhibit medium-sized and either round or elliptic perikarya with their processes within the Acb. Moreover, the projections from the Acb distributed to nuclei in the forebrain, diencephalon, and brainstem. In the forebrain, A2AR neurons from all Acb sub-regions jointly projected to the ventral pallidum, the nucleus of the diagonal band, and the substantia innominata. Heavy projections from the AcbC and the ventral AcbSh, and weaker projections from the medial AcbSh, were observed in the lateral hypothalamus and lateral preoptic area. In the brainstem, the Acb projections were found in the ventral tegmental area, while AcbC and ventral AcbSh also projected to the median raphe nucleus, the dorsal raphe nucleus, and the ventrolateral periaqueductal gray. The results supply a solid base for understanding the roles of the A2AR and A2AR neurons in the Acb, especially in the regulation of sleep.

Neuroprotective effects of neuregulin-1 in rat models of focal cerebral ischemia.

The purpose of this study was to investigate the therapeutic efficacy and mechanism of recombinant human NRG-1 to attenuate ischemia/reperfusion brain injury. NRG-1(3.0 ng/kg) was applied intravascularly 10 min before middle cerebral artery occlusion (MCAO) and then focal cerebral ischemia for 90 min and reperfusion for 24 h. The rats were scored post-reperfusion for neurological deficits and infarct volume in the brain was assessed by 2,3,5-triphenyltetrazolium chloride(TTC). Apoptosis was evaluated by TUNEL staining. Reverse transcription polymerase chain reaction (RT-PCR) was used to measure changes of caspase-3 mRNA. The level of TNF-alpha was determined using enzyme-linked immunosorbent assay (ELISA). Our results demonstrated that recombinant human NRG-1 could reduce cerebral infarct volume by about 71% (P < 0.05) and TUNEL positive cells when given immediately before MCAO, and improved behavior of animals. Furthermore, we also showed that NRG-1 could also decrease the expression of caspase-3 mRNA and production of TNF-alpha protein. These data suggest that pre-administration of NRG-1 attenuates cerebral ischemia and reperfusion injury. This protective effect may be involved in the inhibition of caspase-3 and TNF-alpha.