FGF21 attenuates neuroinflammation following subarachnoid hemorrhage through promoting mitophagy and inhibiting the cGAS-STING pathway.

BACKGROUND: Subarachnoid hemorrhage (SAH) represents a form of cerebrovascular event characterized by a notable mortality and morbidity rate. Fibroblast growth factor 21 (FGF21), a versatile hormone predominantly synthesized by the hepatic tissue, has emerged as a promising neuroprotective agent. Nevertheless, the precise impacts and underlying mechanisms of FGF21 in the context of SAH remain enigmatic. METHODS: To elucidate the role of FGF21 in inhibiting the microglial cGAS-STING pathway and providing protection against SAH-induced cerebral injury, a series of cellular and molecular techniques, including western blot analysis, real-time polymerase chain reaction, immunohistochemistry, RNA sequencing, and behavioral assays, were employed. RESULTS: Administration of recombinant fibroblast growth factor 21 (rFGF21) effectively mitigated neural apoptosis, improved cerebral edema, and attenuated neurological impairments post-SAH. Transcriptomic analysis revealed that SAH triggered the upregulation of numerous genes linked to innate immunity, particularly those involved in the type I interferon (IFN-I) pathway and microglial function, which were notably suppressed upon adjunctive rFGF21 treatment. Mechanistically, rFGF21 intervention facilitated mitophagy in an AMP-activated protein kinase (AMPK)-dependent manner, thereby preventing mitochondrial DNA (mtDNA) release into the cytoplasm and dampening the activation of the DNA-sensing cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway. Conditional knockout of STING in microglia markedly ameliorated the inflammatory response and mitigated secondary brain injuries post-SAH. CONCLUSION: Our results present the initial evidence that FGF21 confers a protective effect against neuroinflammation-associated brain damage subsequent to SAH. Mechanistically, we have elucidated a novel pathway by which FGF21 exerts this neuroprotection through inhibition of the cGAS-STING signaling cascade.

MicroRNA-144 represses gliomas progression and elevates susceptibility to Temozolomide by targeting CAV2 and FGF7.

Malignant gliomas are the most common tumor in central nervous system with poor prognosis. Due to the limitation of histological classification in earlier diagnosis and individualized medicine, it is necessary to combine the molecular signatures and the pathological characteristics of gliomas. Lots of microRNAs presented abnormal expression in gliomas and modulated gliomas development. Exploration the miRNAs profile is helpful for the diagnosis, therapy and prognosis of gliomas. It has been demonstrated that miR-144 plays important roles in solid tumors. However, the detail mechanisms remained unrevealed. In this study, we have demonstrated the level of miR-144 decreased in glioma tissues from patients, especially in gliomas with higher grades. MiR-144 was also validated have lower expression in glioma cell lines compared with cortical neuron cell by using qRT-PCR. The in vitro functional experiment indicated miR-144 improved gliomas progression through repressing proliferation, sensitizing to chemotherapeutics and inhibiting metastasis. We further identified fibroblast growth factor 7 (FGF7) and Caveolin 2 (CAV2) were target genes of miR-144 by luciferase reporter assay and western blotting. The mechanisms study suggested forced FGF7 expression elevated Akt activation and decreased reactive oxygen species (ROS) generation. The MTT and cell cycle assay indicated miR-144 suppressed glioma cells proliferation through modulating FGF mediated Akt signaling pathway. Meanwhile, miR-144 promoted Temozolomide (TMZ) induced apoptosis in glioma cells via increasing ROS production by using FACS. On the other hand, CAV2, as another target of miR-144, accelerated glioma cells migration and invasion via promoting glioma cells EMT progress. Retrieved expression of FGF7 or CAV2 rescued the proliferation and migration function mediated by miR-144. Furthermore, the in vivo experiments in PDX models displayed the anti-tumor function of miR-144, which could be retrieved by overexpression of FGF7 and CAV2. Taken together, these findings indicated miR-144 acted as a potential target against gliomas progression and uncovered a novel regulatory mechanism, which may provide a new therapeutic strategy and prognostic indicator for gliomas.

Silencing lncRNA FOXD2-AS1 inhibits proliferation, migration, invasion and drug resistance of drug-resistant glioma cells and promotes their apoptosis via microRNA-98-5p/CPEB4 axis.

OBJECTIVE: This study was conducted to elucidate the long non-coding RNA FOXD2-AS1 (lncRNA FOXD2-AS1) expression in glioma and its mechanism on the biological features of glioma cells and the drug resistance of temozolomide (TMZ). RESULTS: Highly expressed FOXD2-AS1 was found in glioma. There was more powerful chemotherapeutic resistance of TMZ resistant cell lines than that of the parent cell lines. Silence of FOXD2-AS1 suppressed proliferation and drug resistance and promoted apoptosis of drug-resistant glioma cells. Overexpressed FOXD2-AS1 presented an opposite trend. FOXD2-AS1 could be used as a competing endogenous RNA to adsorb miR-98-5p, thereby up-regulating CPEB4. CONCLUSION: Our study suggests that down-regulated FOXD2-AS1 repressed invasion, proliferation, migration and drug resistance of drug-resistant glioma cells while stimulating their apoptosis via increasing miR-98-5p and inhibiting CPEB4 expression. METHODS: FOXD2-AS1, microRNA-98-5p (miR-98-5p) and cytoplasmic polyadenylation element binding (CPEB4) expression in glioma tissues were tested. Expression of E-cadherin, N-cadherin and Vimentin in glioma cells were explored. A series of assays were conducted to detect the function of FOXD2-AS1 in migration, proliferation, apoptosis, and invasion of glioma cells. Changes in drug-resistance of cells under TMZ treatment were examined, and tumor formation in nude mice was performed to test the changes of drug resistance in vivo.

The Effects of Intelectin-1 on Antioxidant and Angiogenesis in HUVECs Exposed to Oxygen Glucose Deprivation.

Objective: Ischemic stroke leads to cellular death and tissue damage by depriving the areas of glucose and oxygen supplies. The effective treatment of stroke remains a challenge for modern medicine. This study used an oxygen-glucose deprivation (OGD) model of human umbilical vein endothelial cells (HUVECs) to mimic ischemic injuries and explored the role and mechanism of intelectin-1. Methods: Intelectin-1 was transduced into the HUVECs using a lentiviral vector. The PI3K/Akt signaling was examined in intelectin-induced eNOS phosphorylation. The PI3K inhibitor LY294002 was dealed in HUVECs. Results: Our results demonstrated an increase in capillary density, decrease in apoptotic cells, and increase in HIF-1α protein expression following intelectin-1 treatment. Real-time PCR and Western blotting revealed the increased intelectin-1 expression alongside eNOS and Akt phosphorylation with enhanced bcl-2 expression under OGD. Capillary density decreased significantly after LY294002 treatment. Conclusion: These results suggest intelectin-1 promotes angiogenesis, inhibits oxidative stress and reduces apoptosis by stimulating the Akt-eNOS signaling pathway in response to ischemia in vitro.

Anti-GABAB receptor encephalitis in a patient with gastric adenocarcinoma.

Anti-γ-aminobutyric acid-B (GABAB) receptor encephalitis is an autoimmune encephalitis associated with antibodies against neuronal cell surface antigens, which are primarily observed with small-cell lung cancer, melanoma, and thymoma. Here, we first report a case on the association between a relatively frequent cancer, gastric adenocarcinoma (GAC), and a rare GABAB receptor antibody limbic encephalitis. The patient was treated with immunotherapy and combined-drug chemotherapy, which were partially effective in terms of stabilizing the tumor and relieving neurological symptoms. This report indicates that, when patients present with GABAB receptor antibody limbic encephalitis, regular and broad screening for tumors including gastric adenocarcinoma should also be considered.

miR-183-96-182 clusters alleviated ox-LDL-induced vascular endothelial cell apoptosis in vitro by targeting FOXO1.

OBJECTIVE: To investigate the role of FOXO1 and miR-183-96-182 clusters in ox-LDL induced endothelial cell apoptosis. METHODS: FOXO1 overexpression (OE) and knockdown (KD) as well as AKT1 OE in human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs) were achieved by lentiviral transduction. Upregulation of miR-183-5p, miR-182-5p or miR-96-5p was mimicked by agomir treatment. FOXO1 gene transcription was monitored by FOXO1 promotor reporter assay. Cell apoptosis in culture was monitored by TiterTACS in situ detection. Regulation of FOXO1 gene expression by an miRNA targeting mechanism was monitored by AGO2-RNA immunoprecipitation assay. RESULTS: FOXO1 mRNA and protein expression levels in ox-LDL treated HUVECs or HAECs were significantly upregulated due to transcriptional and miRNA targeting mechanisms. MiR-183-5p, miR-182-5p and miR-96-5p expression levels in HUVECs or HAECs were significantly reduced by ox-LDL treatment, the overexpression of which by agomir treatment partially reduced the FOXO1 mRNA/protein expression levels and cell apoptosis which was upregulated by ox-LDL treatment. FOXO1 overexpression antagonized the effect of the agomir treatment indicated above. MiR-183-5p, miR-182-5p and miR-96-5p agomir treatment partially rescued the FOXO1 pSer256/total FOXO1 protein ratio and the AKT1 pSer473 level that were reduced by ox-LDL treatment in the HUVECs or HAECs. AKT1 overexpression significantly reduced FOXO1 protein expression, increased miR-182-5p and miR-183-5p expression, and partially alleviated ox-LDL induced HUVEC or HAEC apoptosis in an miR-183-5p and miR-182-5p-dependent manner. CONCLUSION: miR-183-96-182 clusters could partially alleviate ox-LDL-induced apoptosis in HUVECs or HAECs by targeting FOXO1.

Suppression of SIRT6 by miR-33a facilitates tumor growth of glioma through apoptosis and oxidative stress resistance.

microRNA-33a (miR-33a) belongs to the miR-33 family that is implicated in the progression of various types of cancers. Aberrant expression of miR-33a has been detected in several human cancers, and has been shown to regulate the migration and invasion as well as proliferation and apoptosis of tumor cells. However, the clinical significance and precise mechanisms underlying the dysfunction of miR-33a in glioma have not been well investigated in previous studies. In this study, overexpression of miR-33a was observed in clinical glioma specimens and cell lines. Clinicopathological detection revealed that miR-33a highly expressing patients showed large tumor sized and advanced World Health Organization (WHO) grade as well as reduced overall survival. Furthermore, the results of in vitro experiments confirmed that loss of miR-33a resulted in reduced proliferation and enhanced apoptosis in U251 cells, while miR-33a restoration showed opposite effects in U87 cells. Further studies indicated that miR-33a knockdown restrained tumor growth of glioma in vivo. miR-33a negatively regulated the expression of sirtuin 6 (SIRT6) at both mRNA and protein levels via targeting the 3'UTR of SIRT6 mRNA. SIRT6 was underexpressed and inversely correlated with miR-33a expression in the glioma tissues. Mechanistically, SIRT6 overexpression increased the levels of lactate dehydrogenase (LDH) and reactive oxygen species (ROS) while it reduced cell survival under H2O2 treatment. In addition, SIRT6 restoration led to apoptosis with alterative expression of Bax, Bcl-2, cleaved caspase-8, and inhibition of Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway in glioma. Thus, our studies demonstrated that the deregulation of miR-33a may promote tumor development in human glioma by regulating the expression of its target gene, SIRT6.

Anti-apoptotic and angiogenic effects of intelectin-1 in rat cerebral ischemia.

Ischemic stroke is an acute life-threatening disease, which causes neurological dysfunction. The formation of new blood vessels around the infarct is vital to the restoration of perfusion and healing of brain tissue. Studies have shown that intelectin-1 (omentin) promotes endothelial cell function and angiogenesis in response to ischemia and inhibits apoptosis in rats with unilateral hind limb surgery. In the present study, we investigated the neuroprotective role of intelectin-1 following focal cerebral ischemia. We specifically assessed the effect of increased expression of intelectin-1 in promoting angiogenesis and reducing apoptosis. The treatment was administered using a lentiviral vector, 7 days prior to surgery. The surgery was performed using the established middle cerebral artery occlusion (MCAO) model in rats, and the outcome was evaluated 7 days after injury. Our results demonstrated a significant reduction in brain infarction volume following LV-intelectin-1 treatment. Additionally, CD34 and capillary density were increased in the cerebral ischemic penumbra. Real-time PCR and Western blot revealed an increased expression of intelectin-1, and phosphorylation of eNOS and AKT with enhanced expression of bcl-2 in brain tissues. These data suggest that the successful delivery of LV-intelectin-1 ameliorated ischemic brain injury. It promoted endothelial cell function and revasc ularization, and inhibited apoptosis in response to ischemia by stimulating the Akt-eNOS signaling pathway.

Anti-inflammatory and antiapoptotic effects of mesenchymal stem cells transplantation in rat brain with cerebral ischemia.

BACKGROUND: Excessive inflammation and apoptosis contribute to the pathogenesis of ischemic brain damage. Nuclear factor-kappa B (NF-κB) is considered to be a key protein complex involved in this cascade of events. The aim of the present study was to clarify the protection mechanism of the mesenchymal stem cells (MSCs). METHODS: Lewis rats (N = 90) were randomly assigned to three groups: (1) the sham-operated group; (2) the saline group, in which the animals underwent rat transient middle cerebral artery occlusion (tMCAO, for 2 hours) and were treated with saline through the tail vein; and (3) the MSCs group, in which the animals underwent tMCAO (for 2 hours) and were infused with cultured human MSCs (4 × 10(6)/0.4 ml PBS) through the tail vein. At days 1 and 3 post-MSCs infusion, real-time PCR, and Western blot, immunohistochemical analyses were applied for tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and P-IKKß, p53, and B-cell lymphoma 2 (Bcl-2) expression levels. RESULTS: TNF-α, IL-1ß messenger RNA (mRNA) and P-IκB-α, P-IKKß, p53 protein expression levels were significantly increased in the saline group compared with the sham group. However, IκB-α and Bcl-2 protein expression levels were markedly decreased in the saline group. After injection of BrdU(+) MSCs, the expression levels of TNF-α, IL-1ß mRNA and P-IκB-α, P-IKKß, p53 protein were significantly decreased. Contrary to these findings, IκB-α, Bcl-2 protein expression levels were markedly increased. In addition, we found that infarct area was significantly reduced in MSCs group. CONCLUSIONS: These results suggest that MSCs' neuroprotection is attributable to its anti-inflammatory and antiapoptotic effect through inhibition of NF-κB.

Shuanghuanglian injection downregulates nuclear factor-kappa B expression in mice with viral encephalitis.

A mouse model of viral encephalitis was induced by intracranial injection of a Coxsackie virus B3 suspension. Quantitative real-time reverse transcription-PCR and western blot assay were applied to detect mRNA and protein expression of intelectin-2 and nuclear factor-kappa B in the viral encephalitis and control groups. Nuclear factor-kappa B and intelectin-2 mRNA and protein expression were significantly increased in mice with viral encephalitis. After intraperitoneal injection of Shuanghuanglian at a dose of 1.5 mg/kg for 5 successive days, intelectin-2 and nuclear factor-kappa B protein and mRNA expression were significantly decreased. To elucidate the relationship between intelectin-2 and nuclear factor-kappa B, mice with viral encephalitis were administered an intracerebral injection of 107 pfu recombinant lentivirus expressing intelectin shRNA. Both protein and mRNA levels of intelectin and nuclear factor-kappa B in brain tissue of mice were significantly decreased. Experimental findings suggest that Shuanghuanglian injection may downregulate nuclear factor-kappa B production via suppression of intelectin production, thus inhibiting inflammation associated with viral encephalitis.

Mesenchymal stem cell transplantation increases expression of vascular endothelial growth factor in papain-induced emphysematous lungs and inhibits apoptosis of lung cells.

BACKGROUND: Pulmonary emphysema is characterized by loss of alveolar structures. We have found that bone marrow (BM) mesenchymal stem cell (MSC) transplantation ameliorates papain-induced pulmonary emphysema. However, the underlying mechanism is not completely understood. It has been shown that blocking the vascular endothelial growth factor (VEGF) signaling pathway leads to apoptosis of lung cells and pulmonary emphysema, and MSC are capable of secreting VEGF. We hypothesized that MSC transplantation may have a protective effect on pulmonary emphysema by increasing VEGF-A expression and inhibiting apoptosis of lung cells. METHODS: We examined the morphology and expression of VEGF-A in rat lung after papain treatment and MSC transplantation. We also used a co-culture system in which MSC and cells prepared from papain-treated lungs or control lungs were cultured together. The levels of VEGF-A in cells and culture medium were determined, and apoptosis of cultured lung cells was evaluated. RESULTS: VEGF-A expression in rat lungs was decreased after papain treatment, which was partly rescued by MSC transplantation. MSC production of VEGF-A was increased when MSC were co-cultured with cells prepared from papain-treated lungs. Furthermore, the apoptosis of papain-treated lung cells was inhibited when co-cultured with MSC. The induction of MSC production of VEGF-A by papain-treated lung cells was inhibited by adding anti-tumor necrosis factor (TNF)-alpha antibody to the medium. CONCLUSIONS: The protective effect of MSC transplantation on pulmonary emphysema may be partly mediated by increasing VEGF-A expression and inhibiting the apoptosis of lung cells. TNF-alpha released from papain-treated lung cells induces MSC to secret VEGF-A.

Intelectin is required for IL-13-induced monocyte chemotactic protein-1 and -3 expression in lung epithelial cells and promotes allergic airway inflammation.

Asthma is characterized by airway inflammation, mucus overproduction, airway hyperreactivity, and peribronchial fibrosis. Intelectin has been shown to be increased in airway epithelium of asthmatics. However, the role of intelectin in the pathogenesis of asthma is unknown. Airway epithelial cells can secrete chemokines such as monocyte chemotactic protein (MCP)-1 and -3 that play crucial roles in asthmatic airway inflammation. We hypothesized that intelectin plays a role in allergic airway inflammation by regulating chemokine expression. In a mouse allergic asthma model, we found that mRNA expression of intelectin-2 as well as MCP-1 and -3 in mouse lung was increased very early (within 2 h) after allergen challenge. Expression of intelectin protein was localized to mucous cells in airway epithelium. Treatment of MLE12 mouse lung epithelial cells with interleukin IL-13, a critical mediator of allergic airway disease, induced expression of intelectin-1 and -2 as well as MCP-1 and -3. When IL-13-induced intelectin-1 and -2 expression was inhibited by RNA interference, IL-13-induced extracellular signal-regulated kinase 1/2 phosphorylation and MCP-1 and -3 production by MLE12 cells was inhibited. Furthermore, inhibition of intelectin expression by airway transfection with shRNA targeting intelectin-1 and -2 attenuated allergen-induced airway inflammation. We conclude that intelectin, a molecule expressed by airway epithelial cells and upregulated in asthma, is required for IL-13-induced MCP-1 and -3 production in mouse lung epithelial cells and contributes to allergic airway inflammation.

Mesenchymal stem cells transplantation protects against rat pulmonary emphysema.

Pulmonary emphysema is characterized by loss of alveolar structure. Bone marrow mesenchymal stem cells (MSCs) have been shown to differentiate into alveolar epithelial cells. However, the effect of MSCs transplantation on pulmonary emphysema is unknown. To address this question, cultured bone marrow MSCs from male donor rats were infused into female recipients treated with irradiation and instillation of papain. We found that the emphysematous changes in rats received MSCs transplantation were ameliorated when compared with the rats without MSCs transplantation. Y chromosome fluorescent in situ hybridization (FISH) and immunohistochemical staining for SP-C, confirmed that MSCs engrafted in recipient lungs and differentiated into type II alveolar epithelial cells. Additionally, MSCs transplantation reduced the extent of irradiation and papain-induced alveolar cell apoptosis, likely due to the up-regulation of the expression of Bcl-2 and Bax gene. We conclude that MSCs transplantation protects against the irradiation and papain-induced pulmonary emphysema. The mechanisms of protection may involve the engraftment of MSCs in the lungs, differentiation of MSCs into type II alveolar epithelial cells and suppression of alveolar cell apoptosis.

[Effects of bone marrow mesenchymal stem cells transplantation on the apoptosis of alveolar wall cells in papain and Co60-induced pulmonary emphysema rats].

AIM: To study the effects of bone marrow MSCs transplantation on the apoptosis of alveolar wall cells and the expression of Bcl-2 and Bax of lung tissue in papain and Co60-induced pulmonary emphysema rats. METHODS: Female Lewis rats were randomly divided into three groups: control group, emphysema group, emphysema + MSCs transplantation group. Rats were sacrificed at days 14 and 28 after treatment. Morphologic analysis of the lung tissue was performed. The apoptosis of the lung cells was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The expression of Bcl-2 and Bax were determined by immunohistochemical staining. RESULTS: Emphysematous changes of the lung tissue were observed in emphysema group and emphysema + MSCs transplantation group. However, the emphysematous change in emphysema + MSCs transplantation group was improved compared with the emphysema group. There was significant difference in the number of alveolar counted per unit area (MAN), mean alveoli area (MAA) and mean linear interval(MLI) between emphysema group and emphysema + MSCs transplantation group. The apoptotic index of the alveolar wall cells in emphysema + MSCs transplantation group was less than that in the emphysema group. The percentage of Bcl-2 positive cells in emphysema + MSCs transplantation group was significantly higher than that in the emphysema group. The percentage of Bax positive cells in emphysema + MSCs transplantation group was significantly lower than that in the emphysema group. The ratio of Bcl-2/Bax of emphysema + MSCs transplantation group was significantly higher than that in the emphysema group. CONCLUSION: Bone marrow MSCs transplantation inhibits the apoptosis of alveolar wall cells, upregulates the expression of Bcl-2 and downregulates the expression of Bax. This may be part of the reason that bone marrow MSCs transplantation improves the papain and Co60-induced pulmonary emphysema.