KLF4 protects brain microvascular endothelial cells from ischemic stroke induced apoptosis by transcriptionally activating MALAT1.

In this study, we investigated the expression profile and functional role of Kruppel-like family of transcription factor 4 (KLF4) in mouse cerebral microvascular endothelial cells (MECs) after focal cerebral ischemia and in cultured b.End3 cells after oxygen-glucose deprivation (OGD). Its downstream regulation was also studied. Our data showed that KLF4 was significantly upregulated in the cerebral MECs after transient ischemic insult and in cultured b.End3 cells after 24 h OGD exposure. In in vitro b.End3 cell model, KLF4 shRNA significantly increased OGD-induced activation of caspase-3 and also increased OGD-induced b.End3 cell death. KLF4 shRNA substantially enhanced OGD induced Bim and Bax expression at mRNA and protein levels and also aggravated OGD induced upregulation of E-selectin, MCP-1 and IL-6. Bioinformatic analysis suggested that MALAT1 promoter has a possible KLF4 binding site. In this study, we validated this possible binding site and also demonstrated that enforced KLF4 expression increased MALAT1 transcription. Functionally, knockdown of MALAT1 phenocopied the effect of KLF4 shRNA on enhancing OGD induced cell apoptosis and OGD induced upregulation of pro-apoptotic factors and pro-inflammatory cytokines. Based on these findings, we infer that MALAT1 is a transcriptional target of KLF4 in its protective role in cerebral MECs after ischemic insult.

[Yinxingye Capsule Intervened Vascular Endothelial Cell Apoptosis of Hyperhomocysteinemia Rats: an Experimental Study].

OBJECTIVE: To explore targets of Chinese herbal medicine at cellular and molecular leve1s through an experimental study on Yinxingye Capsule (YC) intervening vascular endothelial cell apoptoeis of hyperhornocysteinemia (HHcy) rats. METHODS: The HHcy model was prepared in male Wistar rats. Totally 42 rats were randomly divided into 4 groups, i.e., the control group (n =10), the model group (n = 11), the YC group (n =11), the folic acid group (n =10). Carboxy methyl cellulose (CMC) solution (1%) was administered to rats in the control group by gastrogavage.3% methionine suspension at 1. 5 g/kg was administered to rats in the model group by gastrogavage. 3% methionine suspension at 1. 5 g/kg and folic acid suspension at 0. 06 g/kg was administered to rats in the folic acid group by gastrogavage. 3% methionine suspension at 1. 5 g/kg and YC at 0. 02 g/kg was administered to rats in the YC group by gastrogavage. Morphological changes of aortic tissue were observed by hematoxylin eosin (HE) staining. The plasma homocysteine (Hcy) level was detected in each group. The endothelium-dependent diastolic functions of the thoracic aorta on different concentrations of sodium nitroprusside (SNP) and acetylcholine (Ach) were detected. Gene expressions of Bcl-2-associated X protein (BAX), inducible nitric oxide synthase (iNOS), c-Fos, cellular inhibitor of apoptosis protein 2 (c-IAP2) were detected by real time polymerase chain reaction (RT-PCR). RESULTS: Pathological results showed that thickening aortic endothelium, swollen and desquamated endothelial cells. Few foam cells could be seen in the model group. Myoma-like proliferation of smooth muscle cells in tunica media could also be seen. These pathological changes were milder in the YC group and the folic acid group. Compared with the control group, plasma Hcy levels increased in the model group (P <0. 05). The endothelium-dependent diastolic rates at 10(-6) and 10(-4)mol/L Ach and 10(-7) -10(-3)mol/L SNP all decreased in the model group (P <0. 01, P <0. 05). Gene expressions of Bax, c-Fos, and iNOS increased, but c-IAP2 gene expressions decreased in the model group (all P <0. 05). Compared with the model group, plasma Hcy levels decreased in the YC group and the folic acid group (P <0. 05). The endothelium-dependent diastolic rates increased in the YC group and the folic acid group at various SNP concentrations except 10(-6) mol/L SNP in the folic acid group. The endothelium-dependent diastolic rates increased in the YC group and the folic acid group at 10(-6) and 10(-4)mol/L Ach (all P <0. 05). Gene expressions of Bax, c-Fos, and iNOS decreased in the YC group and the folic acid group, but c-IAP2 gene expression increased in the folic acid group (all P <0. 05). CONCLUSION: YC could reduce plasma Hcy levels, down-regulate gene expressions of Bax, c-Fos, and iNOS, thereby reducing apoptosis of vascular endothelial cells, improving vascular endothelial function, and delaying atherosclerotic process.

Buyang huanwu decoction (BYHWD) alleviates sepsis-induced myocardial injury by suppressing local immune cell infiltration and skewing M2-macrophage polarization.

OBJECTIVE: To investigate the therapeutic effect of Buyang huanwu decoction (BYHWD) on sepsis-induced myocardial injury (SIMI) and explore the mechanism by which BYHWD mitigates SIMI. METHODS: The Lipopolysaccharide (LPS)-induced SIMI mouse model was established to detect the effect of BYHWD-low (1 mg/kg), BYHWD-middle (5 mg/kg), and BYWHD-high (20 mg/kg) on SIMI. The survival of these BYHWD-treated septic mice was investigated. The histology of myocardial tissues was determined by hematoxylin and eosin (H&E) staining. The apoptotic index and inflamed microenvironment of myocardial tissues were assessed by immunofluorescent staining (IF) and flow cytometry analysis. Liquid chromatography-mass spectrometry (LC-MS/MS) was employed to determine the key chemical components in the serum of BYHWD-loaded septic mice. Immunoblotting assay was utilized to detect NF-κB and TGF-ß signaling activity, and M1/M2-macrophage markers using RAW264.7 cells. RESULTS: The high dosage of BYHWD (BYHWD-high, 20 mg/Kg) significantly attenuated SIMI and improved the survival of septic mice. The BYHWD-high solution markedly reduced myocardial cell apoptosis and mitigated the inflamed microenvironment by suppressing CD45+ immune cell infiltration. Importantly, BYHWD decreased macrophage accumulation and promoted an M2-macrophage polarization. Paeoniflorin (PF) and calycosin-7-O-ß-glucoside (CBG) were identified as the key molecules in BYWHD with therapeutic effect. PF (10 µM) and CBG (1 µM) inhibited NF-κB signaling, meanwhile they upregulated the TGF-ß pathway, thereby facilitating an M2-macrophage phenotypic transition in RAW264.7 cells. CONCLUSIONS: BYHWD, with two effective components PF and CBG, can mitigate SIMI by suppressing the inflamed myocardial microenvironment and skewing an immunosuppressive M2-macrophage phenotype.