[Tetramethylpyrazine regulates angiogenesis of endothelial cells in cerebral ischemic stroke injury via SIRT1/VEGFA signaling pathway].

This study aims to investigate whether tetramethylpyrazine(TMP) can stimulate angiogenesis in cerebral microvascular endothelial cells and alleviate cerebral ischemic stroke(CIS) and to explore the underlying mechanisms. In the animal study, adult Sprague-Dawley rats(n=15) were assigned into sham surgery(sham), middle cerebral artery occlusion/reperfusion(MCAO/R), and MCAO/R+TMP(intraperitoneal injection of 20 mg·kg~(-1)) groups. The neurological function was evaluated by the Z-Longa method. The cerebral infarction volume was detected by TTC staining. Enzyme-linked immunosorbent assay(ELISA) was employed to detect the expression of vascular endothelial growth factor(VEGF), angiopoietin(Ang), and platelet-derived growth factor(PDGF). Immunofluorescence staining was employed to detect Ki67 and the expression of vascular endothelial growth factor A(VEGFA) and slient information regulator 1(SIRT1). Western blot was employed to determine the expression levels of VEGFA, SIRT1, angiopoietin-2(Ang-2), and platelet-derived growth factor B(PDGFB). In the cell study, mouse brain-derived endothelial cells(Bend.3) were cultured, and the optimal concentration of TMP was determined. Then, VEGF, Ang, and PDGF were detected by ELISA after the addition of cabozantinib. Western blot was employed to measure the expression of VEGFA, Ang-2, and PDGFB. Immunofluorescence staining was used to detect CD31, CD34, and Ki67, and the proliferation, migration, and tube formation ability of Bend.3 cells were observed in vitro. Western blot and immunofluorescence staining were performed to measure the expression of SIRT1 and VEGFA after addition of the SIRT1-specific inhibitor selisistat(EX-527). The results showed that compared with the sham group, the MCAO/R group had severe neurological function damage, increased infarction volume, up-regulated expression of VEGF, VEGFA, Ang, Ang-2, PDGF, and PDGFB, and down-regulated expression of Ki67 and SIRT1(P<0.01). Compared with the MCAO/R group, the MCAO/R+TMP group presented alleviated neurological function damage, reduced infarction volume, and activated expression of VEGF, VEGFA, Ang, Ang-2, PDGF, PDGFB, Ki67, and SIRT1(P<0.01). The cell experiments showed that compared with the normal group, Bend.3 cells were activated by oxygen glucose deprivation/reoxygenation(OGD/R) treatment(P<0.05, P<0.01). Compared with the OGD/R group, the OGD/R+TMP group upregulated the expression levels of VEGF, VEGFA, Ang, Ang-2, PDGF, PDGFB, SIRT1, Ki67, CD31, and CD34, enhanced the angiogenic ability of Bend.3 cells without being inhibited by BMS or EX-527(P<0.05, P<0.01, P<0.001). The results suggest that TMP can activate the SIRT1/VEGFA signaling pathway to stimulate angiogenesis and alleviate CIS injury.

Hydroxyfasudil regulates immune balance and suppresses inflammatory responses in the treatment of experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a central nervous system (CNS) disease with complicated etiology. Multifocal demyelination and invasion of inflammatory cells are its primary pathological features. Fasudil has been confirmed to improve experimental autoimmune encephalomyelitis (EAE), an animal model of MS. However, Fasudil is accompanied by several shortcomings in the clinical practice. Hydroxyfasudil is a metabolite of Fasudil in the body with better pharmaceutical properties. Therefore, we attempted to study the influence of Hydroxyfasudil upon EAE mice. The results demonstrated that Hydroxyfasudil relieved the symptoms of EAE and the associated pathological damage, reduced the adhesion molecules and chemokines, decreased the invasion of peripheral immune cells. Simultaneously, Hydroxyfasudil modified the rebalance of peripheral T cells. Moreover, Hydroxyfasudil shifted the M1 phenotype to M2 polarization, inhibited inflammatory signaling cascades as well as inflammatory factors, and promoted anti-inflammatory factors in the CNS. In the end, mice in the Hydroxyfasudil group expressed more tight junction proteins, indirectly indicating that the blood-brain barrier (BBB) was protected. Our results indicate that Hydroxyfasudil may be a prospective treatment for MS.

[Role of ubiquitin ligase Ring2 in DNA damage induced by benzo[a]pyrene].

OBJECTIVE: To investigate the role of ubiquitin ligase Ring2 in the DNA damage induced by benzo[a]pyrene (B[a]P). METHODS: The expression of Ring2 in human bronchial epithelial (16HBE) cells was inhibited by small interfering RNA (siRNA) to obtain siRNA-Ring2 16HBE cells. The siRNA-Ring2 16HBE cells, as well as normal 16HBE cells, were exposed to B[a]P (0, 1, 2, 4, 8, 16, and 32 µmol/L) for 24 h; other siRNA-Ring2 16HBE cells and normal 16HBE cells were exposed to B [a]P (16 µmol/L) for 0, 1, 2, 4, 8, 12, and 24 h. The levels of DNA damage were evaluated by alkaline single cell gel electrophoresis assay. RESULTS: After being treated with siRNA for 36 h, the siRNA-Ring2 16HBE cells showed a 72% decrease in Ring2 expression compared with normal 16HBE cells. The analysis of covariance showed that whether to be treated with siRNA and concentration of B[a]P had impacts on Olive tail moment (OTM) (P = 0.032 and P < 0.001); the adjusted mean of OTM was significantly higher in siRNA-Ring2 16HBE cells than in normal 16HBE cells. Whether to be treated with siRNA and B[a]P exposure time had impacts on OTM (P = 0.031 and P < 0.001); the adjusted mean of OTM was significantly higher in siRNA-Ring2 16HBE cells than in normal 16HBE cells. CONCLUSION: The DNA of 16HBE cells with decreased Ring2 expression has increased susceptibility to B[a]P, which may be due to reduced H2A monoubiquitination following decrease in Ring2 expression.

[Clinical significance of protein Z alteration in patients with cardio-cerebral thrombotic diseases].

OBJECTIVE: To study the alteration of protein Z (PZ) in patients with cardio-cerebral thrombotic diseases, its clinical significance and relations with FX. METHODS: PZ and FX:Ag were measured by ELISA, and plasma FX:C by first stage method. In 170 patients with acute ischemic stroke (AIS), 40 acute myocardial infarction (AMI) and 60 healthy adults as contrast, PZ, FX:C and FX:Ag were measured and compared between incipience and recurrence, different ages and genders. RESULTS: In AIS and AMI groups, PZ levels decreased significantly to (940.02 +/- 229.82) microg/L and (1071.44 +/- 180.52) microg/L, respectively \[the contrast group was (2257.97 +/- 479.76) microg/L, P < 0.001\]. But FX:C and FX:Ag raised to (136.73 +/- 34.93)% and (135.54 +/- 54.39)% in AIS group; and to (139.53 +/- 29.18)%, (129.75 +/- 21.91)% in AMI group, respectively, while in the contrast group they were (94.33 +/- 22.00)% and (77.22 +/- 13.19)% (P < 0.001). In the comparative research between the AIS group, AMI group and the contrast group, PZ level was clearly found to negatively relate to the level of FX:C and FX:Ag (P < 0.001). Meanwhile, PZ level, FX:C and FX:Ag in recur-AIS group and recur-AMI group exhibited significant differences (P < 0.05) from those in the primary AIS and AMI groups, suggesting that the decrease of PZ levels reflected the pathological process of the disease. In addition, PZ level gradually decreased with the increase of age (P < 0.05), while FX:C and FX:Ag had no relations with age (P > 0.05). No correlation was found in sex with PZ level, FX:C, FX:Ag (P > 0.05). CONCLUSION: PZ level was significantly decreased in AIS and AMI patients and was negatively related to FX:C and FX:Ag. The mechanism leading to FX increase may partially related with the decreased of PZ. PZ level was different in the primary and recurrent disease and was gradually decreased with the increase of age. Lack of PZ might be a etiological factor of cardio-cerebral thrombotic diseases.