ObjectiveTo investigate the effects of Astragali Radix polysaccharides （APS） on the polarization of BV2 microglial cells in an oxygen-glucose deprivation/reoxygenation （OGD/R） model through regulation of the Toll-like receptor 4 （TLR4）/nuclear factor-κB （NF-κB） signaling pathway. MethodsThe OGD/R injury model of BV2 microglia was established and divided into blank group， OGD/R group and APS group （0.4 g·L^-1 APS）. Neuroinflammatory injury was induced by lipopolysaccharide （LPS） and treated with APS. The cells were divided into blank group， LPS group （1 mg·L^-1 LPS） and APS group （0.4 g·L^-1 APS+1 mg·L^-1 LPS）. Cell viability was detected using the cell counting kit-8 （CCK-8） assay. Cell morphology was observed under an inverted microscope. Nitric oxide （NO） content in the cell supernatant was determined by the Griess assay. The secretion levels of tumor necrosis factor-α （TNF-α）， interleukin （IL）-6， IL-10， and IL-4 were measured by enzyme-linked immunosorbent assay （ELISA）. Immunofluorescence （IF） was used to detect the double-positive rates of ionized calcium-binding adapter molecule-1/inducible nitric oxide synthase （Iba-1⁺/iNOS⁺） and ionized calcium-binding adapter molecule-1/arginase 1 （Iba-1⁺/Arg1⁺）， as well as the nuclear translocation rate of nuclear factor-κB p65 （NF-κB p65）. Protein expression levels of Iba-1， iNOS， Arg1， TLR4， and NF-κB p65 were detected by Western blot. ResultsIn the OGD/R injury model， compared with the blank control group， BV2 microglial cells in the OGD/R group were activated and exhibited amoeboid morphological changes. The secretion levels of NO， TNF-α， and IL-6 were significantly increased （P<0.01）. The double-positive expression rate of Iba-1⁺/iNOS⁺ and the protein expression of Iba-1 and iNOS were significantly increased （P<0.01）. The nuclear translocation rate of NF-κB p65 and the protein expression levels of TLR4 and NF-κB p65 were significantly increased （P<0.01）. The levels of IL-10 and IL-4 were significantly decreased （P<0.01）， and the double-positive expression rate of Iba-1⁺/Arg1⁺ and Arg1 protein expression were significantly decreased （P<0.01）. Compared with the OGD/R group， the APS group （0.4 g·L^-1） showed reduced cell activation， significantly decreased secretion levels of NO， TNF-α， and IL-6 （P<0.01）， significantly decreased double-positive expression rate of Iba-1⁺/iNOS⁺ and relative protein expression of Iba-1 and iNOS （P<0.01）， significantly decreased nuclear translocation rate of NF-κB p65 and protein expression levels of TLR4 and NF-κB p65 （P<0.01）， significantly increased levels of IL-10 and IL-4 （P<0.01）， and significantly increased double-positive expression rate of Iba-1⁺/Arg1⁺ and Arg1 protein expression （P<0.01）. In the LPS-induced neuroinflammation model， compared with the blank control group， the LPS group showed increased cell activation， significantly increased levels of NO， TNF-α， and IL-6， significantly increased Iba-1⁺/iNOS⁺ double-positive expression rate， NF-κB p65 nuclear translocation rate， and protein expression levels of Iba-1， iNOS， TLR4， and NF-κB p65 （P<0.01）， while IL-10 and IL-4 levels， Iba-1⁺/Arg1⁺ double-positive expression rate， and Arg1 protein expression were significantly decreased （P<0.01）. Compared with the LPS group， the APS group showed reduced cell activation， significantly decreased levels of NO， TNF-α， and IL-6， Iba-1⁺/iNOS⁺ double-positive expression rate， NF-κB p65 nuclear translocation rate， and protein expression levels of Iba-1， iNOS， TLR4， and NF-κB p65 （P<0.01）， while IL-10 and IL-4 levels， Iba-1⁺/Arg1⁺ double-positive expression rate， and Arg1 protein expression were significantly increased （P<0.01）. ConclusionAPS may reduce microglial activation and promote their polarization toward the M2 phenotype by inhibiting activation of the TLR4/NF-κB signaling pathway， thereby alleviating the neuroinflammatory response induced by OGD/R.

OBJECTIVE: To construct a recombinant lentiviral expression vector pCDH-Daxx-EGFP to investigate the effect of Daxx on the proliferation of vascular smooth muscle cells (VSMCs). METHODS: The recombinant lentiviral expression vector pCDHDaxx-EGFP was constructed using PCR-based accurate synthesis method. After identification by sequencing and enzyme digestion, the recombinant lentiviral vector was contransfected into 293T cells with lentivirus packaging vector. The recombinant lentivirus particles were collected and purified to infect VSMCs, whose expression of Daxx was detected with Western boltting. The cells infected with the empty vector pCDH-EGFP or pCDH-Daxx-EGFP were incubated in serum-free medium or in the presence of angiotensin Ⅱ (AngⅡ). The cell viability was determined with MTT assay, and the cell cycle changes were analyzed with flow cytometry. The cell migration ability was assessed using a scratch wound healing assay. The expression of p-Akt protein in the cells was detected using Western blotting. RESULTS: Double enzyme digestion and sequencing confirmed successful construction of the recombinant plasmid. Compared with the cells infected with the empty vector, the cells infected with pCDH-Daxx-EGFP exhibited significantly increased expressions of Daxx protein ( < 0.05). AngⅡ treatment of the cells infected with the pCDH-Daxx-EGFP, as compared with the cells infected with the empty vector, significantly lowered the cell viability, S phase cell ratio and cell migration ability ( < 0.05), and significantly decreased the expression level of p-Akt protein ( < 0.05). CONCLUSIONS We successfully constructed the recombinant lentiviral vector pCDH-Daxx-EGFP and overexpressed Daxx in primary cultured VSMCs using this vector. Daxx overexpression can inhibit AngⅡ-induced proliferation and migration in VSMCs probably by regulating p-Akt protein.

Objective: To study the efficacy of 1 mg·kg^-1·d^-1 and 2.0 mg·kg^-1·d^-1 of propranolol in the treatment of infantile hemangiomas, so as to provide an ideal dosage for clinical treatment. Methods: From September 2015 to October 2016, there were 89 patients in accordance with the inclusion criteria of infantile hemangiomas. According to randomized and controlled principle, the patients were assigned to receive two propranolol regimens, Group A(n=45): propranolol at a dose of 1 mg·kg^-1·d^-1; Group B(n=44): propranolol at a dose of 2 mg·kg^-1·d^-1. 1 or 2 mg of propranolol base per kilogram per day, divided into two doses.The first dose was taken at 9, next at 15.The effective rate, cure rate and adverse effect rate were compared at the six months after treatment. The duration of the treatment was compared after 12 months′ treatment, and the recurrence rate was compared at the three months after being cured.The data were statistically processed with SPSS 22.0. The sample rate was compared with χ² test, and the data were compared with t test. P<0.05 was statistically significant difference. Results: Group A: Treatment for 6 months, 45 children were evaluated as follows: the effective rate was 91.1%(41/45), the cure rate was 60%(27/45). The incidence of adverse reactions was 13.3%(6/45). The cure rate of group A at 12 months was 86.7%(39/45). The duration of treatment was 7.6±2.7 months.The recurrence rate was 9.5%(4/42). Group B: Treatment for 6 months, 44 children were evaluated as follows: the effective rate was 90.9%(40/44), the cure rate was 72.7%(32/44). The incidence of adverse reactions was 15.9%(7/44). The cure rate of group B at 12 months was 88.6%(39/44). The duration of treatment was 6.2±1.9 months. The recurrence rate was 11.9%(5/42). The effective rate of the two groups at 6 months was not statistically significant(χ²=2.583, P=0.461). The cure rate of the two groups at 6 months was statistically significant(χ²=8.339, P=0.004). The incidence of adverse effects was not statistically significant(χ²=0.118, P=0.731). The cure rate of the two groups at 12 month was not statistically significant(χ²=0.080, P=0.778). The duration of treatment between the groups was statistically significant (t=0.290, P=0.009). The recurrence rate of the two groups after 3 months withdrawal was no statistical difference(χ²=0.124, P=0.724). Conclusions The propranolol doses at 1 mg·kg^-1·d^-1 and 2 mg·kg^-1·d^-1 in the treatment of infantile hemangiomas are safe and effective. The propranolol dose at 1 mg·kg^-1·d^-1 doesn′t decrease the effective and cure rate, not increase the recurrence rate in infantile hemangiomas. Low dose at 1 mg·kg^-1·d^-1 can be used as a common dose of propranolol in infantile hemangiomas.