1.Effects of autophagy on viral structures and expression of functional proteins in dorsal root ganglia in a guinea pig model of varicella-zoster virus infection
Xiaojie LAN ; Yang ZHAO ; Shifang WAN ; Zhicheng CAI ; Xingwang WANG ; Huilan YANG
Chinese Journal of Dermatology 2022;55(6):494-500
Objective:To investigate effects of the autophagy inducer rapamycin and autophagy inhibitor 3-methyladenine on viral structures and biosynthesis of functional proteins in dorsal root ganglia in a guinea pig model of varicella-zoster virus (VZV) infection, and to explore their possible mechanisms.Methods:VZV was cultured and proliferated in human embryonic lung fibroblasts (HELFs) , and peripheral blood mononuclear cells (PBMCs) were isolated from guinea pigs. VZV-HELFs and PBMCs were co-cultured for 18-20 hours, and VZV-PBMCs were collected by centrifugation. Thirty-two guinea pigs were randomly and equally divided into 4 groups (8 mice in each group) : blank control group was injected with autologous PBMCs via the medial canthal venous plexus; autophagy inhibition group, autophagy induction group, and VZV infection group were intraperitoneally injected with 3 mg/kg 3-methyladenine solution, 0.5 mg/kg rapamycin solution, and the same volume of 0.9% NaCl solution respectively, followed 2 hours later by injections with 50 μl of VZV-PBMCs via the medial canthal venous plexus. Fourteen days later, the guinea pigs in each group were sacrificed, and dorsal root ganglion tissues were collected. The transmission electron microscope was used to observe the morphology of virus particles, as well as the morphology and number of autophagic vesicles, Western blot analysis was performed to determine the expression of VZV nucleocapsid protein (NCP) , immediate-early protein 62 (IE62) , and autophagy-related proteins Beclin-1 and p62, and immunohistochemical study to determine the expression of anti-VZV antibodies in VZV-infected dorsal root ganglia. Statistical analysis was carried out by using two-independent-sample t test, one-way analysis of variance, least significant difference- t test or Kruskal-Wallis H test. Results:Nucleocapsid-containing virions and scattered autophagosomes were seen in the dorsal root ganglia in the VZV infection group under the transmission electron microscope. The number of autophagic vesicles significantly differed among the blank control group, VZV infection group, autophagy induction group and autophagy inhibition group ( M[ Q1, Q3]: 0, 5[4, 6], 7[5, 9], 0, respectively; H = 135.60, P < 0.01) , and was significantly higher in the VZV infection group than in the blank control group and autophagy inhibition group (both P < 0.05) , as well as in the autophagy induction group than in the autophagy inhibition group ( P<0.05) , but there was no significant difference between the VZV infection group and autophagy induction group ( P>0.05) . Western blot analysis showed that the expression level of IE62 protein was significantly higher in the VZV infection group (1.49 ± 0.06) than in the blank control group (0.50 ± 0.09, t = 9.17, P < 0.05) ; the expression of anti-VZV antibodies was significantly lower in the autophagy inhibition group than in the autophagy induction group and VZV infection group ( t = 9.24, 7.78, respectively, both P < 0.01) , while there was no significant difference between the autophagy induction group and VZV infection group ( P > 0.05) . Conclusion:Autophagy occurred in the dorsal root ganglia of guinea pigs after VZV infection; the inhibition of autophagy could affect the structure of VZV and decrease the expression of VZV functional proteins in the dorsal root ganglia of guinea pigs.
2.Effect of Huanglian Wendantang on Inflammatory Response and Hepatocyte Burn Death in IGT Rats
Han LI ; Yun-lei HUANG ; Wan-ru DONG ; Bo-yan MA
Chinese Journal of Experimental Traditional Medical Formulae 2021;27(8):25-32
Objective:To investigate whether there is inflammatory reaction and cell pyroptosis in impaired glucose tolerance (IGT) rats induced by high-fat diet and the intervention effect of Huanglian Wendantang. Method:Healthy male SD rats were fed with 45% fat content diet for 20 weeks to replicate the IGT model. The rats in line with the model establish criteria were randomly divided into 3 groups, with 10 rats in each group, another 10 rats were selected as the blank control group. Huanglian Wendantang group was given 7.8 g·kg-1·d-1 compound decoction of Huanglian Wendantang, and the positive control group was given 0.05 g·kg-1·d-1 aqueous solution of metformin hydrochloride, with the dose volume of 10 mL·kg-1·d-1. The blank group and the model group were given the same volume of distilled water. After continuous intragastric administration for 4 weeks, the body weight, body length and abdominal circumferences were measured, the Lee's obesity index was calculated, and the levels of fasting plasma glucose (FPG) and 2-hour plasma glucose (2 h PG) were detected in each group. Serum interleukin-6 (IL-6) content was detected by enzyme-linked immunosorbent assay (ELISA). The mRNA and protein expressions of nuclear factor kappa B (NF-