Study Of The Anti-inflammatory Properties And Mechanism Of Action Of Corilagin In HSV-1 Induced Encephalitis

Aims:1.Assessment of regulation and expression of cytokines IL-1β,IL-10,TNF-αand NO in HSV-1 infected microglia 2. Effect of Corilagin on microglial production of cytokines Methods:Microglial cells were divided in 5 groups namely,1. control group,2.HSV-1 infected group,3.HSV-1 infected+ Coraligin (20ng/ml)group,4. HSV-1 infected+ Dexamethasone (0.5ug/ml)group,5.HSV-1 infected+APS(100ug/ml) group. ELISA was used to detect IL-1β,IL-10,TNF-αexpression and Griess reagent NO to detect NO.Results:HSV-1 infected group showed increase in expression of all cytokines named above.HSV-1 infected group with Coraligin showed significant decrease in IL-1β,TNF-αand NO expressions(P<0.01).while IL-10 increase was insignificant. Conclusions:Coraligin has inhibitory effects on microglial chemokines production following HSV-1 central nervous infectionAims:To study the effects of Corilagin on the apoptosis of HSV-1 stimulated BV2 cells and HSV-1 infected PC 12 cells. Methods:PC 12 and BV2 cells were divided into 5 groups namely,1.Normal control group (non-HSV-1 infected),2.HSV-1 infected group 3.HSV-1 infected+ Corilagin (20ng/ml))group 4. HSV-1 infected+ APS(100ug/ml) group.5. HSV-1 infected+ Dexamethasone(0.5ug/ml) group. Apoptotic rate of these BV2 and PC12 cells were determined at 12h,24h,48h intervals. Flow cytometry assay for examining apoptosis rate. Results:1. Compared to the control group, the HSV-infected group showed significant BV2 cells apoptosis(P<0.05).When the same HSV-infected group was compared to the Corilagin group and Dexamethasone group, the latter 2 groups displayed even more significant BV2 apoptosis(P<0.05).Nevertheless, comparison between APS group and HSV-infected group showed little difference of BV2 cells apoptosis; hence, this group served as a negative control in this experiment.All results were uniformly present during each of the 12h,24h and 48 h cells viability testing. 2. Following infection with HSV-1, PC12 cells death in the HSV-1 infected group was significant as compared to the control group(P<0.05).However, PC12 cells death was significantly reduced in the Corilagin group and Dexamethasone group (P<0.05)as compared to the HSV-1 infected group. These results were present at the 24h and 48h cells count intervals.At the 12h cells count interval no significant difference in the apoptotic ratee of the PC12 cells was noted between all the treated groups and the HSV-1-infected group.Conclusions:Corilagin can induce apoptosis of HSV-1 stimulated microglial BV2 cells and at the same time can inhibit HSV-1 induced PC12 cells apoptosisAims:To investigate the apoptotic pathway involved in Hsv-1 stimulated microglias following corilagin intervention. Methods:Model of HSV-1 stimulated BV2 cells was set-up.These BV2 cells were then divided into the following groups:1.Normal control group,2.HSV-1 infected group,3.HSV-1 infected+ Corilagin (20ng/ml)group,4. HSV-1 infected+ APS (100ug/ml)) group,5.HSV-1 infected+Dexamethasone(0.5ug/ml) group.A control group consisting of normal BV2 cells was also included. After a time lapse of 24h, cysteine proteases:caspase-3,caspase-8,caspase-9 and caspase-12 activities were analysed.using Flow cytometry assay., cytochrome c level was also determined by western-blot. Results:1. In the HSV-1 infected group, caspase-3 expression was markedly increase as compared to the control group (P<0.05).In the Corilagin group and Dexamethasone group expression of caspase-3 was more than the HSV-1 infected group(P<0.05).Acting as a negative control caspase 3 expression in the APS group was decreased.2. Caspase-8 expression between the control group and HSV-1 infected group was insignificant(P>0.05)In the Corilagin group and Dexamethasone group, expression of caspase-8 was significantly increased as compared to that of the HSV-1 infected group(P<0.05). Acting as a negative control caspase 8 expression in the APS group was again decreased.3. In the HSV-1 infected group caspase-9 expression was markedly increase as compared to the control group (P<0.05).By contrast, in the Corilagin group, Dexamethasone group and APS group expression of caspase-9 was increased as compared to that of the HSV-1 infected group(P<0.05).In each group, the level of cytochrome c was consistent with its respective caspase-9 expression level.4. Caspase-12 expression between the HSV-1 infected group and control group did not show any significant increase. Caspase-12 expression between the HSV-1 infected group and Dexamethasone was also insignificant. By contrast, in the Corilagin group, expression of caspase-12 was increased as compared to that of the HSV-1 infected group(P<0.05). Furthemore, in the APS group, expression of caspase-12 was also increased as compared to that of the HSV-1 infected group(P<0.05).Conclusions:Alone HSV-1 stimulated microglias apoptosis follows the cytc and caspase-9 initiated mitochondrial pathway.However,following Corilagin intervention, all the 3 main apoptotic pathways(mitochondrial,death receptor and endoplasmic) are involved. Aim:To provide laboratory evidence for possible clinical use of Coraligin through its application on HSV-1 infected murine brain. Methods:Murine model of HSV-1 encephalitis was set up following injection of HSV-1 samples in Balb/c mice brains.These subjects were then randomly divided in the following groups according to the various additional treatments given:1.virus group 2.infected+ Corilagin(0.4mg/per mouse) group 3. infected+ Dexamethasone(2ug/per mouse) group 4. infected+ APS (0.8mg/per mouse) group. A fifth group of uninfected mice acting as control was also included. Following sacrifice of a number of mice in each group at day 4 of experiment, ELISA method was used to detect TNF-a, NO level in the mice brains. HE stain was also used to study brain samples. Brain cells necrosis was observed using TUNEL method. Results:1.Of those sacrificed animals on day 4, levels of TNF-a in the infected group, Corilagin group, Dexamethasone group, APS group were greater when compared to the control group(P<0.01);when Corilagin group,Dexamethasone group were compared to the infected group, TNF-a levels were lower(P<0.01,P<0.05)2. Levels of NO in the infected group, Corilagin group Dexamethasone group,APS group were also greater when compared to the control group(P<0.01);when the Corilagin group, Dexamethasone group,and APS group were compared to the infected group,NO levels were lower(P<0.01,P<0.05)3.In all the intervened groups(Corilagin,APS,Dexamethasone), those sacrificed pathological changes in the brain tissues were minimum. When compared to the infected group, these intervened groups TUNEL positive cells were also lesser.Conclusions:Corilagin can effectively prevent HSV-1 induced brain damage and hence improve prognosis of HSV-1 encephalitis in this study of murine model.