Study Of Effect Of Danba-Ⅰ On Fever Inhibition And Anti-inflammatory In Mice Infected Tick-borne Encephalitis Virus

Objective:basing on the establishment of mice infected Tick-born Encephalitis virus(TBEV)model,this paper analyzes the effects that Mongolian medicine Danba-Ⅰ have on the temperature of mice which infected Tick-born Encephalitis(TBE),on neuroethology,on brain tissue morphology and on brain tissue ultrastructure;and it also gives a further discussion on the regulating effect,which produces by Mongolian medicine Danba-Ⅰ,on TLR3/TRIF signaling pathways of the brain tissue of mice which infected TBE,and a further discussion on the fever inhibition and anti-inflammatory mechanism.Method:144 male BalB/C mice were randomized into 6 groups:blank group,model group,Danba-Ⅰ high dose group,middle dose group,low dose group and dexamethasone group.Each group was separately assigned into 2 subgroups basing on 4 days and 7 days.Set up an infectious animal model by virus intracranial inoculation,12 hours later,gavaged the 3 different doses Danba-Ⅰ groups by using double of an adult dose Danba-Ⅰ,equal to an adult dose Danba-Ⅰ and half of an adult dose Danba-Ⅰ;diluted dexamethasone injection by glucose solution,then carried out intraperitoneal injection to the mice in dexamethasone group;gavaged the blank group and model group by distilled water with the dose equal to an adult dose of Danba-Ⅰ.Observed the general condition and recorded the temperature of mice after gavaging;assessed the neural functional injury of the lab mice according to neurological sign scoring method;observed the brain tissues pathomorphism of lab mice by HE staining method;observed the lab mice brain tissues ultrastructure electron microscopy;measured the content of TLR-3,IL-1β,IL-6 and TNF-α in brain tissues and serum by enzyme linked immune sorbent assay(ELISA);detected brain tissues TLR-3,TRIF and NF-κB protein by Western-blot method;detected the expression of brain tissues TLR-3,TRIF and NF-κB mRNA in the method of Real-time PCR.Result:a)General state:comparing with the model group,the Danba-Ⅰ high and middle dose can significantly improve the general state of mice which infected TBE.b)Temperature:the temperature of mice in model group began to increase on the 3rd day after model established,and peaked on the 4th day,then kept in a high level afterwards;the peak and point-in-time average temperature of mice in the Danba-Ⅰ high and middle dose groups were all lower than the model group(P<0.05).c)Neuroethology:various neural functional injuries was observed after the 4th day in model group and had become the most obviously in the 7th day;the neural functional injuries got a clear alleviation(P<0.05)in the Danba-Ⅰ high and middle dose groups comparing with model group at different time points.d)The result of HE staining method:there was a clear pathomorphism change of the mice brain tissues in model group,a large number of inflammatory cell infiltration and gliocyte proliferation could be observed,and the brain tissues structural damage was very obvious;the Danba-Ⅰ high and middle dose could effectively improve the pathomorphism change of the mice brain tissues which infected TBE.e)The result of electron microscopy method:the neurons organelle of mice brain tissues were damaged clearly in model group,some neurons were even necrotic;the ultrastructure change of the mice brain tissues in Danba-Ⅰ high and middle dose groups was alleviated comparing to model group.f)The result of ELISA method:there were a few TLR-3,IL-1β,IL-6 and TNF-α expressions in the brain tissue and serum of blank group;it is meaningful on statistics comparing the difference between model group and blank group at different time points(P<0.05);the TLR-3,IL-1β,IL-6 and TNF-α expressions have reduced obviously(P<0.05)comparing Danba-Ⅰ high and middle groups with model group at different time points.A correlation analysis indicates that the TLR-3 expression has a high positive correction to IL-1 β,IL-6 and TNF-α expression.g)The result of Real-time PCR method:the brain tissues had TLR-3,TRIF,NF-κB and mRNA expressions at different time points in each group.There were TLR-3,TRIF,NF-κB and a few mRNA expressions in blank group;the ad TLR-3,TRIF,NF-κB and mRNA had clearly high expressions in model group,and there was a statistic difference(P<0.05)at different time points comparing with blank group;the mice brain tissues TLR-3,TRIF,NF-κB and mRNA expressions reduced(P<0.05)in Danba-Ⅰ high and middle dose groups comparing to model group.h)The result of Western blot method:there were TLR-3,TRIF and NF-κB protein expressions at different time points of each group.TLR-3,TRIF and NF-κB protein weak positive staining could be observed in blank group;TLR-3,TRIF and NF-κB protein expressions increased clearly in model group and there was a statistic difference(P<0.05)at different time points comparing with blank group;the TLR-3,TRIF and NF-κB protein expressions at different time points could be reduced in Danba-Ⅰ high and middle dose groups,and it is meaningful on statistics(P<0.05).Conclusion:a)A high and middle dose Danba-Ⅰ can improve the general state of TBE-infected mice,reduce the increasing level of temperature,improve the nerve function and alleviate the change of brain tissue pathology and ultrastructure.b)A high and middle dose Danba-Ⅰ can reduce TLR-E and the content of inflammatory factor,such as IL-β,IL-6 and TNF-α in brain tissues and serum,it can also alleviate the inflammatory injury in immune-mediated acute phase of TBE-infected mice.c)By regulating and controlling the TLR-3,TRIF and NF-κB gene and protein expressions in TLR3/TRIF signaling pathways,the Danba-Ⅰ plays a role in protecting brains.