| Brucellosis is a common zoonotic diseases by the Brucella bacteria,and that infection annually increasing in recent years, developing a serious disease, and the incidence has been extended to some developed countries, Brucella disease has caused serious impediment to the development of China’s animal husbandry as well as import and export trade in China.And this is a serious threat for human public health,and brucellosis currently spread rapidly, weak control, mainly in the Mediterranean countries, Mongolia, China, India and other countries.Now some effectiveness against brucellosis vaccine is weak, not enough to completely control brucellosis. Brucella is a gram-negative bacteria, belonging to facultative intracellular parasites, strong invasion, spread wide, and can cause heat waves, arthritis and other symptoms of human infection with Brucella; easily lead to abortion of females when infected with the disease, sire was infected with the disease orchitis. Brucella have their own unique biological activity, has a unique biological function, and exist some relation between the host immune,and have complex regulatory networks signal. However, the understanding of the Brucella infection of host cells of the immune system as well as the specific mechanisms of intracellular survival People is not very clear So understanding of the Brucella how to weaken immunity and pathogenic mechanisms of the host cell is important, we will look for new brucellosis control strategy to provide new direction and theoretical basis. So we carried out the following research work for cattle Brucella: Objective: This study is based on the smooth Brucella, 2308 rough Brucella RB51 as the research object, To explore NF kappa B and JAK2 / STAT3 signaling pathway in the pathogenesis of Brucella and intracellular survival mechanism in the infection of Brucella virulent and avirulent strains.(1) Brucella 2308 smooth, rough type Brucella abortus RB51 infect macrophages to determine NF kappa B and JAK2 / STAT3 signaling pathway activation.(2) analysis of different times and different infection MOI effect on NF-κB, JAK2 / STAT3 signaling pathway activation(3) after add inhibitor to analyse and compare with the differences of smooth Brucella 2308,dan rough Brucella abortus RB51 stimulated cytokine expression.(4) after add inhibitor to analyse the different of slip type 2308 inhibitor after Brucella, rough Brucella RB51 survive in intracellular.(5) compared with the after add inhibitors the effect on cell apoptosis by effect on Brucella slide 2308, the effects of rough Brucella RB51. Methods: 1(1) 2308 smooth Brucella and rough Brucella RB51 with multiplicity of infection 100, after infected macrophages 4 h, 8 h, 24 h and 48 h hours abandoning the culture medium, cell supernatant protein was added into cell and absorb supernatant protein, then western Blot detection.This method was developed for the simultaneous detection in different time of infection and the infection of different complex(MOI) By brucella 2308 and RB51 infect macrophages on NF kappa B signaling pathway activation.(2) firstly, it was observed that the inhibitor BAY11-7082 whether had toxic to effect the cells by means of the blue staining. Incubation of macrophages with different concentrations of inhibitorsUse ELISA kit to detect cytokines TNF alpha and IL-1 beta and IL-6 expression, At the same time, add signal pathway inhibitor BAY11-7082 for CFU counts and find influence on Brucella virulent and avirulent strains in survival of the intracellular.(3) after 1h incubation by inhibitors, rough Brucella infect macrophages, flow cytometry. Flow cytometry was used to detect apoptosis.2.(1) 2308 smooth Brucella and rough Brucella RB51 with multiplicity of infection 100, after infected macrophages 4 h, 8 h, 24 h and 48 h hours abandoning the culture medium, cell supernatant protein was added into cell and absorb supernatant protein, then western Blot detection.This method was developed for the simultaneous detection in different time of infection and the infection of different complex(MOI) By brucella 2308 and RB51 infect macrophages on JAK2/STAT3 signaling pathway activation.3. Using the purified Brucella LPS and Brucella lps-o chain deletion strains to infect macrophages, and then were detected the activity of NF kappa B and JAK2 / STAT3 signal pathway. Results: 1.(1) the rough Brucella abortus can strong activation of NF kappa B signaling pathway, smooth Brucella 2308 is very weak even does not activate NF kappa B signaling pathway.Rough type Brucella abortus RB51 infect macrophages after 8 hours, the NF kappa B signaling pathway activity was the strongest, the multiplicity of infection of Brucella effect on NF kappa B signaling pathway activation which relate to concentration dependent manner.RB51 and 2308 were the strongest in the activation of NF- kappa B at 80 h in the infection time of 8 h.(2) the inhibitor BAY11-7082 was observed to have no side effects on the cells. The production of cytokines can be inhibited by the addition of inhibitors, And the inhibitor in a concentration dependent manner; after inhibitor treating macrophages,bacteria have increased significantly, significantly higher than the number of the untreated group.(3)The apoptosis rate of cells decreased from 8.22% to 7.51%, small drop.2.(1) Brucella virulent and avirulent strains can activate NF- kappa B and JAK2/STAT3 signaling pathway. In the infection time was 8h and MOI 80 strongly activated, and had the concentration dependence of infection.(2) the inhibitor AG 490 had no side effects on the cells by the blue staining. And after adding inhibitor, smooth Brucella 2308, rough type Brucella RB51 can affect TNF- alpha, IL-6. And does not affect the expression of IL-1β.(3) and after adding inhibitor, apoptosis decreased from 27.76 to 19.34% smooth type is not chang.3. With Brucella abortus virulent and avirulent strains of purified LPS infected macrophages, the activity of NF kappa B and JAK2 / STAT3 signal pathway without significant difference; when using Brucella M5 and purification of M5- Delta wboa LPS infect macrophage,it does not activated NF kappa B and JAK2/ STAT3 signaling pathway. Conclusion: Brucella virulent and avirulent strains can activation of NF kappa B and JAK2 / STAT3 signal pathway at different degrees, so can affect Brucella intracellular survival and apoptosis. But The Brucella LPS activate NF-κB and JAK2/STAT3 signaling pathway which it not havelarge effection. |
