| Objective: TLR7 plays an important role in viral infection.However,its expression,effect,and mechanism in RAW 264.7 macrophages infected by Mycobacterium tuberculosis(Mtb)are still not clear.Here,we evaluate the effect and mechanism of TLR7 on Mtb survival in RAW 264.7 macrophages.Methods: TLR7 m RNA and protein expression in RAW 264.7 macrophages infected by Mtb were detected by RT-PCR and Western blot.Effect of regulation of TLR7 on Mtb: up-regulation of TLR7 experiment grouped as follows: infection control group,starvation(autophagy positive)control group,isoniazid positive control group,and ss RNA group;down-regulation of TLR7 experiment grouped as follows: infection control group,scramble control group,and si RNA group;after treated as above,cells were stained by acid fast staining and the number of Mtb in 100 macrophages was counted.Effect of regulation of TLR7 on cell viability: after treated as above,cell viability was examined by Microplate Reader at 450 nm after incubated with Cell Counting Kit-8(CCK8)for 5 h at 37℃.Cell ultrastructure analysis via transmission electron microscopy(TEM): up-regulation of TLR7 experiment grouped as follows: normal control group,infection control group,starvation(autophagy positive)control group,and ss RNA group;down-regulation of TLR7 experiment grouped as follows: normal control group,infection control group,and si RNA group;cell were digested with trypsin,centrifuged at 2 000 rpm for 15 min,washed with PBS for 3 times,and fixed with 3% glutaraldehyde at 4 ℃ overnight,after dehydration,embedding,sectioning and staining,cells were observed via TEM.After cells were treated as above,LC3(Microtubule-associated protein light chain 3)protein was analyzed by Western blot.Results:(1)PCR showed that TLR7 m RNA expression of macrophages infected by Mtb at 6 h(0.092±0.015),12 h(0.36±0.001)and 24 h(0.371±0.019)was higher than that in normal(0.037±0.001)(P<0.001);TLR7 protein expression of macrophages infected by Mtb at 12 h(0.282±0.018)and 24 h(0.303±0.033)was significantly higher than the normal(0.019±0.002)(P<0.001)detected by Western blot,while 6 h had no difference(0.046±0.001)(P>0.05).(2)Effect of regulation of TLR7 on Mtb: effect of up-regulation of TLR7 on Mtb: Mtb number of ss RNA group(37.67±3.79%)was significantly less than infection control group(127.67±6.66%)(P<0.001)and starvation(autophagy positive)control group(72±8.19%)(P<0.05),but there was no difference with isoniazid positive control group(32.67±3.79%)(P>0.05);effect of down-regulation of TLR7 on Mtb: compared with infection control group(136.33±2.52%),there was no difference of Mtb number in si RNA group(133.00±3.61%)(P>0.05),but had downward trend.(3)Effect of regulation of TLR7 on cell viability: effect of up-regulation of TLR7 on cell viability: the viability of macrophages of ss RNA group increased at 12 h(126.69±5.33%),24 h(117.09±0.10%),and 48 h(143.08±1.16%)compared with the normal(100%)(P<0.001);it was no difference at 12 h(P>0.05),increased significantly at 24 h(P<0.05)and 48 h(P≤0.001)compared with infection control group(12 h,124.19±2.50%;24 h,112.42±1.07%;48 h,135.01±0.71%);it increased at 12 h(P<0.05),24 h and 48 h(P<0.001)compared with starvation(autophagy positive)control group(12 h,112.643±3.774%;24 h,93.713±0.709%;48 h,58.61±0.636%);while there was no difference at 12 h(P>0.05),and it reduced significantly at 24 h and 48 h(P<0.001)compared with isoniazid positive control group(12 h,121.95±4.137%;24 h,125.3±0.708%;48 h,158.26±0.565%);effect of down-regulation of TLR7 on cell viability: si RNA induced obvious decrease of cell viability at 12 h(65.23±2.46%),24 h(58.35±0.21%),and 48 h(21.74±0.17%)compared with the normal(100%)or the infection(12 h,124.19±2.50%;24 h,112.42±1.07%;48 h,135.01±0.71%)(P<0.001).(4)Cell ultrastructure analysis via TEM: up-regulation of TLR7: normal cells mitochondria were clear,1-2 autophagosomes were found in cytoplasm;mitochondria of infected cell were not clear,and nuclear chromatin was pyknotic and edge setted;in cells treated by starvation,mitochondrial matrix was deeply stained,and several autophagosomes were found in cytoplasm;however,mitochondria were relatively clear and autophagosomes significantly increased in cells treated with ss RNA;down-regulation of TLR7: none autophagosome was found in cells treated with si RNA,besides,rough endoplasmic reticulum was dilated,and nuclear week gap was widened.(6)TLR7 induced the formation of LC3-II: ss RNA significantly induced LC3-II formation(3.56±0.091)compared with infection control group(0.692±0.04)(P<0.001),however,it reduced obviously induced by si RNA(0.946±0.055)compared with infection control group(2.400±0.029)(P<0.001).Conclusions:(1)TLR7 expression in RAW 264.7 macrophages infected by Mtb increased and in a time-dependent manner.(2)TLR7 could affect the survival of Mtb in macrophages through autophagy.(3)TLR7 could be used as a therapeutic target for TB. |
PDF Full Text Request