Effect of Yuehua Capsule on Autophagy of Macrophage Infected with Multi-drug Resistant Mycobacterium Tuberculosis / 中国实验方剂学杂志

Objective::This study aims to explore the effect and mechanism of Yuehua capsule serum for autophagy of macrophages infected with multi-drug resistant mycobacterium tuberculosis. Method::The rats were undertaken intragastric gavage with Yuehua capsule by 3.02 g·kg-1 once a day which was produced through low temperature condensation drying method. After 7 days, blood of abdominal aorta of rats was collected to prepare Yuehua capsule serum. RAW264.7 andmultidrug resistant tuberculosis were cultured in vitro.According to cell counting kit-8(CCK-8), 10% drug-containing serum was considered as the effective concentration. The cultured cells were divided into four groups: model groups(10% fetal bovine serum). Yuehua capsule serum(10% Yuehua capsule serum). Autophagy inhibitor group+ 3-MA+ Yuehua capsule medicated serum(3-MA+ 10% Yuehua capsule serum). Rapamycin (Rap) positive control group(200 mg·L-1 Rap+ 10% Yuehua capsule serum). Except for the normal group, the cells of each group were cultured for 24 h and infected for 4 h according to cell-bacteria 1∶10.Testing index: observation of autophagosomes under transmission electron microscope, the test of expression of microtubule-associated protein light chain-3Ⅱ(LC-3Ⅱ), microtubule-associated protein LC 3-Ⅱ/microtubule-associated protein light chain 3-Ⅰ(LC3-Ⅰ) and Beclin-1 with Western blot, indirect immunofluorescence staining for LC3B, and mRNA of Beclin-1 as well as LC3 with real-time fluorescent quantitative polymerase chain reaction(Real-time PCR). Result::Compared with normal group, model group did not see autophagy body cells, cells in the LC-3 Ⅱ, LC-3 Ⅱ/LC-3 Ⅰ, Beclin-1 protein and LC3, Beclin-1 mRNA gene expression level had no significant change, the cells without fluorescent particles, spots, no fluorescence intensity.Compared with model group, Yuehua capsules serum group and Rap positive control group can be observed the formation of phage, mRNA andprotein expression levelof LC-3 Ⅱ, LC-3 Ⅱ/LC-3 Ⅰ, Beclin-1 and LC3, Beclin-1 were significantly increased (P<0.05). Autophagy inhibitor group+ 3-MA+ Yuehua capsule medicated serum did not see autophagy, the mRNA and protein expression level of LC-3 Ⅱ, LC-3Ⅱ/LC-3Ⅰ, Beclin-1 and LC3, Beclin-1 were no significantly increased. Conclusion::Yuehua capsule medicated serum could induce autophagy of macrophages of RAW264.7.The mechanism was probably accomplished through regulating the expression level of autophagy key protein LC3, autophagosome mature protein Beclin-1 and relevant gene, meanwhile the conversion of LC3-I to LC3-Ⅱ was accelerated.

Evaluation of the Performance of GenoType(R) MTBDRplus Assay for Rapid Detection of Multi-drug Resistant Mycobacterium tuberculosis in Sputum Specimens

BACKGROUND: GenoType(R) MTBDRplus assay (Hain Lifescience, Germany) enables detection of the mutations prevalent in rpoB, katG, and inhA genes and identification of Mycobacterium tuberculosis complex (MTB). We evaluated the performance of the MTBDRplus assay in detecting multidrug resistant M. tuberculosis in sputum specimens by directly comparing it to the performance of conventional drug susceptibility testing (DST) with M. tuberculosis culture isolates. METHODS: From December 2007 to July 2008, 40 patients with acid-fast bacilli (AFB) smear-positive and AFB culture-positive sputa, including 19 patients with rifampin (RIF)- or isoniazid (INH)-resistant MTB isolates, were enrolled. The MTBDRplus assay was performed using DNA extracted from respiratory specimens. DST of the culture isolates was performed using an absolute concentration method. RESULTS: The result of the AFB smear test was +/-1 for 7 specimens, +1 for 8 specimens, +2 for 9 specimens, +3 for 9 specimens, and +4 for 7 specimens. The MTBDRplus assay revealed that 37 of the 40 specimens were positive for an MTB-specific band, 12 specimens were RIF-resistant, and 16 specimens were INH-resistant. The rpoB S531L mutation was detected in 58.3% of the RIF-resistant specimens, and the katG S315T1 and inhA C15T mutations were detected in 56.3% and 31.3% of the INH-resistant specimens, respectively. Compared to the sensitivity and specificity of DST, both sensitivity and specificity of MTBDRplus assay for RIF resistance were 100%, and the corresponding values for INH resistance were 82.4% and 90.0%. Discrepant MTBDRplus assay and DST results were obtained in 3 INH-resistant isolates without mutation and 2 INH-susceptible isolates with katG S315T1 and inhA C15T mutations. CONCLUSIONS: The MTBDRplus assay can be applied for AFB smear-positive specimens with positivity +/- to 4+. The assay was reliable for predicting the RIF resistance of culture isolates, but DST was required for confirming INH resistance.

Phagocytosis of Drug-Resistant Mycobacterium Tuberculosis by Peripheral Blood Monocytes / 결핵

BACKGROUND: Phagocytosis is probably the first step for mycobacteria to be virulent in host because virulent strains are more readily phagocytosed by macrophage than attenuated strains. According 13 the traditional concept, multi-drug resistant strains have been regarded as less virulent. However, this concept has been challenged, since recent studies(reported) showed that the degree of virulence and drug-resistance is not related. The purpose of this study is to evaluate whether the phagocytic activity of M. tuberculosis by peripheral blood mononuclear cells(PBMC) is different according to drug-resistance or host factor. To evaluate this, we estimated the difference of phagocytic activity of drug-resistant and drug-sensitive M. tuberculosis and also estimated the phagocytic activity of PBMC from intractable tuberculosis patients and healthy controls. METHODS: PBMC from ten intractable tuberculosis patients and twelve healthy control and three different strains of heat-killed M. tuberculosis, ie, ADS(all drug sensitive), MDR(multi-drug resistant), and ADR(all drug resistant) were used. After incubation of various strains of M. tuberculosis with PBMC, the phagocytic activity was evaluated by estimating proportion of PBMC which have phagocytosed M. tuberculosis. RESULTS: Drug-resistant strains of M. tuberculosis were phagocylosed easily than drug sensitive strains(Percentage of PBMC phagocytosed M. tuberculosis in healthy control : ADS : 32.3α2.9%, ADR : 49.6α3.4%, p=0.0022, Percentage of PBMC phagocytosed M. tuberculosis in intractable tuberculosis patients : ADS : 34.9α3.6%, ADR : 50.7α4.5%), p=0.0069). However, there was no difference in phagocytic activity of PBMC from healthy control and intractable tuberculosis patients. CONCLUSION: Drug-resistant strains of M. tuberculosis were phagocytosed easily than drug sensitive strains and host factors does not seems to influence the phagocytosis of M. tuberculosis.