circRNA_SLC8A1 promotes the survival of mycobacterium tuberculosis in macrophages by upregulating expression of autophagy-related protein SQSTM1/p62 to activate the NF-κB pathway.

Macrophages act as the first immune defense line of the host against Mycobacterium tuberculosis (Mtb). A previous study showed that circRNA_SLC8A1 was significantly upregulated in Mtb-infected macrophages, but its regulatory mechanism in anti-tuberculosis infection is unclear. Therefore, this study aimed to investigate the role of circRNA_SLC8A1 in the anti-tuberculosis activity of macrophages. We showed that circRNA_SLC8A1 was upregulated in tuberculosis patients. Moreover, the binding sites of miR-20b-5p on circRNA_SLC8A1 and Sequestosome 1 (SQSTM1/p62) mRNA were predicted by StarBase and verified by the double luciferase reporter gene assay. Next, we found that miR-20b-5p expression was decreased, while SQSTM1 protein expression was increased in a time- and dose-dependent manner in the human macrophage U937 in response to Mtb infection. Furthermore, circRNA_SLC8A1 overexpression vector (circRNA_SLC8A1) or shRNA (sh-circRNA_SLC8A1) and/or miR-20b-5p mimic or inhibitor and/or SQSTM1 overexpression vector (SQSTM1) or small interfering RNA (si-SQSTM1) or its corresponding control were transfected into Mtb-infected macrophages. Results showed that overexpression of circRNA_SLC8A1 or miR-20b-5p inhibitor promoted the secretion of pro-inflammatory factors IL-1ß, IL-6, and TNF-α, increased Nitric Oxide (NO) content and inducible nitric oxide synthase (iNOS) expression, inhibited Reactive oxygen species (ROS) production. Cleaved-caspase-3 protein expression, and cell apoptosis, and promoted Mtb survival. Silencing SQSTM1 inhibited secretion of pro-inflammatory factors and activation of the NF-κB pathway. Overexpression of miR-20b-5p blocked the promoting of circ-SLC8A1 on SQSTM1 protein expression. In summary, circRNA_SLC8A1 sponged miR-20b-5p to upregulate SQSTM1/p62 expression and promoted Mtb survival in macrophages through the NF-κB signaling pathway.

MicroRNA-20a-3p regulates the host immune response to facilitate the mycobacterium tuberculosis infection by targeting IKKß/NF-κB pathway.

BACKGROUND: Mycobacterium tuberculosis (M.tb) has evolved to utilize different mechanisms to evade the host immune response. Several microRNAs (miRNAs) have been found to regulate innate immune response in M.tb replication and infection, but the roles and detailed molecular mechanisms of miRNAs in M.tb infection remain to be clarified. METHODS: Previously published dataset GSE94007 from GEO database was used for screening differential-expressed miRNAs, and a significant up-regulated miR-20a-3p was chosen for further investigation. Cells were transfected with miR-20a-3p mimics, inhibitors, IKKß siRNA, or their controls to verify the role of miR-20a-3p and IKKß in M.tb infection and host immune response. IL-ß, IL-6 and TNF-α contents in supernatant were measured by ELISA kits. The expression level of IKKß/NF-κB pathway were also detected by western blot. RESULTS: We found that miR-20a-3p was dose-and time-dependently increased during M.tb infection. Subsequently, our results demonstrated that upregulation of miR-20a-3p promoted intracellular growth of bacterial, and suppressed the release of proinflammatory cytokines in both M.tb-infected RAW264.7 and BMDM cells, while downregulation of miR-20a-3p had an opposite effect. Moreover, miR-20a-3p suppressed the activity of NF-κB pathway by directly targeting IKKß, resulting in the suppression of pro-inflammatory cytokines, attenuation of immune response and promotion of M.tb survival. CONCLUSION: Our findings uncover a role of miR-20a-3p and its target IKKß in regulating M.tb induced immune responses and provide a better understanding of pathogenesis of M.tb infection.

NK cell-derived exosomes improved lung injury in mouse model of Pseudomonas aeruginosa lung infection.

BACKGROUND: Pseudomonas aeruginosa (PA) is one of the most common bacteria that causes lung infection in hospital. The aim of our study is to explore the role and action mechanism of NK cells in lung PA infection. METHODS: In this present study, 2.5 × 108 CFU/mouse PA was injected into murine trachea to make lung PA infection mouse model. Anti-asialo GM1 was used to inhibit NK cell. The percentage of NK cells was ensured by flow cytometry, and the M1- and M2-polarized macrophages were determined by flow cytometry, qRT-PCR, and ELISA assay. Besides, H&E staining was performed to ensure the pathological changes in lung tissues. Transmission electron microscopy and western blot were carried out to identify the exosome. RESULTS: Here, in the mouse model of PA lung infection, NK cell depletion caused M2 polarization of lung macrophage, and exacerbated PA-induced lung injury. Next, our data shown that M2 macrophage polarization was enhanced when the generation of NK cell-derived exosome was blocked in the co-culture system of NK cells and macrophages. Subsequently, we demonstrated that NK cells promoted M1 macrophage polarization both in PA-infected macrophage and the mouse model of PA lung infection, and attenuated lung injury through exosome. CONCLUSION: Overall, our data proved that NK cell may improve PA-induced lung injury through promoting M1 lung macrophage polarization by secreting exosome. Our results provide a new idea for the treatment of PA lung infection.

Suppression of miR-147b contributed to H37Rv-infected macrophage viability and migration in tuberculosis in vitro.

BACKGROUND: Tuberculosis (TB) is a severe infectious disease. It was reported that microRNAs played important roles in tuberculosis. However, the role of miR-147b in the disease remained unveiling. METHODS: Tuberculosis cell model was established using macrophage THP-1 cells infected with H37Rv strain. RT-qPCR was first for examination of miR-147b relative expression. Cell viabilities were then measured with MTT. Cell transfection was to interfere the relative expression of miR-147b or C11orf87 in infected cells. RT-qPCR was adopted to confirm the transfection efficiency. Luciferase assay verified the binding sites between miR-147b and C11orf87. Migration was examined by scratch and relative protein expression of EMT biomarkers and phosphorylation of Pi3K and AKT were assessed via Western blot. RESULT: MiR-147b expression was higher and cell viability decreased in H32Rv-THP-1 cells. Cell viability was shown higher after miR-147b downregulation. Luciferase assay confirmed the binding. RT-qPCR found C11orf87 expression was lower in the H32Rv-THP-1 cells. MTT suggested that cell viability fell with the decrease of C11orf87 in infectious cells. Moreover, when H32Rv-THP-1 cells were co-transfected with miR-147b inhibitor and si-C11orf87, cell viability, migration and EMT and activation of Pi3K/AKT pathway was partially reversed compared with mere downregulation of miR-147b. CONCLUSION: miR-147b might regulate macrophage proliferation and migration through targeting C11orf87 via Pi3K/AKT pathway in Tuberculosis in vitro, which calls for in-depth inter-cellular researches and animal researches to further support that miR-147b/C11orf87 axis might be a potential therapeutic target for the molecular treatment of Tuberculosis in the future.

Liver kinase B1 overexpression controls mycobacterial infection in macrophages via FOXO1/Wnt5a signaling.

Mycobacterium tuberculosis (Mtb) is a primary cause of tuberculosis (TB), which has infected more than one-third of the world's population. Mtb survival and subsequent inflammation in macrophages are important components of TB. Liver kinase B1 (LKB1) has demonstrated anti-inflammation effects, but its function and underlying mechanism in mycobacteria-infected macrophages remains unknown. In the current study, we discovered that LKB1 was markedly decreased in Mtb-infected THP-1 and U937 macrophages. Moreover, LKB1 overexpression inhibited Mtb survival in macrophages. Mtb infection increased expression of nitric oxide, inducible nitric oxide synthase, and inflammation-related cytokines interleukin (IL)-6, tumor necrosis factor-α, and IL-1ß, whereas pcDNA3-LKB1 transfection inhibited the release of these cytokines in THP-1 and U937 cells. Furthermore, LKB1 overexpression significantly decreased protein expression of Wnt5a, which is dependent on the elevation of forkhead box protein O1 (FOXO1). Generally, we show that interruption of FOXO1 or overexpression of Wnt5a can reverse the effects of LKB1 on mycobacterial intracellular survival, nitric oxide, inducible nitric oxide synthase expression, and inflammatory cytokine release. These findings indicate important roles for LKB1, FOXO1, and Wnt5a in controlling mycobacteria and cell inflammation.

[Clinical observation on diabetic peripheral neuropathy treated with electroacupuncture and acupoint injection].

OBJECTIVE: To compare the differences in the therapeutic effect on diabetic peripheral neuropathy between the combined therapy of electroacupuncture and acupoint injection and the simple acupoint injection. METHODS: Under the satisfactory control of blood glucose, 60 cases of diabetic peripheral neuropathy were divided randomly into two groups, 30 cases in each one. In electroacupuncture plus acupoint injection group (group A), electroacupuncture and acupoint injection with Methylcobalamin were administered. Penetrating acupuncture was applied from Gongsun (SP 4) to Quanzhong (Extra) and from Yongquan (KI 1) to Taichong (LR 3) mainly. Acupoint injection was administered on Sanyinjiao (SP 6). In acupoint injection group (group B), only acupoint injection with Methylcobalamin was provided on Sanyinjiao (SP 6). After 2 sessions of treatment, the conduction velocity of ulnar nerve and tibial nerve was measured. The scores of Chinese medicine syndrome and diabetic peripheral neuropathy were recorded before and after treatment in two groups. RESULTS: The effective rates were 90.0% (27/30) and 63.3% (19/30) in group A and group B respectively, presenting significant statistical difference (P < 0.05). After treatment, the motor nerve conduction velocity (MCV) and sensory nerve conduction velocity (SCV) of ulnar nerve and tibial nerve in group A were higher than those in group B (P < 0.05, P < 0.01). After treatment, the score of Chinese medicine syndrome in group A was lower than that in group B (14.36 +/- 1.88 vs 26.58 +/- 3.52, P < 0.01), the score of diabetic peripheral neuropathy in group A was lower than that in group B (12.86 +/- 4.28 vs 17.89 +/- 4.35, P < 0.01). CONCLUSION: Electroacupuncture and acupoint injection with Methylcobalamin achieve a significant clinical efficacy on diabetic neuropathy and its efficacy is superior to that of simple acupoint injection with Methylcobalamin. This therapy can effectively increase nerve conduction velocity, control and relieve the symptoms of diabetic peripheral neuropathy.