Effect of the Deletion of icl1 Gene and icl2 Gene on the Growth Rate of Mycobacterium tuberculosis and the Specific Regulatory Mechanism Involved.

Objective: To explore the effect of the deletion of the icl1 gene and icl2 gene on the growth rate of Mycobacterium tuberculosis (Mtb) and the specific regulatory mechanism involved. Methods: H37Rv was purchased from the Tuberculosis Prevention and Control Institute, and H37Rv was grown in Middlebrook 7H9 broth. Macrophages THP-1 cells were purchased by our researchers from the Cell Bank of the Chinese Academy of Sciences, which were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS), at 37°C and 5% CO2. The experiment was divided into 3 groups: the control group (H37Rv infected with THP-1 cells), the icl1/2 deletion group (H37Rv infected with icl1/2 deleted THP-1 cells) and the icl1/2 complementation group (H37Rv infected with icl1/2 deletion, icl1/2 complementary THP-1 cells). Absorbance was measured with a microplate spectrophotometer and the bacterial growth rate was calculated. The colony-forming units (CFU) obtained from the dilution was used to calculate the total number of CFU per milliliter and the percentage of survival of mycobacteria. The protein levels of isocitrate lyase 1 (ICL1), ICL2, p-mTOR and p-Akt were analyzed by Western blot. The CD4+ level was analyzed by flow cytometry. The mRNA expression levels of CCL20, CXCL2, CXCL8, interferon gamma (IFN-γ), interleukin (IL)-17 and IL-22 were analyzed using the quantitative reverse transcription polymerase chain reaction (RT-qPCR) method. Stably transformed monomeric red fluorescent protein (mRFP)-green fluorescent protein (GFP)-LC3 reporter THP-1 cells were used to monitor the aggregation of LC3B in autophagosomes and autophagolysosomes. Results: The Mtb growth rate and CFU of the icl1/2 deletion group were decreased in comparison with the control group (P < .05). When compared with the icl1/2 deletion group, however, the Mtb growth rate and CFU of the icl1/2 complementation group were associated with increased results (P < .05). The protein levels of ICL1 and ICL2 in the icl1/2 deletion group were significantly decreased compared with the control group (P < .05), which were evidently increased in the icl1/2 complementation group when compared with the icl1/2 deletion group (P < .05). In addition, compared with the control group (25.16 ± 2.18), the level of CD4+ appeared to be increased in the icl1/2 deletion group (62.37 ± 5.46) (P < .05), while it was decreased in the icl1/2 complementation group compared with the icl1/2 deletion group (28.33 ± 1.32) (P < .05). The expression levels of chemokine (C-C motif) ligand 20 (CCL20), chemokine (C-X-C motif) ligand 2 (CXCL2), chemokine (C-X-C motif) ligand 8 (CXCL8), IL-17, IFN-γ, and IL-22 mRNA were increased in the icl1/2 deletion group compared with the control group (P < .05), which were significantly decreased in the icl1/2 complementary group compared with the icl1/2 deletion group (P < .05). A comparison between the control group and the icl1/2 deletion group showed that the latter increased the formation of autophagosomes and autophagolysosomes in H37Rv-infected cells (P < .05). However, compared with the icl1/2 deletion group, the icl1/2 complementation group decreased the formation of autophagosomes and autolysosomes in H37Rv-infected cells (P < .05). Moreover, the expression levels of phosphor-mammalian target of rapamycin (p-mTOR) and p-Akt in the icl1/2 deletion group were significantly reduced compared with the control group (P < .05), and were increased in the icl1/2 complementation group compared with the icl1/2 deletion group (P < .05). Conclusion: Loss of icl1/2 was believed to increase the expression of CD4 and CCL20, CXCL8 as well as CXCL2 in the immune system, which increased autophagy. Furthermore, it exerted potential in inhibiting the growth of intracellular Mtb in macrophages.

The Dual Dose-Dependent Effects of Corticosterone on Hippocampal Cell Apoptosis After Traumatic Brain Injury Depend on the Activation Ratio of Mineralocorticoid Receptors to Glucocorticoid Receptors.

In our recent studies, we reported that mineralocorticoid receptor (MR) had the opposite effects of glucocorticoid receptor (GR) on neural cell survival after traumatic brain injury (TBI). However, whether short-term use of high-dose natural glucocorticoids, which are mixed agonists of both MR and GR, leads to neurotoxic effects by inducing excessive GR activation is unclear, as is the threshold GR activation level and the possible signaling pathways remain unclear. In this study, we examined the dual dose-dependent effects of corticosterone (CORT) on spatial memory, hippocampal cell survival and receptor-mediated downstream signaling pathways after TBI. We found that different doses of CORT exhibited dual effects on hippocampal cell survival and rat spatial memory. Low doses of CORT (0.3 and 3 mg/kg) significantly increased MR activation, upregulated Akt/CREB/Bad phosphorylation and Bcl-2 concentration, reduced the number of apoptotic neural cells, and subsequently improved rat spatial memory. In contrast, a high dose of CORT (30 mg/kg) exerted the opposite effects by overactivating GR, upregulating P53/Bax levels, and inhibiting Erk/CREB activity. The results suggest that the neuroprotective and neurotoxic effects of endogenous GC depend on a threshold level and that a higher dose of GC, even for short-term use, should be avoided after TBI.

Corticosteroid receptor rebalancing alleviates critical illness-related corticosteroid insufficiency after traumatic brain injury by promoting paraventricular nuclear cell survival via Akt/CREB/BDNF signaling.

BACKGROUND: We previously found that high-dose methylprednisolone increased the incidence of critical illness-related corticosteroid insufficiency (CIRCI) and mortality in rats with traumatic brain injury (TBI), whereas low-dose hydrocortisone but not methylprednisolone exerted protective effects. However, the receptor-mediated mechanism remains unclear. This study investigated the receptor-mediated mechanism of the opposite effects of different glucocorticoids on the survival of paraventricular nucleus (PVN) cells and the incidence of CIRCI after TBI. METHODS: Based on controlled cortical impact (CCI) and treatments, male SD rats (n = 300) were randomly divided into the sham, CCI, CCI + GCs (methylprednisolone 1 or 30 mg/kg/day; corticosterone 1 mg/kg/day), CCI + methylprednisolone+RU486 (RU486 50 mg/kg/day), and CCI + corticosterone+spironolactone (spironolactone 50 mg/kg/day) groups. Blood samples were collected 7 days before and after CCI. Brain tissues were collected on postinjury day 7 and processed for histology and western blot analysis. RESULTS: We examined the incidence of CIRCI, mortality, apoptosis in the PVN, the receptor-mediated mechanism, and downstream signaling pathways on postinjury day 7. We found that methylprednisolone and corticosterone exerted opposite effects on the survival of PVN cells and the incidence of CIRCI by activating different receptors. High-dose methylprednisolone increased the nuclear glucocorticoid receptor (GR) level and subsequently increased cell loss in the PVN and the incidence of CIRCI. In contrast, low-dose corticosterone but not methylprednisolone played a protective role by upregulating mineralocorticoid receptor (MR) activation. The possible downstream receptor signaling mechanism involved the differential effects of GR and MR on the activity of the Akt/CREB/BDNF pathway. CONCLUSION: The excessive activation of GR by high-dose methylprednisolone exacerbated apoptosis in the PVN and increased CIRCI. In contrast, refilling of MR by corticosterone protects PVN neurons and reduces the incidence of CIRCI by promoting GR/MR rebalancing after TBI.

Corticosterone Replacement Alleviates Hippocampal Neuronal Apoptosis and Spatial Memory Impairment Induced by Dexamethasone via Promoting Brain Corticosteroid Receptor Rebalance after Traumatic Brain Injury.

The balance of mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) is indispensable for maintaining the normal function and structure of the hippocampus. However, changes in GR/MR and their effect on the survival of hippocampal neurons after traumatic brain injury (TBI) are still unclear. Previous studies have indicated that high-dose glucocorticoids (GC) aggravate hippocampal neuronal damage after TBI. We hypothesize that the imbalance of GR/MR expression and activation caused by injury and irrational use of dexamethasone (DEX) aggravates post-traumatic hippocampal apoptosis and spatial memory dysfunction, but that restoration by refilling MR and inhibiting GR promotes the survival of neurons. Using rat controlled cortical impact model, we examined the plasma corticosterone (CORT), corticosteroid receptor expression, apoptosis, and cell loss in the hippocampus, and, accordingly, the spatial memory after TBI and GC treatment within 7 days. Plasma CORT, MR, and GR expression level were significantly reduced at 2 days after TBI. Accordingly, the number of apoptotic cells also peaked at 2 days. Compared with the TBI control group, DEX treatment (5 mg/kg) significantly reduced plasma CORT, upregulated GR expression, and increased the number of apoptotic cells and cell loss, whereas CORT replacement (0.3 mg/kg) upregulated MR expression, inhibited apoptosis, and improved spatial memory. The deleterious and protective effects of DEX and CORT were counteracted by spironolactone and mifepristone respectively. The results suggest that inhibition of GR by RU486 or the refilling of MR by CORT protects hippocampal neurons and alleviates spatial memory impairment via promoting GR/MR rebalancing after TBI.

Ginsenoside Rg1 attenuates okadaic acid induced spatial memory impairment by the GSK3ß/tau signaling pathway and the Aß formation prevention in rats.

Ginsenoside Rg1, one of the major active ingredients isolated from Panax Ginseng, has been shown notable neuroprotective effects in memory impairment animals. However, the role of ginsenoside Rg1 on cognition capacity damaged by neurofibrillary tangles (NFTs) is still poorly understood, and the underlying mechanism remain to be fully elucidated. Okadaic acid (OKA), a potent phosphatase inhibitor, often apply to imitate Alzheimer's disease-like symptom damaged by neurofibrillary tangles, was used to investigate the effects of ginsenoside Rg1 on memory impairment and the related mechanisms in Sprague Dawley (SD) rats. The anti-dementic drug donepezil was used as a positive contrast. The results showed that OKA intracerebroventricular (i.c.v.) injection induced memory impairment, including changes in the ability of orientation navigate, spatial probe and relearning memory in behavioral test of Morris water maze (MWM). However, treatment with Rg1 and donepezil remarkably alleviated these changes. Also OKA treated rats showed memory impairment including increasing of phospho-tau, decreasing of phospho-GSK3ß and the formation of ß-amyloid in special brain regions, which were reversed by Rg1 (20 mg/kg) and donepezil (1 mg/kg) administration. All these indicating that ginsenoside Rg1 protects rats against OKA-induced neurotoxicity. The possible neuroprotective mechanism may be that Rg1 decreases OKA-induced memory impairment through GSK3ß/tau signaling pathway and/or attenuating Aß formation. Thus, these studies indicate that ginsenoside Rg1 might be a potential preventive drug for Alzheimer's disease.