Effects Of Chronic Glucocorticoids Treatment On NLRP-1 Inflammasome Activation And Neuronal Damage In Mice And The Regulation Of Ginsenoside Rg1

Neuroinflammation plays an important role in the pathogenesis of neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD). Chronic glucocorticoids (GCs) exposure has deleterious effects on the structure and function of neurons and is associated with generation and development of AD. However, little is known about the proinflammatory effects of chronic GCs exposure on neurodegeneration in brain. This study can provide new ideas and targets for prevention and treatment of neurodegenerative diseases.ObjectivesThe purpose of this study is to observe the effects of chronic glucocorticoids treatment on activation of NLRP-1 inflammasome and neuronal damage in male mice and the regulation of ginsenoside Rgl, and to explore the possible mechanisms of chronic glucocorticoid on hippocampal neurons. This study can provide new ideas and targets for prevention and treatment of neurodegenerative diseases, and to provide experimental evidence for the neuronal protection of ginsenoside Rgl.Methods1. The male mice were divided into control group and dexamethasone (DEX,5 mg/kg) group, each group was further divided into four time points of 7d,14d,21d and 28d. In the DEX treatment groups, mice were treated with DEX at 5 mg/kg/d (s.c), while in control groups, mice were injected with equal volume of normal saline (NS). The change of body weight was observed weekly. The open field test was used to observe the effects of DEX treatment on motor activity and exploratory behavior. Histological examination was conducted to examine neuronal degeneration and apoptosis in the frontal cortex and hippocampus. The Hoechst 33258 staining was used to observe neuronal apoptosis. Immunohistochemistry was used to detect the expression of MAP2. Western blot was used to observe the expression level of GR, NLRP-1, NLRP3, ASC, Caspase-1, Caspase-5, IL-1? and IL-18. Q-PCR was used to detect the mRNA levels of GR, ASC, NLRP-1, Caspase-1 and IL-1?. The ELISA was used to detect the effects of DEX treatment for 28 d on expressions of IL-1?, IL-6 and TNF-a protein in the frontal cortex and hippocampus brain tissues.2. The male mice were divided into control group, DEX model group, RU486 group, ginsenoside Rgl (1,2,4mg/kg) group for 28 days. The open field test was used to observe the effects of DEX treatment on motor activity and exploratory behavior. Histological examination was conducted to examine neuronal degeneration in the frontal cortex and hippocampus. Immunohistochemistry was used to test the expression of MAP2. Western blot was used to observe the expression of GR, NLRP-1, ASC, Caspase-1, Caspase-5, IL-1? and IL-18.Results1. Compared with control group, DEX treatment for 7,14,21 and 28d significantly decreased the body weight in male mice. The HE results showed that no significant neuronal abnormalities were observed in either the frontal cortex or the hippocampus CA1 and CA3 in control group. In DEX treatment 7 d and 14 d groups, there were slight neuronal damages in the frontal cortex and hippocampus CA1 and CA3. However, in DEX treatment 21 d and 28 d groups, the neurons in the cortex and hippocampus CA3 showed significant degeneration. The neurons manifested acidophilia degeneration, nuclear condensation and disorder of the array. The open field test showed that chronic DEX treatment for 21 d and 28d significantly decreased the spontaneous motor activity and exploratory behavior in mice. The immunohistochemistry results showed that the expression of MAP2 in the cortex and hippocampus CAl and CA3 significantly decreased after DEX treatment for 21d and 28d. Western blot results showed that the expression of GR significantly decreased, and the expression of the NLRP1, Caspase-1, Caspase-5, ASC, IL-1? and IL-18 significantly increased after DEX treatment for 21d and 28d. Nevertheless, the expression of the NLRP3 has no obvious influence. Q-PCR results showed that DEX treatment for 21 d and 28 d could increase the NLRP1, Caspase-1, ASC and IL-1? mRNA levels and reduce the GR mRNA levels. Compared with control group, the levels of IL-1??IL-6 and TNF-? significantly increased after DEX treatment for 28d. 2. The HE staining results showed that there were no abnormal changes in the frontal cortex and hippocampal neurons in control group mice. While in the model group, there were significant acidophilia degeneration, nuclear condensation and disorder of the array in the frontal cortex and hippocampal neurons. And the RU486 and ginsenosides Rgl (2,4mg/kg) treatment could improve the neuronal damage induced by DEX. The open field test results showed that, compared with control group, the moving distance, the mean moving speed, the number of lines crossing and the number of stand up were significantly decreased in DEX model group. While compared with the model group, RU486 and ginsenosides Rgl (2,4mg/kg) treatment dramatically increased the moving distance, the mean moving speed, the number of lines crossing and the number of stand up. The object recognition test results showed that chronic DEX treatment significantly reduced the the number of entries into the new area and the behavior time of entries into the new area.While the RU486 and ginsenosides Rgl (2,4mg/kg) treatment could improve the number of entries into the new area and the behavior time of entries into the new area. The immunohistochemistry results showed that the RU486 and ginsenosides Rgl (2,4mg/kg) treatment could increase the expression of MAP2 CA1 and CA3. Western blot results showed that the RU486 and ginsenosides Rgl (2,4mg/kg) treatment could increase the expression of GR and decrease the expression of NLRP-1, ASC, Caspase-1, Caspase-5, IL-1? and IL-18 in the frontal cortex and hippocampus tissue.ConclusionThe experimental results showed that chronic GCs exposure may damage the motor activity and exploratory behavior, increase the expression of NLRP-1 inflammasome and inflammatory cytokines, and induce neuronal damage in the frontal cortex and hippocampus. The ginsenosides Rgl could improve the motor activity, exploratory behavior, the behavior of exploring new things and decrease the expression of NLRP-1 inflammasomes and inflammatory cytokines. It suggests that chronic GCs exposure may increase brain inflammation via NLRP-1 inflammasome activation and induce neurodegeneration. The ginsenosides Rgl could improve the neuronal damage via inhibiting the activation of NLRP-1 inflammasome.