Effects of cordycepin on the microglia-overactivation-induced impairments of growth and development of hippocampal cultured neurons

Abstract

Microglial cells are normally activated in response to brain injury or immunological stimuli to protect central nervous system (CNS). However, over-activation of microglia conversely amplifies the inflammatory effects and mediates cellular degeneration, leading to the death of neurons. Recently, cordycepin, an active component found in Cordyceps militarisa known as a rare Chinese caterpillar fungus, has been reported as an effective drug for treating inflammatory diseases and cancer via unclear mechanisms. In this study, we attempted to identify the anti-inflammatory role of cordycepin and its protective effects on the impairments of neural growth and development induced by microglial over-activation. The results indicate that cordycepin could attenuate the lipopolysaccharide (LPS)-induced microglial activation, evidenced by the dramatically reduced release of TNF-α and IL-1β, as well as the down-regulation of mRNA levels of iNOS and COX-2 after cordycepin treatment. Besides, cordycepin reversed the LPS-induced activation of NF-κB pathway, resulting in anti-inflammatory effects. Furthermore, by employing the conditioned medium (CM), we found cordycepin was able to recover the impairments of neural growth and development in the primary hippocampal neurons cultured in LPS-CM, including cell viability, growth cone extension, neurite sprouting and outgrowth as well as spinogenesis. This study expands our knowledge of the anti-inflammatory function of cordycepin and paves the way for the biomedical applications of cordycepin in the therapies of neural injuries.

Fig 1. Effects of different doses of cordycepin exposure on BV2 microglia viability.

(A) Representative photographs of microglia exposed to different doses of cordycepin (0, 1, 10, 20, 40 μg/ml). Scale bar = 200 μm. (B) Relative cell viability of the microglia to the control following cordycepin treatment, measured by MTT assay. (C) Relative LDH release to control in the experimental groups. The data were presented by the mean ± SEM. * p < 0.05.

Fig 2. Cordycepin attenuated the LPS-induced inflammatory responses.

(A) Representative images of microglia following 10 μM cordycepin treatment for 1 day with or without LPS treatment. Cells were stained green for tubulin, blue for nucleus. Scale bar = 100μm. The contents of cytokines, including TNF-α (B) and IL-1β (C) in the experimental groups were measured by the ELISA assay. Note the obviously lower concentrations of TNF-α and IL-1β in cordycepin treatment group compared to untreated group after LPS stimulation. (D-E) Relative mRNA levels of iNOS and COX-2 in the experimental groups assessed by real time PCR using the 2^-ΔΔCT method. Data are expressed as the fold change in gene expression normalized to an endogenous gene (β-actin) and relative to the cells in control group without LPS stimulation. Data are presented as mean ± SEM (n = 6 in triplicate). # p < 0. 01.

Fig 3. Cordycepin (10 μg/ml) inhibited NF-кB signaling pathway activated by LPS treatment.

(A) The p65, IкBα and p-IкBα protein expression in the cytoplasm in the cultures under cordycepin treatment with or without LPS stimulation. (B-D) The relative optical densities of p65, IкBα and p-IкBα protein bands to control, normalized to β-actin. (E) The p65 expression in the nucleus in the experimental groups. (F) The relative optical densities of p65 protein bands to control, normalized to HDAC1. Data were presented by mean ± SEM. *p < 0.05, #p < 0.01.

Fig 4. Cordycepin (10 μg/ml) rescued LPS-induced cell death in the hippocampal neurons.

The cells were cultured for 7 days in the different conditioned mediums of the experimental groups. (A) Relative cell viabilities to control in the experimental groups, examined by MTT assay. (C) Relative LDH release to control in the experimental groups. Data were presented by mean ± SEM. *p < 0.05.

Fig 5. Cordycepin (10 μg/ml) rescued the LPS-induced impairments of GCs.

The hippocampal neurons were cultured for 1 day in the different conditioned mediums of the experimental groups. (A) Representative fluorescence images of the hippocampal GC stained for actin, tubulin and merge of the two staining. Scale bar = 20 μm. (B) Relative area of GCs to control. (C) Average number of filopodia emerging from GCs. (D) Average filopodium lengths from the tip of each filopodia to the edge of the GCs. (E) Ratio of number of filopodia and area of GC in GCs. Data were presented by mean ± SEM. * p < 0.05.

Fig 6. Cordycepin treatment (10 μg/ml) could repair the LPS-induced injuries of neurite sprouting and outgrowth.

The hippocampal neurons were cultured for 7 day in the different conditioned mediums of the experimental groups. (A) Typical hippocampal neurons with extending neurites in the culture in different experimental groups, stained for MAP-2. The neurite sprouting and outgrowth were characterized by the number of primary dendrites per cell (B), number of dendritic end tips (C) and the average neurite length (D) in the experimental groups. (E) Western blot analysis of GAP-43 expression in the cultures of different groups. (F) Relative optical densities of GAP-43 show in (E) (n = 3/group). Data were presented by mean ± SEM. * p < 0.05, #p < 0.01.

Fig 7. Effects of cordycepin treatment on spine morphology and density in the hippocampal neurons.

(A) The cultured hippocampal neurons were transfected with mCherry-actin and imaged. The spine in the experimental groups could be clearly observed at high magnification. Scale bar = 2 μm. (B) Proportion of different spine types expressed as percentage of total spines in the neurons in the experimental groups. (C) Spine density expressed as number of spines per 10 μm of dendrites of the neurons in the experimental groups. Data were presented by mean ± SEM. * p < 0.05.