Pre-treatment of rapamycin transformed M2 microglia alleviates traumatic cervical spinal cord injury via AIM2 signaling pathway in vitro and in vivo.

BACKGROUND: Traumatic spinal cord injury (SCI) is still devastating. It was suggested that the inhibition of mTOR may alleviate neuronal inflammatory injury but its underlying mechanism remained to be elucidated. AIM2 (absent in melanoma 2) recruits ASC (apoptosis-associated speck-like protein containing a CARD) and caspase-1 to form the AIM2 inflammasome, activate caspase-1, and elicit inflammatory responses. We designed this study to elucidate whether pre-treatments of rapamycin could suppress SCI induced neuronal inflammatory injury via AIM2 signaling pathway in vitro and in vivo. METHODS: We performed oxygen and glucose deprivation / re-oxygenation (OGD) treatment and rats clipping model to mimic neuronal injury after SCI in vitro and in vivo. Morphologic changes of injured spinal cord were detected by hematoxylin and eosin staining. The expression of mTOR, p-mTOR, AIM2, ASC, Caspase-1 and et al were analyzed by fluorescent staining, western blotting or qPCR. The polarization phenotype of microglia was identified by flow cytometry or fluorescent staining. RESULTS: We found BV-2 microglia without any pre-treatment cannot alleviate primary cultured neuronal OGD injury. However, pre-treated rapamycin in BV-2 cells could transform microglia to M2 phenotype and protects against neuronal OGD injury via AIM2 signaling pathway. Similarly, pre-treatment of rapamycin could improve the outcome of cervical SCI rats through AIM2 signaling pathway. CONCLUSIONS: It was suggested that resting state microglia pre-treated by rapamycin could protect against neuronal injury via AIM2 signaling pathway in vitro and in vivo. Pre-inhibition of mTOR pathway may improve neuronal protection after SCI.

Blood Pressure Variability Associates with Six-Month Outcomes in Acute Cervical Spinal Cord Injury: An Analysis of 105 Patients.

OBJECTIVE: Blood pressure variability (BPV) has been shown to correlate with poor outcomes in patients with intracerebral hemorrhage (ICH) and traumatic brain injury. However, this association has not been elucidated in patients with traumatic cervical spinal cord injury (cSCI). We hypothesized that 24-hour BPV from time of admission is associated with worse outcomes in patients with cSCI. METHODS: We performed a retrospective chart review analysis of adult patients at Huashan Hospital Fudan University between January 2006 and September 2022. We included isolated patients with traumatic cSCI within 6 hours of injury. Outcomes of patients with cSCI were assessed using 6-month American Spinal Injury Association (ASIA) impairment scale grade, and were dichotomized into poor (ASIA grade A-C, or decreasing ASIA grade compared with baseline) and good (ASIA grade D and E, or increasing ASIA grade compared with baseline) outcome groups. Blood pressures (BPs) were recorded during the first 24 hours of hospital course. BP was analyzed in the hyperacute period, from 0 to 4-5 hours; and in the acute period, from 4-5 to 24-25 hours after admission. BPV was analyzed by standard deviation (SD), coefficient of variation (CV), and successive variation (SV) of systolic BP (SBP). RESULTS: We analyzed 105 patients' charts. The first BP assessment, on emergency department arrival, at median 267 minutes (interquartile range, 152-312 minutes) after onset of injury was mean 152.2 mm Hg (SD, 51.8 mm Hg). The second BP assessment, on neurosurgical intensive care unit arrival, was mean 148.1 mm Hg (53.2 mm Hg). Poor outcomes occurred in 63 patients (60%). In univariate analysis, univariate quintile analysis or multivariate analysis, SBPSD, SBPCV, and SBPSV were associated with poor outcomes in both the hyperacute and the acute period. CONCLUSIONS: BPV during the first 24 hours after injury in patients with traumatic cSCI was independently associated with poor functional outcome at 3 months. Stabilization of BPV during the hyperacute and acute period may be a therapeutic target to improve functional outcomes of these patients.

Expression of TNF­α and IL­ß can be suppressed via the PPAR­Î³/mTOR signaling pathway in BV­2 microglia: A potential anti­inflammation mechanism.

Currently, microglia are considered as crucial factors in suppressing inflammatory reactions, but the specific molecular mechanism remains unknown. To elucidate whether peroxisome proliferator­activated receptor­Î³ (PPAR­Î³) can inhibit neuroinflammatory cytokine expression via the mTOR signal pathway, the BV­2 cell line was incubated with lipopolysaccharide (10 mM/ml) to induce an inflammatory injury. PPAR­Î³ was activated by rosiglitazone, and was inhibited by GW9662. The mTOR signal pathway was activated by phosphatidic acid (P.A.), while it was inhibited by rapamycin. Western blotting and reverse transcription­quantitative PCR were used to evaluate the expression levels of PPAR­Î³/mTOR signal pathway related proteins and neuroinflammatory cytokines, including NF­κB, tumor necrosis factor (TNF)­α and interleukin (IL)­1ß. When treated with P.A., the expression levels of phosphorylated (p)mTOR and p­ribosomal protein S6 kinase (pS6K) were significantly increased and the expression levels of TNF­α and IL­1ß were significantly lower. However, the expression of PPAR­Î³ was similar in P.A. treated cells and cells treated with rapamycin. When PPAR­Î³ was activated, pmTOR and pS6K protein expression levels were significantly decreased, and the mRNA expression levels of TNF­α and IL­1ß were significantly reduced, but this inhibition could be alleviated by administrating GW9662. Collectively, it was indicated that the mTOR signal pathway may be located downstream of PPAR­Î³. Furthermore, neuroinflammatory reactions could be inhibited via the activation of PPAR­Î³ by suppressing the mTOR signal pathway in microglia.

Biotransformation of bufadienolides by cell suspension cultures of Saussurea involucrata.

The biotransformation of three bioactive bufadienolides, namely, bufotalin (1), telocinobufagin (2), and gamabufotalin (3) by cell suspension cultures of Saussurea involucrata yielded 11 products. Bufotalin yielded 3-epi-bufotalin (1a), 3-epi-desacetylbufotalin (1b), 3-epi-bufotalin 3-O-ß-D-glucoside (1c), 1ß-hydroxybufotalin (1d), and 5ß-hydroxybufotalin (1e); telocinobufagin yielded 3-dehydroscillarenin (2a), 3-dehydrobufalin (2b), and 3-epi-telocinobufagin (2c); and gamabufotalin yielded 3-epi-gamabufotalin (3a), 3-dehydrogamabufotalin (3b), and 3-dehydro-Δ¹-gamabufotalin (3c), respectively. Among these 11 products, 1a, 1b, 1c, 1d, 3a and 3c are previously unreported. The structures of these metabolites were elucidated based on NMR spectroscopic analyses and mass spectrometry. Most metabolites showed significant cytotoxic activities against human hepatoma (HepG2) and breast cancer (MCF-7) cell lines. In addition, the time course for the biotransformation of 3 was investigated.