Data_Sheet_1_HMG-CoA Reductase Inhibitors Relieve Endoplasmic Reticulum Stress by Autophagy Inhibition in Rats With Permanent Brain Ischemia.pdf

<p>Exploring and expanding the indications of common clinical drugs, such as statins, is important to improve the prognosis of patients with permanent cerebral infarction. It has been suggested that reversing the defects in cellular autophagy and ER stress with statin therapy may be a potential treatment option for reducing ischemic damage. Male Sprague-Dawley rats underwent permanent middle cerebral artery occlusion (PMCAO) by electrocoagulation surgery. Atorvastatin (ATV, 10 mg/kg/day) or vehicle was administered intraperitoneally. Rats were divided into the vehicle-treated (SHAM), ATV pretreatment for MCAO (AMCAO), and 3-methyladenine (3MA) combined with ATV pretreatment (3MAMCAO) groups. Magnetic resonance imaging, as well as immunohistochemical and Western blot assessments, were performed 24 h after MCAO. Each ATV-treated group demonstrated significant reductions in infarct volume compared with that in the vehicle-treated group at 24 h after MCAO, which was associated with autophagy reduction and ER stress attenuation in neurons and neovascularization. Next, Western blotting was used to detect the levels of the autophagy-related proteins LC3B and P62 and of ER stress pathway proteins. However, 3MA significantly partially inhibited the ER stress pathway via limiting the autophagic flux in the AMCAO group. In conclusion, our results imply that the neuroprotective function of ATV depends on autophagic activity to diminish ER stress-related cell apoptosis in rats with PMCAO and suggest that compounds that inhibit autophagic activity might reduce the neuroprotective effect of ATV after brain ischemia.</p

MSS1 gene knockdown reduced the expression of MSS1 at protein levels.

<p>In order to assess the efficacy of gene silencing, western blot analysis was performed after transfection with siRNAs targeting MSS1. The specificity of MSS1 gene silencing was determined by comparing with cells transfected with the scrambled RNA duplex. The BV2 cells were transfected with either MSS1-specfic or control siRNAs. At 24 h post-incubation, cell lysates were analyzed for the protein expression of MSS1 using western blot. Compared with the negative control group, the expression of MSS1 was significantly reduced by incubation with MSS1-targeted siRNAs. Data were obtained from three independent experiments with four to six replicates each. *<i>p</i><0.05 compared with the negative control group.</p

Decreased iNOS expression and NO production by MSS1 gene silencing in LPS-activated microglia.

<p>The BV2 cells were transfected with either MSS1-specfic or control siRNA for 24 h, then cells were stimulated for 24 h with LPS (1000 ng/mL). At 24 h post-LPS incubation, cell lysates were analyzed for the protein production of iNOS using western blot. The Griess assay was performed to measure the production of the NO metabolite, nitrite. Transfection with MSS1-specific siRNA suppressed the LPS-induced iNOS expression at protein levels, along with the production of nitrites Data were obtained from three independent experiments with four to six replicates each. *<i>p</i><0.05 compared with the negative control group.</p

Rifampicin significantly suppressed the expression of MSS1 in LPS-stimulated BV2 microglia.

<p>Cells were treated with the indicated doses of rifampicin for 2 h prior to the addition of LPS (1000 ng/ml). At 24 h post-LPS incubation, cell lysates were analyzed for the protein production of MSS1 using western blot. Rifampicin significantly inhibited the LPS-induced MSS1 expression at protein levels. Data were obtained from three independent experiments with four to six replicates each. *<i>p</i><0.05 compared with untreated cells and cells treated with LPS in the absence of rifampicin.</p

MSS1 gene silencing decreased IkBα protein degradation in LPS-activated microglia.

<p>The BV2 cells were transfected with either MSS1-specfic or control siRNAs for 24 h, then cells were stimulated with LPS (1000 ng/mL) for 30 min before cell lysates were analyzed for IkBα expression using western blot. IkBα protein degradation was significantly reduced after the addition of siRNAs targeting MSS1 in LPS-induced BV2 microglia. Data were obtained from three independent experiments with four to six replicates each. *p<0.05 compared with the negative control group.</p

Differential MSS1 protein expression identified by MALDI-TOF-MS.

<p>Differential MSS1 protein expression identified by MALDI-TOF-MS.</p

MSS1 gene silencing inhibited microglial NF-kB activation in response to LPS stimulation.

<p>The BV2 cells were transfected with either MSS1-specfic or control siRNA for 24 h, then cells were incubated with LPS at 1000 ng/mL for 8 h. After that, cells were transfected with NF-kB-luciferase reporter plasmid and pCMV-gal control vectors using Lipofectamine reagents. NF-kB activation was detected and expressed as relative luciferase activity. Compared with the negative control group, treatment with MSS1-targetd siRNA significantly suppressed the enhancement of NF-kB activity by LPS. Data were obtained from three independent experiments with four to six replicates each. *<i>p</i><0.05 compared with the negative control group.</p