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Total RNA was extracted
from hippocampus using Trizol reagent (Zistabzarpajohan, Iran) according to the
manufacturer’s protocol and treated with DNase I, RNase-Free (Thermo
scientific, Fermentas). The concentration and purification of total RNA was evaluated
by NanoDrop 1000 Spectrophotometer. The
RNA quality was estimated by characterizing 18S and 28S ribosomal RNA bands
using electrophoresis. Then 500 nanograms of total RNA was reverse transcribed
into cDNA using PrimeScript RT reagent kit (Thermo scientific, Fermentas)
according to manufacturer’s protocol. In brief, RNA template, primers and
nuclease free H2O mixture were incubated at 65°C for 5 min then the mixture was
mixed with 2 ?l of 5× PrimeScript buffer solution and incubates at 37 °C, 15
min, 85?C, 5 s and held at -20°C
until further use. The resulting cDNA was then used to quantitatively
measurement of gene expression levels of MMP-9 and GAPDH using SYBR Premix Ex
TaqTM II (TliR-NaseH Plus), Bulk (Takara, Japan) kit. The cycling condition was
set as 95 °C for 30 s, 35 cycles at 95 °C for 10 s; 59 °C for 10 s and 72°C for
35 s. Real-time PCR was performed in a real time qPCR cycler instrument (Rotor-Gene
Q, QIAGEN) and the threshold cycle (Ct) was applied to quantify the mRNA levels
of gene. Relative gene expression levels of MMP-9 were analyzed by 2-??Ct method,
normalized to GAPGD housekeeping gene and relative to control group. Matrix metalloproteinases-9
(MMP), also known as gelatinase
B, is zinc dependent endoprotease and
its expression has been implicated in molecular mechanisms of many
pathological processes in the central nervous system. Considerable researches have been conducted on the role of MMP-9 in neurodegenerative
disease (Helbecque
et
al., 2003; De Luca and Papa
2017). In
Alzheimer’s disease (AD),
higher levels of MMP-9 have been reported in the plasma (Lorenzl et al. 2003 & 2008) and hippocampus (Asahina et al. 2001; Backstrom
et al. 1992). It has been
shown that MMP-9 activity in the frontal and parietal cortex is greater in both
AD and mild cognitive impairment compared to healthy subjects (Bruno et al. 2009). Increased MMP-9 expression
in the hippocampus is involved in the development of cognitive impairment induced by beta-amyloid (Mizoguchi et al. 2009). It has been determined that laminin
degradation by  MMP-9 induces neuronal
damage (Gu et al. 2005).

Furthermore, the clinical
administration of statins (HMG-CoA reductase inhibitors) is associated with
reduction of the AD incidence later in life (Haag et al. 2009). Simvastatin
is a hydrophilic statin, which can cross the blood brain barrier (Saheki et al. 1994) and
has shown that improve memory impairment, and cerebrovascular damage in mice (Roy et al. 2015; Tong
et al. 2012). Simvastatin induced antioxidant proteins (Adeli et al. 2017) and reduced
the expression of neuro-inflammatory mediators (Huang et
al. 2017; McFarland et al. 2017). It
has been shown that simvastatin down-regulated MMP-9 protein expression and mRNA levels
in invitro (Chen and
Chang 2014)
and reduced plasma MMP-9 levels in human (Andrade et al. 2013) studies. Li et
al. (2015) also showed that simvastatin has the
ability to suppress LPS-stimulated MMP-9 release and mRNA expression.

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