The following reagents and kits were used in the present study: Rabbit polyclonal anti-tyrosine hydroxylase (TH; cat. no. 2792; Cell Signaling Technology, Inc., Boston, MA, USA), rabbit polyclonal anti-OX-42 (cat. no. orb11009; Biorbyt, Cambridge, UK), hydroethidine (Molecular Probes, Eugene, OR, USA), LPS (Sigma-Aldrich, St. Louis, MO, USA), heparin (Qianhong Bio-pharma Co., Ltd., Changzhou, China), SST (ProSpec, East Brunswick, NJ, USA), biotinylated goat anti-rabbit secondary antibody (cat. no. A0277) and horseradish peroxidase (HRP)-labeled streptavidin (Beyotime, Shanghai, China), bicinchoninic acid (BCA) kit (Beyotime), Rat TNF-α ELISA kit, Rat IL-1β ELISA kit and Prostaglandin E2 ELISA kit (PGE2; USCN, Wuhan, Hubei, China).

All experiments were performed according to the approved animal protocols and guidelines established by Chung et al (12). 144 female Sprague Dawley rats were provided by the Experimental Animal Centre of China Medical University (Shenyang, China). The animal study protocol was approved by the Animal Experimental Committee of China Medical University, and the mice received humane care according to the Principles of Laboratory Animal Care. They were randomly assigned to six experimental groups and received unilateral administration of 3 μl phosphate-buffered saline (PBS), LPS (5 μg in 3 μl PBS), 2 μl saline + LPS, 2 μl SST (20 μg/kg) + LPS, 2 μl SST (40 μg/kg) + LPS, 2 μl SST (40 μg/kg) respectively, into the right SN as previously described by Chung et al (10). 24 rats of each treated group were separated into three different subgroups for various analyses, including OX-42 and hydroethidine tests of brains after 24 h of treatment, western blot and ELISA assays of SN after 24 h of treatment, as well as immunohistochemical detection of TH and Nissl in the SN after 7 days of treatment. SST was injected 1 h prior to LPS treatment.

Brain tissues were prepared for immunohistochemical staining as previously reported (12). Tissues were dehydrated by using a graded ethanol series of 70% ethanol for 2 h, 80% overnight, 90% for 2 h and 100% for 2 h. Brain tissues were then post-fixed in dimethylbenzene (China National Medicines Corporation Ltd., Beijing, China) for 30 min and embedded in dimethylbenzene-paraffin at 60°C for 2 h, after which samples were embedded in a metal frame. Coronal sections (5 μm) cut by a sliding microtome CM69001 (Leica Microsystems, Mannheim, Germany) were spread in warm water and placed onto glass slides to dry in a 70°C-chamber for 40 min. Sections were dewaxed using ethanol and then boiled in antigen retrieval solution for 10 min. The cooled sections were incubated in 3% H₂O₂ (China National Medicines Corporation Ltd.) for 15 min at room temperature and then blocked with normal goat serum (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) for 15 min. Sections were incubated overnight at room temperature with rabbit anti-OX-42 (1:100; cat. no. orb11009) for microglia and rabbit anti-TH (1:100; cat. no. 2792) for DA neurons. Following removal of unbound primary antibodies by washing, biotinylated goat anti-rabbit secondary antibody (1:200; cat. no. A0277) was added and incubated for 30 min at 37°C. Sections were then washed with PBS and incubated with HRP-labeled streptavidin for 30 min at 37°C. Finally, 100 μl diaminobenzidine (Beijing Solarbio Science & Technology Co., Ltd.) was added for coloration and hematoxylin stain (Beijing Solarbio Science & Technology Co., Ltd.) for counterstaining. Hydroethidine (Molecular Probes) was used for in situ detection of O₂⁻ and O₂⁻ -derived oxidants. For Nissl staining, a number of the SN tissue samples were stained in 0.5% cresyl violet (China National Medicines Corporation Ltd.). Following washing with water and dehydrating with ethanol as well as treating with dimethylbenzene (China National Medicines Corporation Ltd.), stained samples were analyzed under a stereo microscope (BX51; Olympus Corporation, Tokyo, Japan) or viewed with a confocal laser scanning microscope (FV1000S-SIM/IX81; Olympus Corporation).

The total number of TH-positive neurons was counted in the various groups at seven days post-injection (LPS, PBS, SST or a combination) using the stereo microscope BX51 (Olympus Corporation). This unbiased stereological method of cell counting according to a previously described method is not affected by either the counted elements (neurons) or the size of the reference volume (SN) (13).

For western blot analysis, protein was extracted from the SN of eight rats from each group following 24 h of treatment. Following determination of the protein concentration using a BCA kit, 40 μg protein was boiled in PBS with 5X loading buffer (Beijing Solarbio Science & Technology Co., Ltd.) for 5 min. The bands of protein separated by SDS-PAGE (8% gel for iNOS, 10% gel for NF-κB p65 and NF-κB p-p65, and 12% gel for Cox-2) were transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) using an electroblot apparatus (DYCZ-40D, Beijing, China). Filters were blocked in 5% non-fat milk and incubated separately overnight at 4°C with the following primary antibodies: Rabbit polyclonal anti-iNOS (1:1,000; cat. no. bs-2072R; Bioss, Beijing, China), goat polyclonal anti-Cox-2 (1:100; sc-1747), mouse monoclonal anti-NF-κB p-p65 (1:100; cat. no. sc-166748) and rabbit polyclonal anti-NF-κB p65 (1:100; cat. no. sc-372) obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Membranes were then washed with Tris-buffered saline containing Tween-20 (TTBS) (BioSharp, Hefei, China) and incubated with the following corresponding HRP-conjugated secondary antibodies: Goat anti-mouse immunglobulin (Ig)G-HRP (1:5,000; cat. no. A0216), goat anti-rabbit IgG-HRP (1:5,000; cat. no. A0208) and donkey anti-goat IgG-HRP (1:5,000; cat. no. A0181) (Beyotime, Shanghai, China) for 45 min at 37°C. Following washing with TTBS, protein bands were visualized using enhanced chemiluminescence reagent (cat. no. E002-5; Qihai Biotec, Shanghai, China). Protein levels were quantified by gray value analysis using Gel-Pro-Analyzer (Media Cybernetics, Inc., Rockville, MD, USA).

The production of TNF-α, IL-1β and PGE2 from the SN of rats was determined by ELISA. Proteins were extracted through homogenizing tissues and quantitated using the BCA kit. ELISA was then performed according to the manufacturer’s instructions.

Values are expressed as the mean ± standard deviation. All raw data were analyzed by one-way analysis of variance followed by the Bonferroni test for post hoc comparisons. Statistical analyses were performed using GraphPad Prism 5.0 software (GraphPad Software Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference between values.

LPS injection into the SN was previously shown to result in a considerable loss of TH- and Nissl-positive cells, as well as alterations in the morphology of TH-positive neurons to shrunken neuronal cell bodies (12,13). To investigate the potential of SST to prevent LPS-induced neurotoxicity of nigral neurons, SST was administered 1 h prior to LPS injection. SST significantly attenuated the LPS-mediated loss of TH-positive DA neurons in the SN and preserved normal neuronal morphology, as evidenced by an increased number of healthy cell bodies and processes with enhanced staining intensity (Fig. 1A-F). As shown in Fig. 1G, the morphology of the neurons were integrative and well-maintained. In Fig. 1H, the number of healthy neurons was markedly decreased, and the number of dead neurons increased, as characterized by the chromatolysis of Nissl bodies and the presence of shrunken neuronal cell bodies with pyknotic nuclei surrounded by a thin band of cytoplasm. In Fig. 1K, the above pathological damages were notably reduced, following pretreatment with higher concentrations of SST. The results of the LPS + saline group (Fig. 1I) were similar to those exhibited in Fig. 1H. In addition, the results of the LPS + 20 g/kg SST group (Fig. 1J) were similar, but not as obvious, as those exhibited in Fig. 1K, and the results of the 40 μg/kg SST group (Fig. 1L) were similar to the results shown in Fig. 1G, thus indicating that SST had no cytotoxic effects on the neurons. These results showed that SST had neuroprotective effects on LPS-treated SN.

It has been reported that LPS is able to activate rat microglia and lead to neuronal death in vivo (14). Thus, the present study investigated the effect of SST on LPS-induced microglial activation in the SN. SN sections were prepared for immu-nohistochemical staining using antibodies against OX-42 to detect microglial activation. The majority of OX-42-positive microglia exhibited a resting morphology in the PBS-injected SN (Fig. 2A), whereas LPS-treated samples showed activated microglia with enhanced staining intensity and larger cell bodies with short, thick processes (Fig. 2B). Pre-treatment with SST dramatically decreased the number of activated microglia induced by LPS compared that in the saline-pre-treated control (Fig. 2C-E), while SST alone had no effect on microglial activation (Fig. 2F). These findings suggested that SST inhibited the LPS-induced activation of microglia.

Recent studies have suggested that activated microglia are able to produce O₂⁻ and O₂⁻ -derived oxidants and they are thought to mediate the loss of nigral DA neurons in vitro and in vivo (15,16). Hence, the present study investigated whether SST was able to enhance DA neuronal survival by inhibiting LPS-induced production of ROS. Accumulation of ethidium, the fluorescent product of oxidized hydroethidine, was signifi-cantly increased at 48 h in the LPS-treated SN compared with that in the PBS-injected controls, and the LPS-induced oxidant production was dramatically decreased by SST (Fig. 3). These results showed that SST inhibited LPS-induced ROS production.

Several studies have demonstrated that the production of TNF-α, IL-1β and PGE2 are upregulated in LPS-injected SN (14,17). Neuroinflammation is thought to mediate DA neuronal death in the SN (18). Therefore, the present study examined whether SST was able to decrease DA neuronal death by regulating LPS-induced production of TNF-α, IL-1β and PGE2 in the SN. When 20 μg/kg SST was administered prior to LPS injection, the amount of TNF-α, IL-1β and PGE2 was significantly reduced by 24.9% (Fig. 4A), 27.9% (Fig. 4B) and 29.2% (Fig. 4C), respectively. The production of TNF-α, IL-1β and PGE2 was decidedly decreased by 41.8, 43.9 and 41.6%, respectively, when 40 μg/kg SST was administered prior to LPS treatment, while SST had no effect on the production of TNF-α, IL-1β and PGE2 when it was used alone. This confirmed that SST was able to decrease LPS-induced production of TNF-α, IL-1β and PGE2.

Upregulation of Cox-2 and iNOS have been implicated in DA neuronal cell death of patients with degenerative brain diseases (19,20). Inhibition of the activation of NF-κB p65 was able to prevent DA neuronal cell loss in a 1-methyl-4-phenyl-1,2,3,6-tetrahyropyridine-induced mouse model of PD (21). Accordingly, the present study examined the effect of SST on DA neuronal survival through affecting LPS-induced expression of Cox-2, iNOS and the activation of NF-κB in the SN. 24 hours following injection, LPS enhanced the expression of Cox-2, iNOS and activated NF-κB p65 via phosphorylation (Fig. 5). Administration of 20 μg/kg SST prior to LPS injection reduced the expression of Cox-2, iNOS and NF-κB p-p65 by 23.4, 26.1 and 35.9%, respectively. When 40 μg/kg SST was given prior to LPS injection, the expression of Cox-2, iNOS and NF-κB p-p65 was decreased by 40.5, 37.3 and 58.4%, respectively. SST alone had no effects on Cox-2, iNOS and NF-κB p-p65 expression. It was therefore demonstrated that SST was able to inhibit the expression of Cox-2, iNOS and NF-κB p-p65.