SEPS1, p38 mitogen-activated protein kinase (p38 MAPK), phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) and β-actin antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Oligonucleotide primers and dNTP mix were purchased from Bio Basic Inc. (Bio Basic, Toronto, Canada). Protein extraction and BCA protein assay kits were purchased from Promega Co. (Madison, WI, USA). Pyrogen-free water was purchased from Shanghai Yihua Clinical Medicine Technologies Company (Shanghai, China). LPS (from E. coli strain O55:B5) and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Inbred BALB/c mice (male, 17–20 g), were purchased from the Institute of Medical Animal Experimental Center, Peking Union Medical College (Beijing, China). The mice were kept at standard laboratory conditions of temperature and humidity with a 12-h light/dark cycle. All experiments were performed according to the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996) and were treated ethically. The study was approved by the Ethics Committee of PLA General Hospital (Beijing, China).

siRNAs for SEPS1 and the scrambled control were synthesized in vitro using a kit from Ambion (Austin, TX, USA; Silencer^® Negative Control No. 1 siRNA). Primer sequences are provided in Table I (Beijing AuGCT DNA-SYN Biotechnology Co., Ltd., Beijing, China). The mice were transfected with siRNA oligonucleotides (0.5 μg) formulated by Lipofector 2000 (Beyotime Institute of Biotechnology, Jiangsu, China) via tail vein injection. In total, 30 mice were randomly assigned to three groups: i) H group (LPS-induced sepsis group; n=10): Mice with intraperitoneal injection of LPS (10 mg/kg); ii) K group (scrambled siRNA group; n=10): Mice transfected with scrambled control siRNA 12 h prior to injection with LPS; iii) L group (SEPS1 siRNA group; n=10): Mice transfected with SEPS1 siRNA 12 h prior to injection with LPS. The mice in all groups fasted with free access to water following LPS injection and were continuously monitored over a 24-h period. For each time-point (12 and 24 h), five animals were used.

Following the LPS injections at 12 and 24 h, the blood was collected in heparinized tubes by extirpating the left eyeball of five mice in each group. Serum was isolated by centrifugation at 4,000 × g, 4°C for 15 min and maintained at −70°C until it was required for further analysis. The levels of serum alanine transaminase (ALT), aspartate aminotransferase (AST), serum creatinine (Cr), blood urea nitrogen (BUN), myocardial kinase (CK-MB), creatine kinase (CK) and lactic dehydrogenase (LDH) were assessed using an automatic biochemical analyzer (HITACHI 7170, Hitachi, Ltd., Tokyo, Japan) with commercial kits (LDH-Cytotoxicity Assay Kit II; Biosino Biotechnology Co., Ltd., Beijing, China).

At each time-point of assessment, the mice were sacrificed once the blood had been obtained. Under strict aseptic conditions, 0.1–0.2 g of liver tissue was removed and placed into a pyrogen-free homogenizer. Three volume equivalents of pyrogen-free saline was added and the liver tissue was homogenized in an ice bath. The liver homogenate supernatant was prepared by centrifugation at 10,000 × g at 4°C for 15 min and maintained at −30°C until it was required for further analysis. The samples were analyzed for TNF-α and IL-6 using an ELISA kit (Endogen Inc., Woburn, MA, USA) according to the manufacturer’s instructions. The absorbance was measured at 450 nm. The concentrations of TNF-α and IL-6 in the experimental samples were extrapolated from a standard curve.

Once the mice had been sacrificed, the liver tissue samples were maintained in liquid nitrogen for western blot analysis. The liver tissue samples were cut up and ground with pre-cooled lysis buffer. Equal quantities of protein were separated by SDS-PAGE (10% gel) and transferred to nitrocellulose (CN) membranes (Hybond Inc., Escondido, CA, USA). The membranes were blocked for 1 h at room temperature with 10% non-fat dry milk and subsequently incubated at 4°C overnight with anti-mouse antibody (1:300 dilution in blocking buffer). Once the membranes had been washed, they were incubated with a 1:1,000 dilution of horseradish peroxidase-conjugated anti-rabbit immunoglobulin G secondary antibody (ZSGB-Bio Co., Beijing, China) for 1 h at room temperature. The blots were visualized using an Enhanced Chemiluminescence kit (Pierce Biotechnology Inc., Rockford, IL, USA), and data were quantified using the Gel Doc EQ system (Bio-Rad, Hercules, CA, USA)

Total cellular RNA was extracted with TRIzol^® reagent (Takara, Seoul, Korea) according to the manufacturer’s instructions. Reverse transcription was performed at 37°C for 1 h in a reaction mixture containing 2 mg total RNA, 0.5 mg oligo-dT primer, 10 mM dNTPs, 25 U RNase inhibitor and 200 U M-MLV reverse transcriptase (Promega). qPCR was performed using a SYBR FAST qPCR kit (KAPA, Boston, Massachusetts, USA) with 10 ng cDNA according to the manufacturer’s instructions. Cycling conditions were as follows: 95°C for 1.5 min and 40 subsequent cycles of 95°C for 3 sec, 56.4°C for 40 sec and 72°C for 10 sec. Upon the completion of 40 PCR amplification cycles, melting curve analysis was performed. SEPS1 mRNA levels were normalized to actin, a stable housekeeping gene.

Liver and lung tissue samples were deparaffinized three times with xylene for 5 min each, followed by rehydration with a series of absolute, 70% and 50% ethanol for three min each and washed under running tap water for 5 min. Tissue samples were subsequently blocked with 3% hydrogen peroxide for 5 min, placed in distilled water for 5 min, followed by 10 min incubation with a 1:20 dilution of goat serum. Subsequently, tissue samples were incubated with anti-SEPS1 polyclonal serum at 4°C overnight and washed five times with phosphate-buffered saline and Tween 20 (PBST), for two min each. Tissue samples were then incubated with a 1:1,000 dilution of horseradish peroxidase-conjugated antibody (Sigma-Aldrich) for 30 min and washed again with PBST. Following washing, the samples were developed with 3,3′-diaminobenzidine substrate solution for 3 min and washed again with PBST. Finally, the tissue samples were counterstained with Harris’ hematoxylin (Sigma-Aldrich) for 1 min, followed by washing, differentiation with ethanol containing 1% acid alcohol solution, bluing with ammonia in water, another washing step, dehydration with increasing series of alcohols, clearance with xylene and subsequent mounting with dibutyl phthalate xylene (DPX).

Processed liver and lung tissue samples were deparaffinized three times with xylene for two min each, rehydrated with a series of absolute, 95% and 80% ethanol for two min each, followed by a wash with running tap water for 5 min. Subsequently, the tissue samples were stained with Harris’ haematoxylin (Sigma-Aldrich) for 5 min and washed with running tap water. Differentiation with 1% acid alcohol solution was performed for 10 sec, followed by washing and bluing by placing the tissue samples into a solution of ammonia in water for 10 sec. Following a washing step, the samples were counterstained with eosin Y (Sigma-Aldrich) for two min, dehydrated with an increasing series of ethanol for two min each, cleared by three washes with xylene for two min each and finally mounted with DPX.

Values are expressed as the mean ± standard deviation from a minimum of three independent experiments. The statistical significance of the results between each treated group was analyzed by Student’s t-test. P<0.05 was considered to indicate a statistically significant difference.

In order to examine whether the SEPS1 gene was silenced, the expression of the SEPS1 gene was detected by qPCR and western blot analysis. SEPS1 protein expression in liver tissue decreased 12 and 24 h following transfection with SEPS1 siRNA (Fig. 1). The results of the immunohistochemical analysis also revealed that SEPS1 protein expression in liver and lung tissue decreased in the SEPS1 siRNA group (Figs. 2 and 3). qPCR results demonstrated that SEPS1 gene expression in liver tissue decreased significantly compared with the control group (Fig. 4).

In the present study, important biochemical indicators were detected in order to reflect the effects on the liver, kidney and heart following siRNA silencing of the SEPS1 gene. At 12 h following injection with LPS, the concentrations of ALT and BUN exhibited no statistical difference among the three groups (P>0.05); AST and LDH levels exhibited significant differences between the groups except for a non-statistical difference between the H and L groups (P>0.05); the Cr levels showed no statistical difference among the groups except for a significant difference between the H and L groups (P<0.05); CK and CK-MB concentrations exhibited no statistical difference among the groups except for a significant difference between the K and L groups (P<0.05). At 24 h following injection with LPS, the concentration of ALT was significantly higher in the H group than in the other two groups (P<0.05), and demonstrated no statistical difference between the K and L groups; there were significant differences among the three groups in the concentrations of AST and BUN (P<0.05); there was no significant difference among the three groups in LDH levels (P>0.05); the statistical results of difference in Cr, CK and CK-MB concentrations among the three groups at 24 h were the same as that at 12 h. There was a significant increase in the ALT levels and a significant decrease in the Cr levels at 24 h compared with 12 h among the groups (P<0.05). Furthermore, there was a significant increase in the AST levels and a significant decrease in the BUN levels at 24 h compared with 12 h in the H and K groups (P<0.05); however, the difference in group L was not significant. There was no significant difference in CK, LDH and CK-MB concentrations at 24 h compared with 12 h among the groups (P<0.05) (Figs. 5, 6 and 7). The results demonstrated that the blood levels of ALT, AST, BUN, CK-MB, CK and LDH all increased in the SEPS1 siRNA group following silencing of the SEPS1 gene with SEPS1 siRNA (Tables II and III).

To investigate the proinflammatory cytokine levels in mice with sepsis, the effect of SEPS1 siRNA on the circulating levels of primary (TNF-α) and secondary (IL-6) cytokines was examined. At 12 h following LPS injection, IL-6 and TNF-α levels were highest in group K and lowest in group H. However, neither IL-6 nor TNF-α levels exhibited a statistical difference among the three groups (P>0.05). At 24 h, following LPS injection, IL-6 and TNF-α levels were highest in group L and lowest in group K. There was a significant difference in IL-6 levels between all groups except for the difference between groups H and K, whereas no significant difference in TNF-α levels was present between the groups except for a statistically significant difference between groups K and L. There was a significant decrease in the IL-6 and TNF-α levels at 24 h compared with 12 h in the K group (P<0.05). However, there was no significant difference in IL-6 and TNF-α levels at 24 h compared with 12 h in the other groups (P>0.05)(Fig. 8).

MAPKs are involved in signal transduction pathways leading to the regulation of inflammatory mediators. In the present study, the SEPS1 siRNA group enhanced p-p38 protein levels at 12 and 24 h following LPS stimulation (Fig. 9), suggesting that SEPS1 siRNA treatment activated the phosphorylation of p38 MAPK.

Pathological results demonstrated that the liver and lung cells swelled significantly and the inflammatory cells infiltrated the portal area, indicating that cell lesion markedly occurred in the SEPS1 siRNA group compared with the control group, which may correlate with the decrease in SEPS1 gene expression (Figs. 10 and 11).