A total of 40 specific-pathogen-free male Sprague-Dawley (SD) rats (weight, 250–300 g; age, 8–10 weeks) were obtained from the Department of Laboratory Animal Science of Nanchang University (Jiangxi, China). The production license number was 96021. All animal procedures were approved and conducted in accordance with the Guidelines for the Care and Use of Animals of the Ethics Committee of Nanchang University (Nanchang, China). Male Sprague-Dawley rats were housed in individual cages in a temperature-controlled room (18–26°C, 40–60% humidity) under a 12-h light/dark cycle and with ad libitum access to food and water. The rats were sacrificed by tail vein air embolism following anesthetization with 1% pentobarbital sodium (45 mg/kg; Sinopharm Chemical reagent Co., Ltd., Shanghai, China).

All reagents and assay kits were purchased from commercial sources. APS and IBU were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China) and Sigma-Aldrich, Inc. (Merck KGaA, Darmstadt, Germany), respectively. Rat IL-6 ELISA and TNF-α ELISA kits were purchased from Elabscience Biotechnology Co., Ltd. (Wuhan, China). Acetylcholine (ACh) assay kit was purchased from Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China). Furthermore, the iMark microplate absorbance reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and DNM-9602 microplate reader (Perlong Medical Equipment Co., Ltd., Nanjing, China) were used for microplate spectroscopic methods and ELISA. Finally, an optical microscope (BX45; Olympus Soft Imaging Solutions GmbH, Münster, Germany) was used for the observation of tissue sections.

Rats were anesthetized by an intraperitoneal injection of 45 mg/kg pentobarbital sodium. Polymicrobial sepsis was induced by CLP as described previously (12). The whole process was performed under sterile conditions with the abdominal skin sterilized by 75% alcohol. In brief, laparotomy was conducted through a 1.5-cm lower-midline incision. The cecum was exposed and ligated immediately distal to the ileocecal valve to avoid intestinal obstruction, and was then punctured twice with a 16-gauge needle, squeezed gently to force out a small volume of feces and returned to the abdominal cavity. Finally, the abdomen was sutured layer-by-layer.

A total of 40 SD rats were randomly divided into four groups: CLP (n=10), IBU 20 mg/kg (n=10), APS 25 mg/kg (n=10) and IBU+APS (n=10) groups. APS was injected through the abdomen for 4 consecutive days prior to CLP. IBU was administered by injection at 2 and 8 h after CLP.

Blood samples of the rats in each group were collected from the femoral vein prior to and at 2, 8 and 12 h after CLP. The samples were immediately treated with sodium citrate, and then centrifuged at 3,400 × g and 4°C (TGL-16; Changsha Xiangyi Centrifuge Instrument Co., Ltd., Changsha, China) for the separation of serum and plasma. The latter was stored at −80°C for the measurement of ACh, TNF-α and IL-6. The left lungs were removed and rinsed several times in ice-cold saline, then dried with filter paper and stored at −80°C for subsequent mRNA detection. The right lungs were cut into blocks and fixed with 10% neutral formalin. Pathological changes in the upper lobe were observed using a microscope after hematoxylin and eosin staining.

The ACh content of the samples was measured by a microplate spectroscopic method according to the manufacturer's protocol after rewarming the samples. Furthermore, TNF-α and IL-6 were measured using ELISA kits, according to the manufacturer's instructions.

The mRNA expression of the α7 receptor in lung tissues was analyzed by RT-qPCR. The total RNA was isolated from the lung tissues of each group using TRIzol reagent (Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer's instructions. In total, 5 µg total RNA was reverse transcribed into cDNA using the PrimeScript^® 1st Strand cDNA Synthesis kit (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's protocol, and the PCR reaction mixtures were prepared using SYBR-Green qPCR master mix (Thermo Fisher Scientific, Inc., Waltham, MA, USA). β-actin was used as an internal control. The primers were synthesized by Sangon Biotech Co., Ltd., (Shanghai, China). The sequences were as follows: β-actin forward: 5′-AACCCTAAGGCCAACAGTGAAAAG-3′ and reverse: 5′-TCATGAGGTAGTCTGTCAGGT-3′; α7 forward: 5′-AAACTCACAGATGGGCAAGG-3′ and reverse: 5′-CCGTAAGCAACACGACTGAC-3′. The cycling conditions were as follows: 1 cycle at 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec, 55°C for 20 sec and 72°C for 1 min. The relative expression level of α7 receptor mRNA in the lung tissues were determined by the 2^−ΔΔCq method (13). The experiments were performed in triplicate.

SPSS 12.0 (SPSS, Inc., Chicago, IL, USA) was used for data analysis. All data are presented as the mean ± standard deviation and were compared using Dunnett's multiple comparison tests. P<0.05 was considered to indicate a statistically significant difference. The graphs and figures were created using Graphpad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA).

As indicated in Figs. 1 and 2, the TNF-α and IL-6 concentrations in the serum exhibited the same trend in each group after modeling: TNF-α and IL-6 concentrations started to increase 2 h after CLP and peaked at 8 h after which their concentration declined slightly. TNF-α and IL-6 in the serum of the APS, IBU and APS + IBU groups were reduced compared with those of the CLP group. However, only APS combined with IBU inhibited the increase of TNF-α and IL-6 in the serum of CLP-induced septic rats significantly (P<0.05 vs. the CLP group). These results indicate that APS combined with IBU inhibits the generation of the inflammatory mediators TNF-α and IL-6 during sepsis more strongly than either component alone and display superior anti-inflammatory effects.

As shown in Fig. 3, the ACh levels in the serum differed at various time points after modeling. Compared with the ACh levels in the serum of the CLP and the IBU groups, those in the APS + IBU groups were significantly increased (P<0.05). Furthermore, the increase was more evident in the combined therapies group. This suggests that the anti-septic effects of APS combined with IBU may be mediated by the CAP to some extent.

Since nAChR α7 is required for inhibiting cytokine synthesis by the CAP, the expression of nAChR α7 was measured in the lung tissues of rats from the four groups. Compared with the CLP group, the IBU, APS and APS + IBU groups exhibited increased nAChR α7 mRNA expression, and the expression level was significantly increased in the APS + IBU group (P<0.05; Fig. 4). These results indicate that the CAP may be associated with the anti-inflammatory effect of APS combined with IBU.

Hematoxylin and eosin stained lung tissues in the CLP group showed collapsed alveoli, evident exudation, enlargement of the alveolar septum, and infiltration with numerous interstitial lung inflammatory cells when observed under a microscope (Fig. 5A). However, in the IBU and APS groups, the lung cells exhibited alveolar collapse, exudation, widening of the alveolar septum and some interstitial lung inflammatory cell infiltration (Fig. 5B and C). However, in the IBU + APS group the pathological features were clearly ameliorated (Fig. 5D), indicating a lower degree of pathological damage and a significantly improved lung histology.