Protocatechuic Aldehyde Protects against ... - Wiley Online Library

Basic & Clinical Pharmacology & Toxicology, 2012, 110, 384–389

Doi: 10.1111/j.1742-7843.2011.00827.x

Protocatechuic Aldehyde Protects against Experimental Sepsis In Vitro and In Vivo Yong Xu1, Wang-Lin Jiang1, Shu-Ping Zhang1, Hai-Bo Zhu2 and Jian Hou2 1

School of Pharmaceutical Sciences, Binzhou Medical University, Yantai, China, and 2State Key Laboratory of Long-acting and Targeting Drug Delivery Technologies, Yantai, China (Received 22 August 2011; Accepted 19 October 2011)

Abstract: Recent studies have demonstrated that nuclear factor-jB (NF-jB) and high-mobility group box 1 (HMGB1) are associated with the pathophysiology of sepsis. The present study was carried out to investigate the effects of protocatechuic aldehyde (PA) on an experimental model of sepsis induced by caecal ligation and puncture (CLP) in rats and to elucidate the potential mechanism in the cultured murine macrophage cell line, RAW264.7 cells. Treatment of RAW 264.7 cells with PA blocked TNF-a-induced NF-jB phosphorylation and decreased HMGB1 expression. Septic rats received doses of 50 mg of PA alone or plus Imipenem by intravenous bolus injection into the tail vein. The results showed that PA reduced serum levels of HMGB1 and triggering the receptor expressed on myeloid cells, it attenuated myeloperoxidase in the lung, liver and small intestine, while it up-regulated serum level of IL-10. Meanwhile, PA alone or plus Imipenem reduced CLP-induced lethality in septic rats. These data indicate that the anti-septic effect of PA is mediated by decreasing local and systemic levels of a wide spectrum of inflammatory mediators. The protective effects of PA might block the inflammatory cascades through HMGB1 and NF-jB signalling pathway. Our studies enhance the case for the use of PA in sepsis, and PA therefore seems promising in the treatment of sepsis in human beings.

Sepsis is a significant health problem and a major cause of death in intensive care units worldwide. Approximately 750,000 people are afflicted with sepsis annually in the United States alone [1]. Despite improved medical care, the mortality rate still ranges from 30% to 60% [2]. The pathogenesis of sepsis is characterized by inflammatory and immune responses that lead to downstream tissue damage, organ failure and ultimately death. Unfortunately, clinical trials using therapeutic strategies that target proinflammatory mediators such as TNF-a and IL-1b have failed to improve sepsis-related survival [3]. Many studies have indicated that nuclear factor kappaB (NF-jB) is activated in sepsis, and this activation is mediated by Toll-like receptor 4 (TLR4) [4]. This suggests that inhibition of NFjB activation may represent a treatment strategy in sepsis. Inhibition of NF-jB activation improves survival of septic mice [5,6]. High-mobility group box 1 (HMGB1) is an evolutionarily conserved protein present in the nucleus of almost all eukaryotic cells where it functions to stabilize nucleosomes and acts as a transcription factor [7]. HMGB1 is an important danger-associated molecular pattern [8], which enhances the inflammatory response in septic shock [9,10], interacts with TLR4 and increases TLR4-mediated NF-jB activation [11]. Inhibition of HMGB1 may be a potential target in sepsis therapy.

Author for correspondence: Wanglin Jiang, School of Pharmaceutical Sciences, Binzhou Medical University, Yantai 264003, China (fax +86 535 6706060, e-mail [email protected]).

Protocatechuic aldehyde (PA) is a naturally occurring compound, extracted from the degradation of phenolic acids. Many bioactivities of PA have been reported amongst these anti-tumour and antioxidant activity [12], suppression of TNF-a-induced ICAM-1 and VCAM-1 expression [13]. The purpose of the current study was to determine the effect of PA administered after induction of caecal ligation and puncture (CLP) sepsis in the presence and absence of Imipenem on inflammation, bacterial burden and lethality. Experiments were also conducted on the effect of PA on LPS-induced inflammatory response in RAW 264.7 cells and whether NF-jB activation was regulated by HMGB1 during sepsis. Materials and Methods Materials. Protocatechuic aldehyde (PA, formula: C7H6O3, molecule weight: 138.12, CAS number: 139-85-5) was obtained from Shandong Engineering Research Center for Nature Drug, Yantai, China. The purity of PA was 99.3%. Imipenem was purchased from Maigao Technology Company (Shenzhen, China) with a purity of 99.7%. RPMI 1640 medium was purchased from Gibco. Foetal calf serum was purchased from Sijiqing Company (Hangzhou, China). LPS was from Escherichia coli serotype 055: B5, purchased from the Second Army Medicine University (Shanghai, China). Enzyme-linked immunosorbent assay (ELISA) kits of high-mobility group box 1 protein (HMGB1), TNF-a and IL-10 were purchased from Xitang Biology Technology Company (Shanghai, China). The triggering receptor expressed on myeloid cells 1 (TREM-1) ELISA kits were purchased from Jingtian Biology Technology Company (Shanghai, China). Limulus amoebocyte lysate (LAL) was purchased from Xiamen Zhanjiang A & C Biological Company (Shanghai, China). Myeloperoxidase (MPO) kits were purchased from Maisha Biology Technology Company (Shanghai, China).

2011 The Authors Basic & Clinical Pharmacology & Toxicology 2011 Nordic Pharmacological Society

PROTOCATECHUIC ALDEHYDE AND SEPSIS Animals. Adult male Sprague–Dawley rats (200–250g) were obtained from Shandong Luye Pharmaceutical Company (China). All animals were housed individually at 20 € 2C, at a humidity of 50 € 10%, in a 12-hr light:dark cycle and with free access to chew and water. The experimental procedures were approved by the institutional animal ethics committee. Cell culture and western blot analysis. The murine macrophage cell line RAW264.7 cells were maintained in RPMI 1640 medium supplemented with 100 U ⁄ ml penicillin ⁄ streptomycin, 2 mM L-glutamine, 50 lM 2-mercapto ethanol and 10% heat-inactivated foetal calf serum at a concentration of 1 · 106 cells ⁄ ml. Then, it was incubated with complete medium in the absence (unstimulated) or presence of LPS (0.1 lg ⁄ ml), and then, PA was added to the medium at various concentrations (0.33–9 lM). Cell-free supernatants were collected after PA treatment for 24 hr. Cell viability was assessed by measuring lactate dehydrogenase (LDH) in the medium using a 7170-automatic biochemistry analyzer (Tokyo, Japan). The levels of TNF-a, IL-6 and IL-10 in the medium were determined using ELISA kits. For the experiment of TNF-a-stimulated RAW264.7 cells in vitro, RAW264.7 cells were pre-incubated with the HMGB1 inhibitor, glycyrrhizin (the final concentration was 100 lM) or PA (3 or 9 lM) for 30 min. and then incubated with TNF-a (20 ng ⁄ ml) for 30 min. Cells were cultured for 24 hr and then washed twice with ice-cold PBS and lysed in NP40 lysis buffer (Biosource, Camarillo, CA, USA) (50 mM Tris, pH 7.4, 250 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 1% NP-40 and 0.02% NaN3), supplemented with 1 mM PMSF and 1· protease inhibitor cocktail (Sigma, Saint Louis, MO, USA). Equal amounts of cell protein (50 lg) were separated by SDS–PAGE and analysed by western blot using specific antibodies to HMGB1, phosphor-NF-jB and PCNA (loading control). The antibodies were all purchased from Beijing Biosynthesis Biotechnology Company (Beijing, China). Optical densities of the bands were scanned and quantified with a Gel Doc 2000 (Bio-Rad Laboratories, Hemel Hempstead, UK). Data were normalized against those of the corresponding PCNA bands. Results were expressed as fold increase over control. Septic shock was induced by caecal ligation and puncture (CLP). Prior to the CLP surgery, rats were fasted overnight but still with free access to water. CLP surgery was performed as described previously [14]. All animals were anaesthetized by intramuscular injection of ketamine 50 mg ⁄ kg. The abdomen of each rat was shaved and prepared with iodine. Through a midline laparotomy, the caecum was filled with faeces by milking the stools back from the descending colon and then ligated just below the ileocaecal valve with a 3-0 silk ligature. The anti-mesenteric coecal surface was punctured twice with a 21-gauge needle twice and gently squeezed to ensure patency of the perforation sites below the ligature. The bowel was placed back into the peritoneal cavity, and the abdomen was closed in two layers with plain gut surgical suture 4-0 and skin clips. The operative procedure was carried out under aseptic conditions. The sham-operated rats received a midline laparotomy, the caecum was exposed, but not ligated with needle and punctured and then closed using surgical suture. All CLP-operated rats were administered a saline bolus (6 ml) subcutaneously. After surgery, the animals were put back into their cages with free access to food and water. A pilot study was conducted with three different doses of PA (25, 50 or 100 mg ⁄ kg) to determine the dose-dependent effect in CLP surgery rats. It was observed that PA treatment at doses of 50 and 100 mg ⁄ kg significantly (p < 0.05) lowered elevated levels of TREM1 and HMG1, as well as the MPO of lung, liver and small intestine in septic rats. Hence, PA 50 mg ⁄ kg was chosen for our study. A total number of 130 rats were used and divided into two groups for different experiments: the first group was used to assess mortality and the second group for biochemistry analysis. There were 80 rats in the first group, and these rats were divided into four subgroups: (i) CLP group (vehicle-treated group, n = 20); (ii) PA 50 mg ⁄ kg group (n = 20); (iii) Imipenem 20 mg ⁄ kg group (n = 20); (vi) Imipe-

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nem 20 mg ⁄ kg + PA 50 mg ⁄ kg group (n = 20). After CLP surgery for 1 hr, all rats in this group received the above drug by intravenous bolus injection into the tail vein once every 12 hr for 3 continuous days. Mortality was monitored on day 12 after surgery. There were 50 rats in the second group, and these were divided into five subgroups of 10 rats, cf. the above. All rats in this group received only two injections at 1 and 13 hr, and they were anaesthetized with 10% chloral hydrate at 20 hr after CLP surgery to collect blood. In addition, the small intestine, liver and lung were collected for biochemistry measurement. Determination of serum levels of IL-10, TREM-1 and HMGB1, and MPO of lung, liver and small intestine. Serum levels of IL-6, IL-10, HMGB1 and TREM-1 were measured using ELISA kits according to the manufacturer’s instructions. Neutrophil infiltration in the lung, liver and small intestine was monitored by measuring MPO activity as reported previously [15]. The assay was carried out according to the instructions provided by the manufacturer. Western blots analysis in septic rats. For western blot analysis of collected lung tissues, the tissues were defrosted and immersed in icecold lysis buffer (50 mM Tris–HCl, 1 mM EDTA, 1 mM EGTA, 0.5 mM Na3VO4, 0.1% 2-mercaptoethanol, 1% Triton X-100, 50 mM NaF, 5 mM sodium pyrophosphate, 10 mM sodium b-glyceropyrophosphate, 0.1 mM phenylmethanesulfonyl fluoride and protease inhibitor mixture) for 10 min. The supernatant was collected after a shake in 1% p-nitorphenyl phosphate for 10 sec. and centrifuged at 12,000 · g for 30 sec. at 4C. Equal amounts of protein were separated by SDS–PAGE and analysed by western blot using the antibodies (Beijing Biosynthesis Biotechnology Company, Beijing, China) of HMGB1, phosphor-NF-jB and PCNA. Optical densities of the resultant bands were scanned and quantified with a Gel Doc 2000 (Bio-Rad). The data were normalized against those of the corresponding PCNA. Results were expressed as fold increase over sham. Statistical analysis. The survival time of the groups was calculated by the Kaplan–Meier method, the differences being tested with the log rank test (SPSS 16.0). Quantitative data from experiments were expressed as mean € S.D., and significance was determined by oneway analysis of ANOVA followed by Tukey’s test. p < 0.05 was considered statistically significant.

Results Effect of PA on the release of TNF-a, IL-6 and IL-10 in activated RAW 264.7 cells. The effects of PA on the levels of TNF-a, IL-6 and IL-10 were evaluated. Firstly, we evaluated the effect of PA on the levels of several inflammatory mediators and LDH from RAW 264.7 cells, as shown in table 1. PA concentration dependently inhibited TNF-a and IL-6 levels, which were increased by LPS stimulation, while it increased the release of IL-10. LDH release showed no remarkable difference in the LPS-stimulated or PA-treated groups. It indicated that LPS stimulation caused no cell death, and that treatment of PA had no cytotoxicity from 0.33 to 9 lM. In addition, the maximum inhibition concentration to the levels of TNF-a and IL-6 in activated RAW 264.7 cells was 3 and 9 lM. Thus, we selected PA 3 and 9 lM to investigate its effect on NF-jB activation and HMGB1 expression. Effect of PA on NF-jB activation and HMGB1 expression. Both NF-jB and HMGB1 expression were low in RAW264.7 cells, as shown in fig. 1. However, the expres-


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Table 1. Effect of PA on TNF-a, IL-6, IL-10 and LDH release in LPS-stimulated RAW264.7 cells. Group

Concentration (lM)

Control LPS

– – 0.33 1 3 9

PA

TNF-a (ng ⁄ ml)

IL-10 (pg ⁄ ml)

0.42 2.11 1.99 1.52 1.07 1.01

1.60 1.63 1.81 1.95 2.04 2.01

€ € € € € €

0.13 0.34* 0.40 0.29** 0.18*** 0.22***

€ € € € € €

0.31 0.25* 0.27 0.22** 0.25*** 0.26***

IL-6 (ng ⁄ ml) 0.49 1.92 1.58 1.29 1.03 0.95

€ € € € € €

0.13 0.35* 0.32 0.38** 0.22*** 0.27***

LDH (AU) 0.41 0.42 0.43 0.40 0.42 0.43

€ € € € € €

0.11 0.15 0.12 0.13 0.14 0.11

Data are expressed as mean € S.D.; n = 6 in each group. RAW264.7 macrophages at a concentration of 1 · 106 cells ⁄ ml and incubated with complete medium in the absence (control) or presence of LPS 0.1 lg ⁄ ml were subsequently added with PA and treated for 24 hr. The levels of TNF-a, IL-10 and IL-6 were determined by ELISA kits, and LDH was quantified using the kit according to the manufacturer’s instructions. *p < 0.01 versus control group; **p < 0.05, ***p < 0.01 versus LPS-stimulated group. One-way analysis of anova followed by Tukey’s test. PA, protocatechuic aldehyde; LDH, lactate dehydrogenase.

Effect of PA on serum levels of IL-10, HGMB1 and TREM-1. The effects of PA on serum levels of IL-10, HGMB1 and TREM-1 were evaluated, as shown in table 2. Septic rats showed significantly higher levels of HGMB1 and TREM-1 than sham-operated rats. In contrast, i.v. of PA alone or plus Imipenem resulted in a marked reduction of serum levels of HGMB1 and TREM-1, while there was a marked increase in serum level of IL-10. Effect of PA on MPO activities of lung, liver and small intestine. Intravenous of PA alone or plus Imipenem inhibited the increase of MPO activities in lung, liver and small intestine in the septic rats, as shown in table 3. Notably, PA plus Imipenem was mostly more efficient than PA or Imipenem alone.

Fig. 1. Effects of protocatechuic aldehyde (PA) on TNF-a-induced high-mobility group box 1 (HMGB1) and phosphorylation of NFjB expression. RAW264.7 cells were pre-incubated with HMGB1 inhibitor, glycyrrhizin (100 lM) or PA (3 or 9 lM) for 30 min. and then incubated with TNF-a (20 ng ⁄ ml) for 30 min. HMGB1 and phosphor-NF-jB expression were analysed by western blotting. Results are expressed as fold increase over control. PA indicates protocatechuic aldehyde. Data are expressed as mean € S.D., n = 5. *p < 0.05, **p < 0.01 versus TNF-a-induced group; #p < 0.01 versus the control group. Significance was determined by one-way of anova followed by Tukey’s test.

sion of NF-jB and HMGB1 was significantly increased by TNF-a stimulation. We compared the effect of PA on the TNF-a-induced (20 ng ⁄ ml for 30 min.) activation of NFjB and HMGB1 expression in RAW264.7 cells with that of a selective HMGB1 inhibitor, glycyrrhizin. The results showed that pre-treatment of RAW264.7 cells with glycyrrhizin (100 lM) for 30 min. blocked the TNF-a-induced HMGB1 expression and reduced the phosphorylation of NF-jB. Treatment of RAW264.7 cells with PA 3 or 9 lM blocked TNF-a-induced NF-jB phosphorylation and reduced HMGB1 expression.

Effect of PA on NF-jB activation and HMGB1 expression in lung tissue of septic rats. High-mobility group box 1 expression and phosphorylation of NF-jB were low in the sham rats. However, they were high after CLP surgery, as shown in fig. 2. Treatment with PA not only reduced HMGB1 expression but also decreased phosphorylated NF-jB in lung tissue of the septic rats. Effect of PA on the survival time of septic rats. We evaluated the effect of PA on rat survival time and mortality after CLP surgery at 144 hr, as shown in fig. 3. I.v. of PA or PA plus Imipenem resulted in extended rat survival time (p < 0.01). Compared with the PA group, chi-square of PA plus Imipenem group was 0.581 (p value in the log rank test was 0.446); compared with the Imipenem group, chisquare of PA plus Imipenem group was 3.906 (p value in the log rank test was 0.048). PA plus Imipenem was more efficient than Imipenem 20 mg ⁄ kg. Discussion The present study on the RAW 264.7 cell line and a septic rat model had three notable findings. Firstly, post-operative treatment with PA prolonged survival of rats after CLP surgery. Secondly, CLP surgery induced a significant systemic


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Table 2. Effect of PA on serum levels of TNF-a, IL-6, IL-10 and HMGB1 in septic rats after CLP surgery at 20 hr. Dose (mg ⁄ kg)

Group Sham CLP CLP + PA CLP + IMP CLP + Imipenem + PA

– – 50 20 20 + 50

IL-10 (pg ⁄ ml) 97 185 228 196 255

€ € € € €

24 33* 37** 38 5***,a

IL-6 (pg ⁄ ml) 143 548 389 479 301

€ € € € €

25 81* 85*** 77 67***,aa,b

TNF-a (ng ⁄ ml)

HMGB1 (ng ⁄ ml)

TREM (ng ⁄ ml)

26 201 139 176 93

5.3 19.3 14.1 15.6 10.9

43 223 158 144 96

€ € € € €

5 36* 3*** 41 32***,aa,bb

€ € € € €

1.3 3.7* 3.0*** 3.1** 3.4***,aa,b

€ € € € €

12 53* 26*** 30*** 17***,aa,bb

Data are expressed as mean € S.D.; n = 10 in each group. PA or Imipenem was injected intravenously alone or PA plus Imipenem at 1 and 13 hr after surgery. After CLP surgery at 20 hr, serum levels of IL-10, HMGB1 and TREM were quantified using commercial ELISA kits. *p < 0.01 versus sham group; **p < 0.05, ***p < 0.01 versus CLP group; ap < 0.05, aap < 0.01 versus CLP + Imipenem group; bP < 0.05, bb p < 0.01 versus CLP + PA group. One-way analysis of anova followed by Tukey’s test. HMGB1, high-mobility group box 1; TREM, triggering receptor expressed on myeloid cells; CLP, caecal ligation and puncture.

Table 3. Effect of PA on MPO activity of lung, liver and small intestine in septic rats after CLP surgery at 20 hr. MPO (U ⁄ g tissue) Group Sham CLP CLP + Imp 20 CLP + PA 50 CLP + Imp 20 + PA 50

Lung 2.8 18.0 13.4 13.8 9.6

€ € € € €

0.5 3.9* 3.4** 4.1** 3.2***,a,b

Liver 0.13 0.49 0.37 0.37 0.26

€ € € € €

0.04 0.12* 0.12** 0.09** 0.10***,a,b

Small intestine 0.69 3.55 2.70 2.74 2.03

€ € € € €

0.17 0.78* 0.66** 0.61** 0.59***,a,b

Data are expressed as mean € S.D.; n = 10 in each group. PA or Imipenem was injected intravenously alone or PA plus Imipenem at 1 and 13 hr after CLP surgery. MPO activity was carried out according to the instructions provided by the manufacturer. *p < 0.01 versus sham group; **p < 0.05, ***p < 0.01 versus CLP group; ap < 0.05 versus CLP + Imipenem group; bP < 0.05 versus CLP + PA group. One-way analysis of anova followed by Tukey’s test. CLP, caecal ligation and puncture; MPO, myeloperoxidase.

inflammatory response, which was attenuated by treatment with PA. Thirdly, PA suppressed protein expression of HMGB1 and NF-jB in vitro and in vivo. Caecal ligation and puncture surgery is a more realistic sepsis model and has clear parallels to clinical situations with patients as well as analogical symptoms to be observed in human beings. It demonstrated that the hyperdynamic phase persists from 2 to 10 hr, and the hypodynamic phase occurs at 16 hr and lasts till 20 hr after CLP surgery [16]; thus, we chose to analyse the biochemical parameters after CLP surgery at 20 hr. Triggering receptor expressed on myeloid cells 1 is an amplifier of inflammatory response. It is highly expressed by neutrophils, macrophages and mature monocytes. Its expression dramatically increases in tissues infected by grampositive and gram-negative bacteria. Blockade of TREM-1 showed to protect mice from CLP-induced shock [17,18]. In our study, PA inhibited the serum level of TREM-1. This suggests that PA could reduce sepsis severity by blockade of TREM-1. During sepsis, blockade of IL-10 by neutralizing antibodies has been shown to decrease survival and increase neutro-

Fig. 2. Effects of protocatechuic aldehyde (PA) on lung tissue of phosphor-NF-jB and HMGB1 in septic rats by western blot analysis 20 hr after caecal ligation and puncture (CLP) surgery. Rats were subjected to CLP surgery for 20 hr. Total protein extracts were prepared and assayed for phosphor-NF-jB and HMGB1 by western blot analysis, and blots were normalized to PCNA expression. Results are expressed as fold increase over sham group. PA indicates protocatechuic aldehyde. Data are expressed as mean € S.D., n = 5. **p < 0.01 versus vehicle-treated group. #p < 0.01 versus sham group. Significance was determined by one-way analysis of anova followed by Tukey’s test.

phil accumulation [19]. Increase of IL-10 could decrease organ injury during sepsis [20]. We have demonstrated that serum levels of IL-10 increased. Based on our results, inhibition of cytokines might be related to an increase of serum levels of IL-10 resulting from administration of PA at CLPinduced systemic inflammation. Myeloperoxidase is a peroxidase enzyme most abundantly present in neutrophil granulocytes. Neutrophils are the first cells to be activated in the host immune response to infection or injury and are critical cellular effectors in both humoral and innate immunity, which are central to the pathogenesis of sepsis and multi-organ dysfunction. Sepsis-induced neutrophil-mediated tissue injury has been demonstrated in a variety of organs [21]. Our results indicate that PA inhibits MPO activities of lung, liver and small intestine in septic rats


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Fig. 3. Effect of PA on survival of septic rats after caecal ligation and puncture (CLP) surgery. Indicated drugs were injected intravenously at 1, 13, 25, 37, 49 and 73 hr after CLP surgery, n = 20 in each group. Survival rate was monitored at 24, 48, 72 and 144 hr after CLP surgery. PA indicates protocatechuic aldehyde. *p < 0.05 versus vehicle-treated group; **p < 0.01 versus vehicle-treated group; F p < 0.05 versus CLP + Imipenem 20 mg ⁄ kg group. The survival times of the groups were calculated by the Kaplan–Meier method, and the differences tested with the log rank test.

and suggest that PA attenuates sepsis-induced neutrophilmediated tissue injury. Macrophages that migrate to the site of injury serve to amplify the inflammatory response with release of various inflammatory mediators. Excessive and ⁄ or dysregulated inflammatory responses cause delayed suppression of inflammatory response with reduced ability to secrete cytokines leading to inability to clear infection [22,23]. Additionally, peritoneal macrophages are essential for bacterial clearance in CLP-induced sepsis [24], so we selected RAW 264.7 cells to investigate the mechanism of PA on sepsis. The NF-jB pathway plays an important role during the development of septic shock and septic lethality. One study demonstrated that blocking the NF-jB pathway improved survival [25]. After LPS stimulation, NF-jB is phosphorylated and coordinates the induction of production and secretion of proinflammatory cytokines encoded by several genes. Therefore, inhibiting NF-jB activation is crucial for treating inflammation. Our results indicate that PA suppresses NF-jB activation and attenuates phosphorylation of NF-jB in RAW 264.7 cells. This suggests that PA reduces sepsis severity by blockade of the NF-jB pathway. Inhibition of HMGB1 expression is suggested as a novel therapeutic approach for the treatment of sepsis [26]. HMGB-1 promotes chemotaxis and the production of cytokines in a process that involves the activation of the NF-jB [27]. A curb both on HMGB1 and NF-jB signalling pathway by PA is implicated in its molecular regulation on CLP-triggered inflammation, because HMGB1 and phosphorylated NF-jB are both alleviated by western blot analysis in vitro and in vivo. Therefore, we believe that the protective effects of PA might be due to the suppression of the inflammatory cascades through HMGB1 and NF-jB signalling pathway. We compared the anti-sepsis effect of Imipenem with PA plus Imipenem. The results showed that PA plus Imipenem

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