Inhibitory Effect Of Lidocaine On The Expression Of HMGB1 In Septic Rats

Background:Sepsis is defined as a systemic inflammatory response syndrome caused by a microbial infection which results in excessive stimulation of the host immune system and production of various pro-inflammatory cytokines. Furthermore, the overproduction of cytokines leads to the lethal multiple organs damage. Despite advances in modern intensive care, the overall mortality of severe sepsis exceeds 30%, and it has been the third leading cause of death after cardiovascular disease and cancer. HMGB1, a nuclear protein widely studied as a transcription factor and growth factor, has recently been identified as a necessary and critical mediator of severe sepsis. It is released by activated inflammation cells and necrotic tissues cells during endotoxaemia and sepsis with significant delayed kinetics, serum HMGB1 significantly elevated from 8h to at least 72 h, in comparison with TNF-a and IL-1β. Treatment with anti-HMGB1 antibodies, A box of HMGB 1 and ethyl pyruvate (a inhibitor of HMGB 1) beginning as late as 24 h after CLP surgery significantly increases survival. So, the therapeutic window for anti-HMGB1 therapies is significantly wider than TNF-a targeted interventions, it is may now be possible to develop inhibitors of HMGB 1 for treatment of sepsis.Lidocaine, a common local anesthetic agent, has been known to possess anti-inflammatory effects. It has been shown to modulate inflammatory cascades and provide protection from ischemic reperfusion injury and septic peritonitis. It plays a key role of anti-inflammatory effects on various cell types, including monocytes, macrophages, and neutrophils. Previous studies have found that lidocaine suppressed the expression of the T cell derived pro-inflammatory cytokines, such as interleukin (IL)-2, tumor necrosis factor (TNF)-a and interferon (IFN)-y. The anti-inflammatory effects of lidocaine may be mediated by inhibition of NF-κB activation and cytokine release. However, the effect of lidocaine on HMGB1 has not been explored yet.RAW264.7 cells, a murine macrophage cell line, are often used as fair substitutes for macrophages in analyzing the production of inflammatory mediators/cytokines with various stimuli. Similar levels of HMGB1 release by lipopolysaccharide (LPS) or IFN-y-activated macrophage RAW264.7 cells and human peripheral blood monocytes have been reported. Given that HMGB1 is overexpressed and released under various types of inflammatory disorders and the effect of lidocaine involved in the inhibition of NF-κB signaling pathway, we hypothesized that lidocaine may inhibit the upregulation of HMGB1 during inflammatory response. Thus, the first part of our study sought to determine whether lidocaine could inhibit the expression and release of HMGB1 from macrophages RAW264.7 induced by LPS, and ultimately play the role of anti-inflammation.Cecal ligation and puncture (CLP)-induced sepsis is an animal model that has high clinical relevance to humans, as it reproduces many hallmarks of sepsis that occur in patients[13]. In the second part of our study, we employed the CLP model to induce the sepsis of Wistar rats and treaded with different dose of lidocaine injected into peritoneal cavity at 0,1,2 hours after operation to investigate that if lidocaine can inhibit the expression of HMGB1 in the organ of septic rats and provide protection of sepsis induced by CLP.To detect the direct injury of HMGB1 and the protective effect of lidocaine, we administered highly purified rHMGBl to C3H/HeJ mice (intraperitoneal 500μg per mouse) and 100μg HMGB1 intratracheal injection in 50μl sterile water.Methods:RAW 264.7 cells were seeded at 1ml aliquots of 5×106 cells/ml in 96 wells tissue culture dishes, and incubated with LPS (100ng/ml) in the absence or presence of graded concentrations of lidocaine (2,20, and 200μg/ml), or cultured in media alone. Cell-free supernatants were collected after 24h stimulated for HMGB1 determination by enzyme-linked immunosorbent assays (ELISA). Meanwhile, the total RNA was extracted for detecting the HMGB1mRNA with real-time polymerase chain reaction (RT-PCR) and the nuclear fraction extracted for measuring the activity of NF-κB with ActiveMotif NF-κB family kit. The immuocytochemistry was also used to assay the translocation of HMGB1 from nucleus to cytoplasm.Wistar rats, anesthetized by intraperitoneal injection of 50 mg/kg sodium pentobarbital and held under anesthesia during the entire surgical procedures, were assigned into one of the following five groups:sham-operated group (n=15), cecal ligation and double puncture (CLP) treated with normal saline group (n=20), CLP treated with lidocaine 5,10 or 20mg/kg groups (n=15). Normal saline 0.5ml or lidocaine 5,10,20mg/kg were injected into peritoneal cavity at 0,1,2 hour after operation. Twenty-four hours after CLP, the level of HMGB1 mRNA and NF-κB p65 in liver and kidney were assessed with RT-PCR as well as histological alteration, plasma alanine aminotransferase (ALT) and creatinine myeloperoxidase (MPO). Seven days survival was also investigated in another set of 100 rats.C3H/HeJ mice, anesthetized by intraperitoneal injection of 50 mg/kg sodium pentobarbital and held under anesthesia during the entire surgical procedures, were assigned into one of the following three groups:contral group (n=10) treated with normal saline 1ml, i.p.; HMGB1 group (n=10) treated with rHMGBl 100μg/1ml and lidocaine group (n=10) treated with rHMGB1 100μg+lidocaine 20mg/kg to investigate the survival. Another set of C3H/HeJ mice were assigned into three group: contral treated with treated with normal saline 50μl, i.t.; HMGB1 group (n=10) treated with rHMGBl 100μg/50μl and lidocaine group(n=10) treated with rHMGBl 100μg i.t.+ lidocaine 20mg/kg i.p. to investigate the ratio of neutrolphils and protein content of bronchoalveolar lavage fluid, the activity of myeloperoxidase and content of TNF-a and NF-κB in lung.Results:We found that the level of HMGB1 protein in supernatant of RAW264.7 cells was decreased significantly in a dose-dependent manner by administration of lidocaine at 24 hour after incubated with LPS 100ng/ml. Meanwhile, the increasing concentrations of lidocaine had nonsignificant effect on RAW264.7 cell viability by MTT. To gain further insight into the responsible mechanism for the inhibiting effect of lidocaine on HMGB1 release, we next tested the effect of lidocaine on HMGB1 transcription. Data indicated that lidocaine alleviated HMGB1 production at the transcriptional level and inhibited the translocation of HMGB1 from nucleus to cytoplasm shown in immunocytochemical staining. The above results indicated that lidocaine inhibited HMGB1 mRNA expression in LPS-stimulated RAW264.7 cells, so we next aimed to evaluate whether the upstream signal transduction pathway of NF-κB was also affected. Our results showed that lidocaine significantly reduced NF-κB translocation stimulated with LPS in dose-dependent manner. All these data from our study suggest that lidocaine can inhibit HMGB1 production at the transcriptional level through a mechanism that involves, at least partly, the NF-κB signal transduction pathway.Survival investigation is a major index of pharmacological intervation in diseases with high mortality just like sepsis. In the present study, the 7 days survival of septic rats was investigated and the log-rank analysis demonstrated that a significant level of protection was conferred by lidocaine5,10,20mg/kg (X2=5.472, P=0.19; X2=12.859, P<0.001; X2=26.237, P<0.001).We examined the degree of hepatic dysfunction by measuring plasma ALT and the degree of renal dysfunction by measuring creatinine 24 h after CLP. In this model of sepsis, plasma ALT was significantly increased in normal saline treated rats (92.00±13.38 U/1) compared with sham surgery controls (41.60±7.89 U/1;t=7.661, P<0.001). Rats treated with lidocaine 10,20mg/kg had significantly lower plasma ALT at 24 h (71.20±8.76 U/1, t=3.162, P<0.001; 58.00±10.37 U/1,t=5.168, P<0.001) compared with rats treated with normal saline alone Therefore, local anesthetic infusion alleviates CLP-induced renal injury.Similarly,24h after CLP operation plasma creatinine was significantly increased in saline vehicle treated rats (51.00±5.00μmol/l) compared with sham surgery controls (20.40±5.32μmol/l; t=11.279,P<0.001). Rats treated with lidocaine5,10, 20mg/kg had significantly lower plasma creatinine at 24 h (44.80±3.7μmol/l, t=2.285, P=0.033; 34.80±4.44μmol/l, t=5.971, P<0.001; 27.40±2.30<mol/l,t=8.699, P<0.001) compared with rats treated with saline vehicle alone Therefore, local anesthetic infusion alleviates CLP-induced renal injury. CLP caused extensive morphological damage of liver, kidney compared with sham-operated group 24 hours after CLP. These morphological changes were better attenuation in the group treaded with lidocaine 20mg/kg compared with CLP+NS group.The level of organs HMGB1mRNA in rats undergoing CLP with normal saline was significantly increased in comparison with the sham group (P<0.001). However, HMGB1mRNA in rats treaded with lidocaine resulted in significantly decrease of HMGB1mRNA in liver, kidney compared with rats treaded with normal saline in a dose-dependent manner (P<0.001).The immunohistochemisty staining of NF-κB p65 of sham group were mainly in the cytoplasm with tasteless brown color. However, the staining of NF-κB p65 of CLP treaded with normal saline manifested all over the visual fields, and nuclear (blue) were coved by brown and couldn't be seen clearly. But the staining of NF-κB p65 in group treaded with lidocaine 20mg/kg became light and the nuclear could be seen clearly in blue.Within 2 hours of rHMBG1 500μg i.p., the mice developed signs of endotoxemia, including lethargy, piloerection and diarrhea and eighty percent mice died within 48 hours. Meanwhile, only 20 percent of mice reated with lidocaine died (X2=4.736, P=0.03). rHMBG1 100μg i.t. administered to C3H/HeJ mice can induce a increase of ratio of neutrolphils and protein content of bronchoalveolar lavage fluid, as well as the activity of myeloperoxidase and content of TNF-a and NF-κB in lung 24 hours later (P<0.05). By treated with lidocaine, all index above decreased (P<0.05).Conclusions:Our data suggest that lidocaine might play the role of anti-inflammation by inhibition of expression of HMGB1 mRNA, and translocation of both HMGB1 and NF-κB from nucleus to cytoplasm, and the mechanism of these effects might be involved in, at least partly, the inhibition of NF-κB signal pathway. Our study also demonstrates that intraperitoneal injection of lidocaine provides significantly protection of liver and kidneys from CLP-induced acute injury and then result in a higher survival rate by attenuating the expression of HMGB1, a critical mediator of severe sepsis. Intraperitoneal injection of lidocaine also provides significantly protection of lung from HMGB1-induced acute injury and then result in a higher survival rate.