Role And Mechanism Of HMGB1 And Its Inhibitor Glycyrrhizin In Traumatic Pancreatitis

Objective:Although pancreatic trauma is rare, occurring in only 2% to 5% of trauma victims, it is often imperceptible and intractable with a higher complications rate and mortality. The incidence of pancreatic trauma accounts for 5% of closed abdominal trauma and 2%~6% of abdominal penetrating trauma. As early signs and symptoms of pancreatic trauma are not obvious, it is often noticed until trauma-induced acute pancreatitis is presented. Trauma-induced acute pancreatitis, also referred to as traumatic pancreatitis(TP), are often followed by some serious complications, for example, SIRS, shock, multiple organ failure(MOF), etc. Although the pathogenesis and treatments of acute pancreatitis induced by other causes have been widely researched, there are few researches on the pathogenesis and treatment of trauma-induced acute pancreatitis.High mobility group box 1(HMGB1) is an intranuclear highly conserved nonhistone chromosomal protein that functions as a stabilizer of nucleosome structure and regulator of the genes transcription. HMGB1 can be actively or passively released from cells and plays important roles in a large variety of diseases. A strong correlation is found between HMGB1 levels and severity of these diseases. Accordingly, it has been speculated that HMGB1 might be a target for anti-inflammatory treatment and the inhibitors of HMGB1 were investigated to explore potential new treatment strategy for these diseases. However, there were neither the studies on the relationship between HMGB1 and TP, nor the reports about the inhibitors of HMGB1 on treatment of TP.Recently, glycyrrhizin(GL) was recognized as an HMGB1 inhibitor, which binds directly to HMGB1 and inhibits its cytokine activities. GL, a main active ingredient in licorice root, is usually administered to treat patients with chronic hepatitis. This compound is associated with numerous pharmacologic effects, including anti-inflammatory, antiviral, antitumor, and hepatoprotective activities. However, the roles and mechanisms of GL in the treatment of TP were not investigated previously.We have developed an experimental model of isolated traumatic pancreatitis in rats and have been interested in the mechanisms and therapies of traumatic pancreatitis. Therefore, we will investigate the role of HMGB1 in TP and the effect and mechanism of its inhibitor GL in TP.Methods:Part I: The role of HMGB1 in traumatic pancreatitis in ratsA total of 48 Wistar rats were randomly divided into six groups(n=8 in each):(1) sham operation control group(control group),(2) traumatic pancreatitis 6h group(TP 6h group),(3) TP 12 h group,(4) TP 24 h group,(5)TP 48 h group,(6) TP 72 h group. The model of TP rats was made by the biological impact machine-Ⅲ using compressed air with a single 400 kPa pressure to impact the pancreas. All the rats were killed humanely at the the corresponding time point. The blood and pancreas were collected for measuring these indexes: the level of serum amylase, the level of serum HMGB1 and TNF-α, pancreatic macropathology, histopathological analysis and pathology scores, immunohistochemical analysis and Western blot analysis of HMGB1, the correlation between pancreatic pathological score and HMGB1.Part II: The protective effect of HMGB1 inhibitor glycyrrhizin in traumatic pancreatitis in ratsA total of 60 Wistar rats were randomly divided into three groups(n=20 in each):(1) sham operation control group(control group),(2) traumatic pancreatitis 24 h group(TP group),(3) traumatic pancreatitis treatment with glycyrrhizin group(TP-GL group). To determine survival rates during the first 7 days after injury, another 60 rats(n=20 in each) were grouped and treated as mentioned above. The model of TP rats was made as the method in part I. All the rats were killed humanely after the corresponding treatments 24 h. The blood and pancreas were collected for measuring these index: the level of serum amylase, the level of serum HMGB1, TNF-α and IL-6, pancreatic macropathology, histopathological analysis and pathology scores, detection of apoptosis in pancreatic cells with TUNEL and the apoptotic index, Western blot analysis of Caspase 3, detection the activity of superoxide dismutase(SOD) and malondialdehyde(MDA) in pancreatic tissue.Part III: The pathway and molecular mechanism of HMGB1 inhibitor glycyrrhizin in traumatic pancreatitis in ratsA total of 60 Wistar rats were randomly divided into three groups(n=20 in each):(1) sham operation control group(control group),(2) traumatic pancreatitis 24 h group(TP group),(3) traumatic pancreatitis treatment with glycyrrhizin group(TP-GL group). The model of TP rats was made as the method in part I. All the rats were killed humanely after the corresponding treatments 24 h. The blood and pancreas were collected for measuring these index: immunohistochemical analysis of HMGB1, TLR4 and NF-κB in pancreatic tissue, Western blot detection of HMGB1, TLR4 in pancreatic tissue, Real-Time PCR detection of HMGB1, TLR4 mRNA in pancreatic tissue, EMSA assay of NF-κB in pancreatic tissue, ELISA assay of TNF-α and IL-6 in pancreatic tissue,the correlation among HMGB1 and TLR4, NF-κB in pancreatic tissues.Results:Part I: The role of HMGB1 in traumatic pancreatitis in rats1. In TP rats, the levels of serum amylase increased significantly at 6h after impacting, rised further at 12 h, reached a peak at 24 h, declined at 48 h, and dropped to the level of baseline in control group at 72 h.2. In TP rats, the levels of serum TNF-α increased significantly at 6h after impacting, reached a peak at 12 h, declined at 24 h, and dropped to the level of baseline at 48 h and 72 h. Meanwhile, the levels of serum HMGB1 increased significantly at 6h after impacting, rised further at 12 h, reached a peak at 24 h, declined at 48 h and 72 h.3. The pancreatic macropathology, histopathological analysis and pathology scores in TP rats showed that pancreatic tissue injury increased significantly at 6h after impacting, rised further at 12 h, reached a peak at 24 h, declined at 48 h, and dropped at 72 h.4. Pancreatic immunohistochemistry and Western blot analysis of HMGB1 in TP rats indicated that HMGB1 increased significantly at 6h after impacting, rised further at 12 h, reached a peak at 24 h, declined at 48 h and 72 h.5. HMGB1 of serum and pancreatic tissue in TP rats had a positive correlation with pancreatic pathology scores and the correlation coefficient were r=0.841 and r=0.83.Part II: The protective effect of HMGB1 inhibitor glycyrrhizin in traumatic pancreatitis in rats1. Kaplan-Meier survival curve showed TP-GL group 7-day survival rate was significantly higher than TP group(65% versus 45%), which indicating the potential role of GL to improve the survival rate in TP rats.2. Serum amylase levels of TP and TP-GL group were higher than that of the control group, and serum amylase levels of TP-GL group were slightly lower than that of TP group. However, it was not statistical significance, which indicating that GL did not have a role to inhibit pancreatin.3. Serum TNF-α, IL-6 and HMGB1 levels of TP and TP-GL groups were obviously higher than that of the control group. However, comparing with TP group, all the proinflammatory cytokines in TP-GL group showed a significant decrease, which indicating that GL inhibited early and late inflammatory mediators.4. Pancreatic pathological damages of TP and TP-GL groups were obviously higher than that of the control group. However, TP-GL group were much better than TP group, which indicating GL could improve pancreatic pathologic lesions in rats.5. The levels of apoptosis in TP and TP-GL groups were obviously higher than that in the control group. However, the apoptotic levels in TP-GL group were obviously lower than that in TP group, which indicating that GL might inhibit apoptosis of pancreatic acinar cells in rats.6. SOD levels in TP group and TP-GL group were significantly lower than that in the control group, however, the levels of pancreatic tissues SOD in TP-GL group were obviously higher than that in TP group; MDA levels of pancreatic tissues in TP group significantly increased, however, MDA levels of pancreatic tissues in TP-GL group were obviously lower than that in TP group, which suggesting that GL could improve oxidative stress in pancreas of TP rats.Part III: The pathway and molecular mechanism of HMGB1 inhibitor glycyrrhizin in traumatic pancreatitis in rats1. The HMGB1 levels of pancreas tissue in TP rats were significantly increased, while those were significantly reduced in TP-GL group, which showing GL could inhibit HMGB1 of pancreatic tissue in TP rats.2. The TLR4 levels of pancreas tissue in TP rats were significantly increased, while those were significantly reduced in TP-GL group, which showing GL could inhibit TLR4 of pancreatic tissue in TP rats.3. The NF-κB levels of pancreas tissue in TP rats were significantly increased, while those were significantly reduced in TP-GL group, which showing GL could inhibit NF-κB of pancreatic tissue in TP rats.4. The TNF-α and IL-6 levels of pancreas tissue in TP rats were significantly increased, while those were significantly reduced in TP-GL group, which showing GL could inhibit TNF-α and IL-6 of pancreatic tissue in TP rats.5. The positive correlations were shown among the expressions of HMGB1 and TLR4,NF-κB. The protective effects of GL in the pancreas of TP rats were probably by inhibition of HMGB1-TLR4-NF-κB pathway.Conclusion:The HMGB1 levels of serum and pancreatic tissue in TP rats were significantly increased, and were positive correlation with the pancreatic pathological scores in TP rats. The HMGB1 levels could be as an important indicator to assess the severity of TP. Treatment with GL in TP rats could increase 7-day survival rate, reducing serum early and late inflammatory mediators, improving pancreatic pathological damage, decreasing the level of apoptosis in pancreatic tissue, ameliorating the oxidative stress in pancreatic tissue. TP induced the activation of HMGB1-TLR4-NF-κB pathway and the elevation of inflammatory cytokines in rat pancreatic tissue. However, treatment with GL might inhibit activation of this pathway and reduce the levels of inflammatory factors. Therefore, our study indicates that GL has a protective effect in pancreatic tissue of TP rats.