- Email: [email protected]
Baicalein protect pancreatic injury in rats with severe acute pancreatitis by inhibiting pro-inflammatory cytokines expression
Accepted Manuscript Baicalein protect pancreatic injury in rats with severe acute pancreatitis by inhibiting pro-inflammatory cytokines expression Jun Li, Yongtao Wu, Shu Zhang, Jian Zhang, Fanpu Ji, Wangjun Bo, Xiaoyan Guo, Zongfang Li PII:
S0006-291X(15)30611-2
DOI:
10.1016/j.bbrc.2015.09.094
Reference:
YBBRC 34594
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 11 September 2015 Accepted Date: 17 September 2015
Please cite this article as: J. Li, Y. Wu, S. Zhang, J. Zhang, F. Ji, W. Bo, X. Guo, Z. Li, Baicalein protect pancreatic injury in rats with severe acute pancreatitis by inhibiting pro-inflammatory cytokines expression, Biochemical and Biophysical Research Communications (2015), doi: 10.1016/ j.bbrc.2015.09.094. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Baicalein Protect Pancreatic Injury in Rats with Severe Acute Pancreatitis by inhibiting Pro-inflammatory Cytokines Expression Jun Li*#, Yongtao Wu†#, Shu Zhang*‡, Jian Zhang*‡, Fanpu Ji*, Wangjun Bo*, Xiaoyan
*
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Guo*§ and Zongfang Li*‡§
National-local Joint Engineering Research Center of Biodiagnostics and Biotherapy, the Second Affiliated Hospital of Xi’an Jiaotong University College of Medicine, 157
Department of General Surgery, Hong Hui Hospital, Xi’an Jiaotong University
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†
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West 5th Road, Xi'an 710004, Shaanxi Province, PR. China
College of Medicine, 555 East Friendship Road, Xi'an 710054, Shaanxi Province, PR. China ‡
Department of General Surgery, the Second Affiliated Hospital of Xi’an Jiaotong University College of Medicine, 157 West 5th Road, Xi'an 710004, Shaanxi Province,
These authors contributed equally to this work.
Jun Li, Ph.D.
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#
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PR. China
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National Local Joint Engineering Research Center of Biodiagnostics and Biotherapy the Second Affiliated Hospital of Xi’an Jiaotong University College of Medicine 157 West 5th Road, Xi'an 710004, Shaanxi, PR. China e-mail
[email protected]
Yongtao Wu, Ph.D. Department of General Surgery, Hong Hui Hospital, Xi’an Jiaotong University College of Medicine 555 East Friendship Road, Xi'an 710054, Shaanxi, PR. China 1
ACCEPTED MANUSCRIPT e-mail
[email protected]
Shu Zhang, Ph.D. National Local Joint Engineering Research Center of Biodiagnostics and Biotherapy,
University College of Medicine 157 West 5th Road, Xi'an 710004, Shaanxi, PR. China e-mail
[email protected]
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Jian Zhang, Ph.D.
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Department of General Surgery, the Second Affiliated Hospital of Xi’an Jiaotong
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National Local Joint Engineering Research Center of Biodiagnostics and Biotherapy, Department of General Surgery, the Second Affiliated Hospital of Xi’an Jiaotong University College of Medicine
157 West 5th Road, Xi'an 710004, Shaanxi, PR. China [email protected]
Fanpu Ji, Ph.D.
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e-mail
National Local Joint Engineering Research Center of Biodiagnostics and Biotherapy
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the Second Affiliated Hospital of Xi’an Jiaotong University College of Medicine 157 West 5th Road, Xi'an 710004, Shaanxi, PR. China [email protected]
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e-mail
Wangjun Bo, Ph.D.
National Local Joint Engineering Research Center of Biodiagnostics and Biotherapy the Second Affiliated Hospital of Xi’an Jiaotong University College of Medicine 157 West 5th Road, Xi'an 710004, Shaanxi, PR. China e-mail §
[email protected]
Corresponding authors: 2
ACCEPTED MANUSCRIPT Dr. Zongfang Li, M.D., Ph.D. and Dr. Xiaoyan Guo, M.D. National Local Joint Engineering Research Center of Biodiagnostics and Biotherapy, the Second Affiliated Hospital of Xi’an Jiaotong University College of Medicine, 157 West 5th Road, Xi’an 710004, Shaanxi, P.R. China
Email: [email protected], [email protected] Running title
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Telephone: +86-29-87679684, +86-29-87679275 Fax: +86-29-87678634
Baicalein Protect Pancreatic Injury of SAP in Rats
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experimental study
Text word count: 4408
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the date of submission:
Number of references: 27; Tables: 1; Figures: 4
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Abstract word count: 136
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ACCEPTED MANUSCRIPT Abstract Background/Aim: Inflammatory cytokines is a key point in the development of pathogenesis of SAP. Inflammatory mediators TNF-α and IL-6 are up-regulated in serum of patients with SAP and become good discriminators of SAP severity.
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Materials and Methods: In this study, we investigated the treatment effectiveness of Baicalein on SAP rat model. Baicalein was intravenously injected immediately after SAP induction in rats. The mortality, histopathology score, ascites fluid volume, and
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pro-inflammatory cytokine production were evaluated at 12 h after SAP induction.
Results: Baicalein decreased the pancreatic histopathology score, reduced ascites fluid
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production, protected against pancreatic injury, and improved survival in rats with SAP. The serum IL-6 and TNF-α concentrations were also down-regulated by Baicalein. Conclusion: Baicalein demonstrated a well curative capability on rats with SAP. The mechanism may be alleviateing pancreatic injury and inhibiting pro-inflammatory
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cytokines expression.
Key Words: Baicalein, Severe acute pancreatitis, Systemic inflammatory response,
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Pancreatic injury
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ACCEPTED MANUSCRIPT Introduction SAP is a serious necro-inflammatory disease with a high morbidity and mortality rate due to lack of specific therapy. However, the pathogenesis of SAP has not yet been clarified (1,2). More than a century ago, Chiari et al. observed that prematurely
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activated digestive enzymes in the pancreas in SAP patients are responsible for this debilitating disease (3). Inflammatory mediators is vital for progression of SAP and has been receiving increasing attention in recent years. SAP is a potentially lethal
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inflammatory condition of the pancreas that involves both peripancreatic tissue and remote organs. The inflammatory pathways participate in the of this disease from
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early stage to the end. Local inflammatory reaction in pancreas leads to systemic inflammatory response syndrome (SIRS) and multiorgan failure (MOF), which is believed to be the capital cause of mortality (4,5). Inflammatory components include interleukin (IL)-6, tumor necrosis factor (TNF)-α, platelet-activating factor, IL-8, IL-10,
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monocyte chemotactic protein-1 (MCP-1), and IL-1β are considered to be required for SAP development (6). These inflammatory mediators are produced by many different cell types include neutrophils, monocytes, pancreatic acinar cells, macrophages, and
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lymphocytes (4-10). A recent study has shown that proinflammatory stimuli such as
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IL-6 and TNF-α play a central role in the initiation and progression of SAP (11). TNF-α is thought to induce a cascade of other inflammatory cytokines and activate various immune cells, thus inducing the proinflammatory response (12,13). It has been confirmed that the IL-6 level in serum is a very good discriminator of SAP, and when accompanied by multi-organ failure could be used as an early marker of a possible fatal outcome (9). TNF-α and IL-6 aggravate SAP and increase plasma extravasation, inducing leukocyte adherence, resulting in SIRS and MODS (14). Preventing the activity of both cytokines can markedly attenuate the systemic amplification of SAP, 5
ACCEPTED MANUSCRIPT which is responsible for the increased mortality associated with SAP. To enhance therapeutic efficacy, practitioners of traditional Chinese medicine use formulae to treat patients with SAP based on clinical experience. The well-known formula to treat SAP is known as “Qingyi Decoction” therapy and has been advocated
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for 100 years. A major component of the formulae, Scutellaria baicalensis Georgi (“Huang-Qin” in Chinese), is used clinically to treat SAP and exhibits many biological activities, such as antipyretic, antibacterial, and anti-inflammatory properties. Baicalein
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(5,6,7-trihydroxyflavone-7-O-D-glucuronic acid), a flavonoid, with the molecular formula C15H10O5 and a molecular mass of 270.24, is isolated from the Chinese
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traditional medicine herb S. baicalensis Georgi. It is the principal component with bioactivity of S. baicalensis and has excellent antioxidant and anti-inflammatory activities(15-18). Baicalein has been reported to exhibit a property of inhibiting inflammatory mediators production, such as IL-6, interferon-γ, TNF-α, and MCP-1 in
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vitro and in vivo, and could be a validity anti-inflammatory agent (19-21). In our previous study, we have refined the formula Qingyi Decoction based on Chinese medicine theory and devised a Chinese patent medicine “Emodin & Baicalei”. The
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combined use of Baicalein and Emodin showed a therapeutic effect on pancreatic
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injuries in SAP rats (17,18). However, the effectiveness of use of Baicalein alone on SAP and whether it is reasonable to combine Baicalein and Emodin has not been reported. Therefore, we have designed animal experiments to study the therapeutic effects of independent use of Baicalein on SAP and inflammatory cytokine production in rats, thus providing opportunities for the progression of better treatment approaches in SAP. Materials and Methods Materials 6
ACCEPTED MANUSCRIPT 84 male Sprague-Dawley (SD) rats weighing 300–310 g were used in this study. The animals were purchased from the Experimental Animal Center of Xi’an Jiaotong University Health Science Center (Xi’an, China), fed standard laboratory and fasted overnight preoperatively, with free access to water. All procedures in rats were approved
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by the Institutional Animal Use and Care Committee of Xi’an Jiaotong University Health Science Center and performed according to the National Guide for the Care and Use of Laboratory Animals. Sodium taurocholate, Baicalein, and sodium pentobarbital
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were purchased from Sigma-Aldrich (St. Louis, MO, USA). IL-6 and TNF-α enzyme-linked immunosorbent assay (ELISA) kits were obtained from Senxiong
(Nanjing, China). SAP Model Preparation
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Biotech (Shanghai, China). The Amylase Kit was purchased from Jiancheng Biotech
After fasting overnight, 84 rats were divided into sham operation (SO), SAP model
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(SAP), and Baicalein treatment (BA) groups randomly (n=28). Each group was randomly allocated to three subgroups for the time points of 3, 6, and 12 h after the operation (8 rats in 3h and 6h time point groups, 12 rats in the 12h time point group).
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SAP was induced in rats of the SAP and BA groups using the modified protocol of
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Schmidt J et al (22). Surgical anesthesia was induced with an intraperitoneal injection of sodium pentobarbital (50 mg/kg, Sigma-Aldrich) and the abdomen was opened by a midline incision. The duodenum and the common bile duct were identified. The common biliopancreatic duct was advanced with a catheter and the hepatic duct was closed with a small bulldog clamp to prevent backflow. SAP model was induced by retrograde injection into the common biliopancreatic duct of 5% sodium taurocholate (Sigma-Aldrich) at a dose of 0.1 mL/100 g body weight (with 0.1 mL/min injection velocity). Then, the injecting point was pressed for 3 min and the surgery finished with 7
ACCEPTED MANUSCRIPT abdominal stratified closing. Special attention was focused on atraumatic surgical technique. In the SO group, incisions were closed immediately following the turning over of the pancreas. Baicalein (20 mg/kg) was injected via tail vein to fasted rats of the BA group. SO and SAP rats were given a same amount of normal saline. Animals were
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anesthetized with sodium pentobarbital 3, 6, or 12 h after the surgery and a laparotomy performed. Intracardiac blood was collected from the right ventricle, centrifuged at 1800×g for 10 minutes and the serum stored at −20 °C for subsequent analysis. Ascites
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fluid was harvested and weight. The entire pancreas was removed immediately and immersion fixed in 4% paraformaldehyde in anatomic orientation, then embedded in
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paraffin, and sectioned at 4 µm for histologic analysis.
Detection of 12-h Mortality and Ascites Fluid Amount in SAP Rats The mortality of rats was determined by calculating the numbers of surviving and dead rats in every group over the 12 h post-surgery. The ascites fluid amount was
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assayed by the difference of gauze weight before and after being placed in the abdominal cavity to last for 5 min. Histopathologic Analysis removed
entire
pancreatic
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The
tissues
were
immersion
fixed
in
4%
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paraformaldehyde for 24 hours, followed by dehydrating and embedding in paraffin using a routine protocol. The paraffin-embedded tissue samples were cut at 4 µm thick at longitudinal section and stained with hematoxylin and eosin. The slides were scored by two blinded experienced pathologists, and the histopathological changes of the pancreatic tissue were evaluated by light microscopy. Two slides and ten fields/slide were examined for histopathological analysis in each pancreas. The histopathology scoring criteria were: edema, acinar necrosis, hemorrhages, and inflammation. We complied the standardized scoring system defined with Spomam et al (23). as shown in 8
ACCEPTED MANUSCRIPT Table 1, and the final score of each section was the summation of each pathological parameter . Measurement of Amylase Activity and Pro-inflammatory Cytokine Level in Serum The serum amylase activity was determined according to the instruction of
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manufacturer (Jiancheng Biotech, Nanjing, China). Serum IL-6 and TNF-α concentrations were measured by commercially available ELISA kits (Senxiong Biotech, Shanghai, China) complying with the manufacturer’s protocols. Absorbance was
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detected at 450 nm by a microplate mode reader (BioTek Inc., Winooski, VT, USA). Statistical Analysis
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All data were presented as the means ± standard deviation (S.D). Statistical comparisons were made by analysis of variance (SPSS ver. 14.0; SPSS Software, Chicago, USA). A probability of experimental error < 5% (P < 0.05) was considered acceptable for statistical significant difference.
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Results
Baicalein Reduces the 12-h Mortality and Ascites Fluid Amount in SAP Rats The mortality 12 hours after SAP induction in SAP group was 50%, and Baicalein
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markedly decreased this mortality (Fig. 1 A, P < 0.01). The amount of ascites fluid in
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SAP group also increased significantly at 3, 6, and 12 h time points compared with the SO group (Fig. 1 B, P < 0.01). Treatment with Baicalein greatly decreased the amount of ascites fluid at the 12h time point (Fig. 1 B, P < 0.05). However, Baicalein did not affect the ascites fluid at 3, 6 h (Fig. 1 B, P > 0.05). Baicalein Attenuates the Severity of Pancreatic Injury SO group showed entirely normal acinar architecture. SAP group demonstrated pancreatic injury characterize by moderate tissue edema, light hemorrhage and necrosis at 3 h time point after SAP induction compared with SO group. As time progressed, the 9
ACCEPTED MANUSCRIPT severity of pancreatic injury deteriorated and there were grossly edema and sublobular hemorrhages associated with obvious neutrophil infiltration and parenchymal necrosis by 6 h after SAP induction. 12 hours after SAP induction, the pancreas showed massive fat and parenchymal necrosis, inflammatory infiltration, cell lysis, and a reduction in
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cell number plus the aforementioned damage (Fig. 2 a–c). Treatment with Baicalein ameliorated pancreatic injury and decreased the histopathology scores at the 6 h and 12 h time points (Fig. 2, P < 0.05), but exhibited no influence at 3 h (Fig. 2, P >0.05).
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Effect of Baicalein on Serum Amylase Activity in SAP Rats
The serum amylase activity in SAP group increased significantly at 3, 6, and 12 h
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time points compared with SO group (Fig. 3, P < 0.01). However, treatment with Baicalein did not affect the serum amylase activity (Fig. 3, P > 0.05). Baicalein Reduces the Serum Level of Proinflammatory Cytokines Compared with the SO group, serum level of IL-6 and TNF-α in SAP group were
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increased significantly at 3, 6, and 12 h time points (Fig. 4, P < 0.01). Baicalein treatment markedly decreased the level of the pro-inflammatory cytokines at the 6h and
Discussion
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12h time points (Fig. 4, P < 0.05), but showed no action at 3 h (Fig. 4, P >0.05).
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To date, there is no specific treatment for SAP. However, this study highlighted the therapeutic effect of Baicalein against SAP in a rat model. In our study of pancreatitis induced by taurocholate, both rat mortality and pancreatic tissue pathology score were reduced after Baicalein treatment, thus demonstrating the therapeutic benefits of Baicalein in SAP. The pathological changes present in the pancreas during SAP pathogenesis are complicated. This process is associated with pancreas autodigestion by prematurely activated digestive enzymes, acinar rupture, vasodilatation, increased blood flow, and inflammatory cell infiltration, which could all lead to pancreatic injury (4). 10
ACCEPTED MANUSCRIPT The results demonstrated that compared with SO group, the histopathology scores were significantly elevated at 3, 6 and 12h time points in SAP rats. Pancreatitis-specific changes were showed as tissue edema, neutrophil infiltration, parenchymal hemorrhage, and acinar cell necrosis, which aggravated as time progressed. Baicalein administration
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reduced the pathologic scores at all time points, indicating the amelioration of pancreatic injury. Improving survival rate has become a standard criterion to estimate the efficacy of a drug, so the therapeutic effect of Baicalein on SAP is worth further
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study.
The pathogenesis of SAP is poorly understood. At present, it is known that the
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“inflammatory reaction” hypothesis plays a critical role in the pathophysiology of SAP and current research continues to focus on its mechanism. The excessive inflammatory reaction (SIRS) is suggested to be the main cause of high mortality and morbidity in SAP (3). TNF-α and IL-6 are known to participate in the initiation and progression of
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SAP. TNF-α is thought to initiate a cascade of inflammatory reactions and be a reliable predictor of outcome in SAP (24,25). IL-6 is also a principal pro-inflammatory cytokine and has been identified as the most reliable prognostic factor in SAP (26). Previous
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studies have shown that up-regulation of both IL-6 and TNF-α in pancreas and serum
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were observed in SAP induced by taurocholate (24,27). In our study, we similarly found that serum IL-6 and TNF-α were considerably higher than in SO group at all time points and that Baicalein reduced serum IL-6 and TNF-α at 6 and 12 h time points after SAP induction. We first time provided important data showing that Baicalein can attenuate the pro-inflammatory cytokines production in SAP, thus effectively protecting pancreatic tissue injury. There is evidence to show that Baicalein display many biological effects, including antipyretic, antibacterial, and anti-inflammatory properties. Previous studies revealed 11
ACCEPTED MANUSCRIPT Baicalein could suppress the multiple inflammatory mediators generation, including IL-6, TNF-α, INF-γ, and MCP-1 both in vitro and in vivo and could be a potential anti-inflammatory agent (19-21). However, independent use of Baicalein on SAP has not been reported and the effect of Baicalein is still not fully clarified. In our study, we
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first time found Baicalein exhibited inhibitory effects on pro-inflammatory cytokine production, decreasing the serum IL-6 and TNF-α concentrations and attenuating the inflammatory response in SAP.
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In addition to increased migration of inflammatory cells and pro-inflammatory cytokine release, SAP causes damage to vascular endothelial cells and increased plasma
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extravasation, leading to the accumulation of ascites fluid (22). In our study, SAP induced in rats by sodium taurocholate appeared to be coupled with hemorrhagic ascites and increased ascites fluid amount compared with the SO group. Baicalein reduced the ascites fluid volume 12 h after SAP induction, demonstrating the anti-inflammatory
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activity of Baicalein can reduce capillary permeability and protect the capillary walls. The amylase test is a standardization in the clinical diagnosis of SAP. Determination of serum amylase activity reveals inflammation in the pancreas. In this
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study, we found serum amylase activity was elevated in SAP rats at 3h, 6h, and 12h
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after SAP model induction. However, the degree of increased amylase does not necessarily equal the severity of SAP. Some patients with mild pancreatitis may have high amylase activity, while some have moderate amylase activity but acute necrotizing pancreatitis. Thus, the level of amylase activity cannot serve as a diagnostic standard to estimate the prognosis of SAP. This may explain why Baicalein attenuates SAP, but does not decrease the amylase activity. Results in our study indicated that inflammation should be a major target for developing therapy for SAP and remains our greatest challenge in the field of SAP 12
ACCEPTED MANUSCRIPT research. Baicalein demonstrated good therapeutic effects in SAP and has great future potential. Although details of the molecular mechanisms of the anti-inflammatory properties of Baicalain in SAP remain unclear, it demonstrate a good perspective for SAP therapy and deserve to lucubrate.
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Baicalein both significantly suppressed the inflammatory response and reduced pancreatic injury in SAP. It also improved survival in SAP rats. Thus, Baicalein may be a candidate drug for SAP therapy with good perspective. However, the insufficiency of
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our research was that there is a deficiency on specific mechanisms study of Baicalein. Further experiments are therefore necessary to illustrate the pharmacologic mechanisms
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of Baicalein on SAP. As lacking any specific therapy for SAP has been a serious shortcoming of current treatments, Baicalein may provide benefits for SAP patients in the future. Acknowledgments
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This work was supported by the National Natural Science Foundation of China (Grant Nos. 30171167, 30901945) and The Science and Technology Program of Shaanxi Province (No. 2012KTCQ03-15).
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Author Contributions
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Yongtao Wu and Zongfang Li designed the research; Yongtao Wu and Shu Zhang performed the research. Yongtao Wu and Jun Li analyzed the data and wrote the paper. Jun Li, Jian Zhang, Fanpu Ji, and Wangjun Bo were involved in the editing of the manuscript; Xiaoyan Guo and Zongfang Li finalized and revised the manuscript. All authors have read and approved the final version of the manuscript. Disclosures None of the authors have conflicts of interest to disclose. References 13
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ACCEPTED MANUSCRIPT Figure legands Figure 1 a. Effect of Baicalein on the 12 h mortality of SAP rats. Animals were administrated with Baicalein or normal saline immediately after SAP induction.
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The mortality was evaluated by calculating the numbers of surviving and dead rats over the 12 h post-surgery. Data represents mean±S.D (* P < 0.01). b. Baicalein
decreased the ascites fluid amount at 3, 6, and 12 h time points of SAP rats.
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Animals were administrated with Baicalein or normal saline after SAP induction. Ascites fluid amount was assayed by the difference of gauze weight before and
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after being placed in the abdominal cavity to last for 5 min. Data represents mean±S.D (* P < 0.01 compared with the SO group. ** P < 0.05 compared with the SAP group).
Figure 2. Effect of Baicalein on histological changes and histopathology scores
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for pancreatic tissues at 3, 6, and 12 h time points. Animals were administrated with Baicalein or normal saline after SAP induction. Histopathology scores were
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evaluated complied the standardized scoring system described in Materials and Methods. (a) SO group; (b) SAP group; (c) BA group. The SAP group exhibited
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massive parenchymal and fat necrosis, extensive hemorrhaging and cell number decrease, while in the SO group the change was not remarkable. The BA group exhibited few pathological changes, pancreatic tissue showed little edema and no inflammatory infiltrate 12 h after SAP induction; (d): The histopathology scores increased at all tine points after SAP induction. Treatment with Baicalein markedly reduced the histopathology scores at 6-h and 12-h time points. Data represents mean±S.D (* P < 0.01 compared with the SO group. ** P < 0.05 compared with the SAP group). 18
ACCEPTED MANUSCRIPT Figure 3. Effect of Baicalein on serum amylase activity at 3, 6, and 12 h time points. Serum amylase activity was determined as described in Materials and Methods. Serum amylase activity increased significantly after SAP induction at all time points. However, treatment with Baicalein did not alter the serum amylase
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activity. Data represents mean±S.D (* P < 0.01 compared with the SO group).
Figure 4. ELISA showing that Baicalein affects serum level of pro-inflammatory
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cytokines at 3, 6, and 12 h time points. The level of serum IL-6 and TNF-α
increased significantly in the SAP group at all time points investigated. Baicalein
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administration markedly reduced the level of pro-inflammatory cytokines at 6 and 12 h time points. Data represents mean±S.D (* P < 0.01 compared with the SO
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group. ** P < 0.05 compared with the SAP group).
19
ACCEPTED MANUSCRIPT Table 1.
Criteria for microscopical assessment Assessment
Score
Edema
mild
1
moderate
2
severe
3
mild
1
moderate
2
severe
3
<2/section
3
3-5/section
5
>5/section
7
focal (<5%)
3
Parenchymal necrosis
Hemorrhages
and/or sublobular
5
and/or lobular (>20%)
7
mild
3
moderate
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severe
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Fat necrosis
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Inflammatory
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Histological patterns
5
7
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We first time found Baicalein exhibited inhibitory effects on pro-inflammatory cytokine production in severe acute pancreatitis.
Baicalein protect pancreatic injury in rats with severe acute pancreatitis, which may be a candidate drug for severe acute pancreatitis therapy with good perspective.
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Baicalein attenuates severe acute pancreatitis, but does not decrease the amylase
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activity.
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1
S0006-291X(15)30611-2
DOI:
10.1016/j.bbrc.2015.09.094
Reference:
YBBRC 34594
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 11 September 2015 Accepted Date: 17 September 2015
Please cite this article as: J. Li, Y. Wu, S. Zhang, J. Zhang, F. Ji, W. Bo, X. Guo, Z. Li, Baicalein protect pancreatic injury in rats with severe acute pancreatitis by inhibiting pro-inflammatory cytokines expression, Biochemical and Biophysical Research Communications (2015), doi: 10.1016/ j.bbrc.2015.09.094. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Baicalein Protect Pancreatic Injury in Rats with Severe Acute Pancreatitis by inhibiting Pro-inflammatory Cytokines Expression Jun Li*#, Yongtao Wu†#, Shu Zhang*‡, Jian Zhang*‡, Fanpu Ji*, Wangjun Bo*, Xiaoyan
*
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Guo*§ and Zongfang Li*‡§
National-local Joint Engineering Research Center of Biodiagnostics and Biotherapy, the Second Affiliated Hospital of Xi’an Jiaotong University College of Medicine, 157
Department of General Surgery, Hong Hui Hospital, Xi’an Jiaotong University
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†
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West 5th Road, Xi'an 710004, Shaanxi Province, PR. China
College of Medicine, 555 East Friendship Road, Xi'an 710054, Shaanxi Province, PR. China ‡
Department of General Surgery, the Second Affiliated Hospital of Xi’an Jiaotong University College of Medicine, 157 West 5th Road, Xi'an 710004, Shaanxi Province,
These authors contributed equally to this work.
Jun Li, Ph.D.
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#
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PR. China
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National Local Joint Engineering Research Center of Biodiagnostics and Biotherapy the Second Affiliated Hospital of Xi’an Jiaotong University College of Medicine 157 West 5th Road, Xi'an 710004, Shaanxi, PR. China e-mail
[email protected]
Yongtao Wu, Ph.D. Department of General Surgery, Hong Hui Hospital, Xi’an Jiaotong University College of Medicine 555 East Friendship Road, Xi'an 710054, Shaanxi, PR. China 1
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[email protected]
Shu Zhang, Ph.D. National Local Joint Engineering Research Center of Biodiagnostics and Biotherapy,
University College of Medicine 157 West 5th Road, Xi'an 710004, Shaanxi, PR. China e-mail
[email protected]
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Jian Zhang, Ph.D.
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Department of General Surgery, the Second Affiliated Hospital of Xi’an Jiaotong
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National Local Joint Engineering Research Center of Biodiagnostics and Biotherapy, Department of General Surgery, the Second Affiliated Hospital of Xi’an Jiaotong University College of Medicine
157 West 5th Road, Xi'an 710004, Shaanxi, PR. China [email protected]
Fanpu Ji, Ph.D.
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National Local Joint Engineering Research Center of Biodiagnostics and Biotherapy
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the Second Affiliated Hospital of Xi’an Jiaotong University College of Medicine 157 West 5th Road, Xi'an 710004, Shaanxi, PR. China [email protected]
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Wangjun Bo, Ph.D.
National Local Joint Engineering Research Center of Biodiagnostics and Biotherapy the Second Affiliated Hospital of Xi’an Jiaotong University College of Medicine 157 West 5th Road, Xi'an 710004, Shaanxi, PR. China e-mail §
[email protected]
Corresponding authors: 2
ACCEPTED MANUSCRIPT Dr. Zongfang Li, M.D., Ph.D. and Dr. Xiaoyan Guo, M.D. National Local Joint Engineering Research Center of Biodiagnostics and Biotherapy, the Second Affiliated Hospital of Xi’an Jiaotong University College of Medicine, 157 West 5th Road, Xi’an 710004, Shaanxi, P.R. China
Email: [email protected], [email protected] Running title
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Telephone: +86-29-87679684, +86-29-87679275 Fax: +86-29-87678634
Baicalein Protect Pancreatic Injury of SAP in Rats
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experimental study
Text word count: 4408
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the date of submission:
Number of references: 27; Tables: 1; Figures: 4
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Abstract word count: 136
3
ACCEPTED MANUSCRIPT Abstract Background/Aim: Inflammatory cytokines is a key point in the development of pathogenesis of SAP. Inflammatory mediators TNF-α and IL-6 are up-regulated in serum of patients with SAP and become good discriminators of SAP severity.
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Materials and Methods: In this study, we investigated the treatment effectiveness of Baicalein on SAP rat model. Baicalein was intravenously injected immediately after SAP induction in rats. The mortality, histopathology score, ascites fluid volume, and
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pro-inflammatory cytokine production were evaluated at 12 h after SAP induction.
Results: Baicalein decreased the pancreatic histopathology score, reduced ascites fluid
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production, protected against pancreatic injury, and improved survival in rats with SAP. The serum IL-6 and TNF-α concentrations were also down-regulated by Baicalein. Conclusion: Baicalein demonstrated a well curative capability on rats with SAP. The mechanism may be alleviateing pancreatic injury and inhibiting pro-inflammatory
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cytokines expression.
Key Words: Baicalein, Severe acute pancreatitis, Systemic inflammatory response,
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Pancreatic injury
4
ACCEPTED MANUSCRIPT Introduction SAP is a serious necro-inflammatory disease with a high morbidity and mortality rate due to lack of specific therapy. However, the pathogenesis of SAP has not yet been clarified (1,2). More than a century ago, Chiari et al. observed that prematurely
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activated digestive enzymes in the pancreas in SAP patients are responsible for this debilitating disease (3). Inflammatory mediators is vital for progression of SAP and has been receiving increasing attention in recent years. SAP is a potentially lethal
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inflammatory condition of the pancreas that involves both peripancreatic tissue and remote organs. The inflammatory pathways participate in the of this disease from
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early stage to the end. Local inflammatory reaction in pancreas leads to systemic inflammatory response syndrome (SIRS) and multiorgan failure (MOF), which is believed to be the capital cause of mortality (4,5). Inflammatory components include interleukin (IL)-6, tumor necrosis factor (TNF)-α, platelet-activating factor, IL-8, IL-10,
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monocyte chemotactic protein-1 (MCP-1), and IL-1β are considered to be required for SAP development (6). These inflammatory mediators are produced by many different cell types include neutrophils, monocytes, pancreatic acinar cells, macrophages, and
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lymphocytes (4-10). A recent study has shown that proinflammatory stimuli such as
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IL-6 and TNF-α play a central role in the initiation and progression of SAP (11). TNF-α is thought to induce a cascade of other inflammatory cytokines and activate various immune cells, thus inducing the proinflammatory response (12,13). It has been confirmed that the IL-6 level in serum is a very good discriminator of SAP, and when accompanied by multi-organ failure could be used as an early marker of a possible fatal outcome (9). TNF-α and IL-6 aggravate SAP and increase plasma extravasation, inducing leukocyte adherence, resulting in SIRS and MODS (14). Preventing the activity of both cytokines can markedly attenuate the systemic amplification of SAP, 5
ACCEPTED MANUSCRIPT which is responsible for the increased mortality associated with SAP. To enhance therapeutic efficacy, practitioners of traditional Chinese medicine use formulae to treat patients with SAP based on clinical experience. The well-known formula to treat SAP is known as “Qingyi Decoction” therapy and has been advocated
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for 100 years. A major component of the formulae, Scutellaria baicalensis Georgi (“Huang-Qin” in Chinese), is used clinically to treat SAP and exhibits many biological activities, such as antipyretic, antibacterial, and anti-inflammatory properties. Baicalein
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(5,6,7-trihydroxyflavone-7-O-D-glucuronic acid), a flavonoid, with the molecular formula C15H10O5 and a molecular mass of 270.24, is isolated from the Chinese
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traditional medicine herb S. baicalensis Georgi. It is the principal component with bioactivity of S. baicalensis and has excellent antioxidant and anti-inflammatory activities(15-18). Baicalein has been reported to exhibit a property of inhibiting inflammatory mediators production, such as IL-6, interferon-γ, TNF-α, and MCP-1 in
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vitro and in vivo, and could be a validity anti-inflammatory agent (19-21). In our previous study, we have refined the formula Qingyi Decoction based on Chinese medicine theory and devised a Chinese patent medicine “Emodin & Baicalei”. The
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combined use of Baicalein and Emodin showed a therapeutic effect on pancreatic
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injuries in SAP rats (17,18). However, the effectiveness of use of Baicalein alone on SAP and whether it is reasonable to combine Baicalein and Emodin has not been reported. Therefore, we have designed animal experiments to study the therapeutic effects of independent use of Baicalein on SAP and inflammatory cytokine production in rats, thus providing opportunities for the progression of better treatment approaches in SAP. Materials and Methods Materials 6
ACCEPTED MANUSCRIPT 84 male Sprague-Dawley (SD) rats weighing 300–310 g were used in this study. The animals were purchased from the Experimental Animal Center of Xi’an Jiaotong University Health Science Center (Xi’an, China), fed standard laboratory and fasted overnight preoperatively, with free access to water. All procedures in rats were approved
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by the Institutional Animal Use and Care Committee of Xi’an Jiaotong University Health Science Center and performed according to the National Guide for the Care and Use of Laboratory Animals. Sodium taurocholate, Baicalein, and sodium pentobarbital
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were purchased from Sigma-Aldrich (St. Louis, MO, USA). IL-6 and TNF-α enzyme-linked immunosorbent assay (ELISA) kits were obtained from Senxiong
(Nanjing, China). SAP Model Preparation
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Biotech (Shanghai, China). The Amylase Kit was purchased from Jiancheng Biotech
After fasting overnight, 84 rats were divided into sham operation (SO), SAP model
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(SAP), and Baicalein treatment (BA) groups randomly (n=28). Each group was randomly allocated to three subgroups for the time points of 3, 6, and 12 h after the operation (8 rats in 3h and 6h time point groups, 12 rats in the 12h time point group).
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SAP was induced in rats of the SAP and BA groups using the modified protocol of
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Schmidt J et al (22). Surgical anesthesia was induced with an intraperitoneal injection of sodium pentobarbital (50 mg/kg, Sigma-Aldrich) and the abdomen was opened by a midline incision. The duodenum and the common bile duct were identified. The common biliopancreatic duct was advanced with a catheter and the hepatic duct was closed with a small bulldog clamp to prevent backflow. SAP model was induced by retrograde injection into the common biliopancreatic duct of 5% sodium taurocholate (Sigma-Aldrich) at a dose of 0.1 mL/100 g body weight (with 0.1 mL/min injection velocity). Then, the injecting point was pressed for 3 min and the surgery finished with 7
ACCEPTED MANUSCRIPT abdominal stratified closing. Special attention was focused on atraumatic surgical technique. In the SO group, incisions were closed immediately following the turning over of the pancreas. Baicalein (20 mg/kg) was injected via tail vein to fasted rats of the BA group. SO and SAP rats were given a same amount of normal saline. Animals were
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anesthetized with sodium pentobarbital 3, 6, or 12 h after the surgery and a laparotomy performed. Intracardiac blood was collected from the right ventricle, centrifuged at 1800×g for 10 minutes and the serum stored at −20 °C for subsequent analysis. Ascites
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fluid was harvested and weight. The entire pancreas was removed immediately and immersion fixed in 4% paraformaldehyde in anatomic orientation, then embedded in
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paraffin, and sectioned at 4 µm for histologic analysis.
Detection of 12-h Mortality and Ascites Fluid Amount in SAP Rats The mortality of rats was determined by calculating the numbers of surviving and dead rats in every group over the 12 h post-surgery. The ascites fluid amount was
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assayed by the difference of gauze weight before and after being placed in the abdominal cavity to last for 5 min. Histopathologic Analysis removed
entire
pancreatic
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The
tissues
were
immersion
fixed
in
4%
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paraformaldehyde for 24 hours, followed by dehydrating and embedding in paraffin using a routine protocol. The paraffin-embedded tissue samples were cut at 4 µm thick at longitudinal section and stained with hematoxylin and eosin. The slides were scored by two blinded experienced pathologists, and the histopathological changes of the pancreatic tissue were evaluated by light microscopy. Two slides and ten fields/slide were examined for histopathological analysis in each pancreas. The histopathology scoring criteria were: edema, acinar necrosis, hemorrhages, and inflammation. We complied the standardized scoring system defined with Spomam et al (23). as shown in 8
ACCEPTED MANUSCRIPT Table 1, and the final score of each section was the summation of each pathological parameter . Measurement of Amylase Activity and Pro-inflammatory Cytokine Level in Serum The serum amylase activity was determined according to the instruction of
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manufacturer (Jiancheng Biotech, Nanjing, China). Serum IL-6 and TNF-α concentrations were measured by commercially available ELISA kits (Senxiong Biotech, Shanghai, China) complying with the manufacturer’s protocols. Absorbance was
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detected at 450 nm by a microplate mode reader (BioTek Inc., Winooski, VT, USA). Statistical Analysis
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All data were presented as the means ± standard deviation (S.D). Statistical comparisons were made by analysis of variance (SPSS ver. 14.0; SPSS Software, Chicago, USA). A probability of experimental error < 5% (P < 0.05) was considered acceptable for statistical significant difference.
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Results
Baicalein Reduces the 12-h Mortality and Ascites Fluid Amount in SAP Rats The mortality 12 hours after SAP induction in SAP group was 50%, and Baicalein
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markedly decreased this mortality (Fig. 1 A, P < 0.01). The amount of ascites fluid in
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SAP group also increased significantly at 3, 6, and 12 h time points compared with the SO group (Fig. 1 B, P < 0.01). Treatment with Baicalein greatly decreased the amount of ascites fluid at the 12h time point (Fig. 1 B, P < 0.05). However, Baicalein did not affect the ascites fluid at 3, 6 h (Fig. 1 B, P > 0.05). Baicalein Attenuates the Severity of Pancreatic Injury SO group showed entirely normal acinar architecture. SAP group demonstrated pancreatic injury characterize by moderate tissue edema, light hemorrhage and necrosis at 3 h time point after SAP induction compared with SO group. As time progressed, the 9
ACCEPTED MANUSCRIPT severity of pancreatic injury deteriorated and there were grossly edema and sublobular hemorrhages associated with obvious neutrophil infiltration and parenchymal necrosis by 6 h after SAP induction. 12 hours after SAP induction, the pancreas showed massive fat and parenchymal necrosis, inflammatory infiltration, cell lysis, and a reduction in
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cell number plus the aforementioned damage (Fig. 2 a–c). Treatment with Baicalein ameliorated pancreatic injury and decreased the histopathology scores at the 6 h and 12 h time points (Fig. 2, P < 0.05), but exhibited no influence at 3 h (Fig. 2, P >0.05).
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Effect of Baicalein on Serum Amylase Activity in SAP Rats
The serum amylase activity in SAP group increased significantly at 3, 6, and 12 h
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time points compared with SO group (Fig. 3, P < 0.01). However, treatment with Baicalein did not affect the serum amylase activity (Fig. 3, P > 0.05). Baicalein Reduces the Serum Level of Proinflammatory Cytokines Compared with the SO group, serum level of IL-6 and TNF-α in SAP group were
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increased significantly at 3, 6, and 12 h time points (Fig. 4, P < 0.01). Baicalein treatment markedly decreased the level of the pro-inflammatory cytokines at the 6h and
Discussion
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12h time points (Fig. 4, P < 0.05), but showed no action at 3 h (Fig. 4, P >0.05).
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To date, there is no specific treatment for SAP. However, this study highlighted the therapeutic effect of Baicalein against SAP in a rat model. In our study of pancreatitis induced by taurocholate, both rat mortality and pancreatic tissue pathology score were reduced after Baicalein treatment, thus demonstrating the therapeutic benefits of Baicalein in SAP. The pathological changes present in the pancreas during SAP pathogenesis are complicated. This process is associated with pancreas autodigestion by prematurely activated digestive enzymes, acinar rupture, vasodilatation, increased blood flow, and inflammatory cell infiltration, which could all lead to pancreatic injury (4). 10
ACCEPTED MANUSCRIPT The results demonstrated that compared with SO group, the histopathology scores were significantly elevated at 3, 6 and 12h time points in SAP rats. Pancreatitis-specific changes were showed as tissue edema, neutrophil infiltration, parenchymal hemorrhage, and acinar cell necrosis, which aggravated as time progressed. Baicalein administration
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reduced the pathologic scores at all time points, indicating the amelioration of pancreatic injury. Improving survival rate has become a standard criterion to estimate the efficacy of a drug, so the therapeutic effect of Baicalein on SAP is worth further
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The pathogenesis of SAP is poorly understood. At present, it is known that the
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“inflammatory reaction” hypothesis plays a critical role in the pathophysiology of SAP and current research continues to focus on its mechanism. The excessive inflammatory reaction (SIRS) is suggested to be the main cause of high mortality and morbidity in SAP (3). TNF-α and IL-6 are known to participate in the initiation and progression of
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SAP. TNF-α is thought to initiate a cascade of inflammatory reactions and be a reliable predictor of outcome in SAP (24,25). IL-6 is also a principal pro-inflammatory cytokine and has been identified as the most reliable prognostic factor in SAP (26). Previous
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studies have shown that up-regulation of both IL-6 and TNF-α in pancreas and serum
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were observed in SAP induced by taurocholate (24,27). In our study, we similarly found that serum IL-6 and TNF-α were considerably higher than in SO group at all time points and that Baicalein reduced serum IL-6 and TNF-α at 6 and 12 h time points after SAP induction. We first time provided important data showing that Baicalein can attenuate the pro-inflammatory cytokines production in SAP, thus effectively protecting pancreatic tissue injury. There is evidence to show that Baicalein display many biological effects, including antipyretic, antibacterial, and anti-inflammatory properties. Previous studies revealed 11
ACCEPTED MANUSCRIPT Baicalein could suppress the multiple inflammatory mediators generation, including IL-6, TNF-α, INF-γ, and MCP-1 both in vitro and in vivo and could be a potential anti-inflammatory agent (19-21). However, independent use of Baicalein on SAP has not been reported and the effect of Baicalein is still not fully clarified. In our study, we
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first time found Baicalein exhibited inhibitory effects on pro-inflammatory cytokine production, decreasing the serum IL-6 and TNF-α concentrations and attenuating the inflammatory response in SAP.
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In addition to increased migration of inflammatory cells and pro-inflammatory cytokine release, SAP causes damage to vascular endothelial cells and increased plasma
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extravasation, leading to the accumulation of ascites fluid (22). In our study, SAP induced in rats by sodium taurocholate appeared to be coupled with hemorrhagic ascites and increased ascites fluid amount compared with the SO group. Baicalein reduced the ascites fluid volume 12 h after SAP induction, demonstrating the anti-inflammatory
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activity of Baicalein can reduce capillary permeability and protect the capillary walls. The amylase test is a standardization in the clinical diagnosis of SAP. Determination of serum amylase activity reveals inflammation in the pancreas. In this
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study, we found serum amylase activity was elevated in SAP rats at 3h, 6h, and 12h
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after SAP model induction. However, the degree of increased amylase does not necessarily equal the severity of SAP. Some patients with mild pancreatitis may have high amylase activity, while some have moderate amylase activity but acute necrotizing pancreatitis. Thus, the level of amylase activity cannot serve as a diagnostic standard to estimate the prognosis of SAP. This may explain why Baicalein attenuates SAP, but does not decrease the amylase activity. Results in our study indicated that inflammation should be a major target for developing therapy for SAP and remains our greatest challenge in the field of SAP 12
ACCEPTED MANUSCRIPT research. Baicalein demonstrated good therapeutic effects in SAP and has great future potential. Although details of the molecular mechanisms of the anti-inflammatory properties of Baicalain in SAP remain unclear, it demonstrate a good perspective for SAP therapy and deserve to lucubrate.
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Baicalein both significantly suppressed the inflammatory response and reduced pancreatic injury in SAP. It also improved survival in SAP rats. Thus, Baicalein may be a candidate drug for SAP therapy with good perspective. However, the insufficiency of
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our research was that there is a deficiency on specific mechanisms study of Baicalein. Further experiments are therefore necessary to illustrate the pharmacologic mechanisms
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of Baicalein on SAP. As lacking any specific therapy for SAP has been a serious shortcoming of current treatments, Baicalein may provide benefits for SAP patients in the future. Acknowledgments
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This work was supported by the National Natural Science Foundation of China (Grant Nos. 30171167, 30901945) and The Science and Technology Program of Shaanxi Province (No. 2012KTCQ03-15).
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Author Contributions
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Yongtao Wu and Zongfang Li designed the research; Yongtao Wu and Shu Zhang performed the research. Yongtao Wu and Jun Li analyzed the data and wrote the paper. Jun Li, Jian Zhang, Fanpu Ji, and Wangjun Bo were involved in the editing of the manuscript; Xiaoyan Guo and Zongfang Li finalized and revised the manuscript. All authors have read and approved the final version of the manuscript. Disclosures None of the authors have conflicts of interest to disclose. References 13
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23. H. Spomam, A. Sokolowski, G. Letko, Effect of temporary ischema upon development and histological patterns of acute pancreatitis in the rats, Pathol. Res. Pract. 184 (1989) 507-513.
24. D.Q. Zhang, H. Feng, W.C. Chen, Effects of Hydrogen-Rich Saline on Taurocholate-Induced Acute Pancreatitis in Rat, Evid. Based. Complement. Alternat. Med. 2013 (2013) 731932. 25. N. Pooran, A. Indaram, P. Singh, S. Bank, Cytokines (IL-6, IL-8, TNF): early and reliable predictors of severe acute pancreatitis, J. Clin. Gastroenterol. 37 (2003) 16
ACCEPTED MANUSCRIPT 263-266. 26. H. Zhang, P. Neuhöfer, L. Song, B. Rabe, M. Lesina, M.U. Kurkowski, M. Treiber, T. Wartmann, S. Regnér, H. Thorlacius, D. Saur, G. Weirich, A. Yoshimura, W. Halangk, J.P. Mizgerd, R.M. Schmid, S. Rose-John, H. Algül, IL-6 trans-signaling
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promotes pancreatitis-associated lung injury and lethality, J. Clin. Invest. 123 (2013) 1019-1031.
27. L. Wu, B. Cai, S. Zheng, X. Liu, H. Cai, H. Li, Effect of Emodin on Endoplasmic
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Reticulum Stress in Rats with Severe Acute Pancreatitis, Inflammation. 36 (2013)
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1020-1029.
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ACCEPTED MANUSCRIPT Figure legands Figure 1 a. Effect of Baicalein on the 12 h mortality of SAP rats. Animals were administrated with Baicalein or normal saline immediately after SAP induction.
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The mortality was evaluated by calculating the numbers of surviving and dead rats over the 12 h post-surgery. Data represents mean±S.D (* P < 0.01). b. Baicalein
decreased the ascites fluid amount at 3, 6, and 12 h time points of SAP rats.
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Animals were administrated with Baicalein or normal saline after SAP induction. Ascites fluid amount was assayed by the difference of gauze weight before and
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after being placed in the abdominal cavity to last for 5 min. Data represents mean±S.D (* P < 0.01 compared with the SO group. ** P < 0.05 compared with the SAP group).
Figure 2. Effect of Baicalein on histological changes and histopathology scores
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for pancreatic tissues at 3, 6, and 12 h time points. Animals were administrated with Baicalein or normal saline after SAP induction. Histopathology scores were
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evaluated complied the standardized scoring system described in Materials and Methods. (a) SO group; (b) SAP group; (c) BA group. The SAP group exhibited
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massive parenchymal and fat necrosis, extensive hemorrhaging and cell number decrease, while in the SO group the change was not remarkable. The BA group exhibited few pathological changes, pancreatic tissue showed little edema and no inflammatory infiltrate 12 h after SAP induction; (d): The histopathology scores increased at all tine points after SAP induction. Treatment with Baicalein markedly reduced the histopathology scores at 6-h and 12-h time points. Data represents mean±S.D (* P < 0.01 compared with the SO group. ** P < 0.05 compared with the SAP group). 18
ACCEPTED MANUSCRIPT Figure 3. Effect of Baicalein on serum amylase activity at 3, 6, and 12 h time points. Serum amylase activity was determined as described in Materials and Methods. Serum amylase activity increased significantly after SAP induction at all time points. However, treatment with Baicalein did not alter the serum amylase
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activity. Data represents mean±S.D (* P < 0.01 compared with the SO group).
Figure 4. ELISA showing that Baicalein affects serum level of pro-inflammatory
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cytokines at 3, 6, and 12 h time points. The level of serum IL-6 and TNF-α
increased significantly in the SAP group at all time points investigated. Baicalein
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administration markedly reduced the level of pro-inflammatory cytokines at 6 and 12 h time points. Data represents mean±S.D (* P < 0.01 compared with the SO
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group. ** P < 0.05 compared with the SAP group).
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ACCEPTED MANUSCRIPT Table 1.
Criteria for microscopical assessment Assessment
Score
Edema
mild
1
moderate
2
severe
3
mild
1
moderate
2
severe
3
<2/section
3
3-5/section
5
>5/section
7
focal (<5%)
3
Parenchymal necrosis
Hemorrhages
and/or sublobular
5
and/or lobular (>20%)
7
mild
3
moderate
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severe
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Fat necrosis
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Inflammatory
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Histological patterns
5
7
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We first time found Baicalein exhibited inhibitory effects on pro-inflammatory cytokine production in severe acute pancreatitis.
Baicalein protect pancreatic injury in rats with severe acute pancreatitis, which may be a candidate drug for severe acute pancreatitis therapy with good perspective.
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Baicalein attenuates severe acute pancreatitis, but does not decrease the amylase
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activity.
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1