| Objective: To clarify specific roles of apoptosis of pancreatic and lung tissues in the pathological process of severe acute pancreatitis (SAP) complicated by acute lung injury(ALI), the regulation mechanism of apoptosis in process of SAP associated with ALI is explored to identify the therapeutic target and regulation of emodin on the disease by focusing on Notch/Hes signal transduction pathway.Methods: 144 rats were randomly divided into a model group (N=136) and a sham operation group (SO group, N=8) after 1 week of acclimation. Model group: 3.5% sodium taurocholate (lml/kg of body weight) was administrated by retrograde injection through pancreaticobiliary duct to establish the SAP lung injury model; SO group: the pancreas was only exposed and slightly flipped, the rest of surgical procedure was as same as the model group. The model group was randomly divided to 4 groups according to 4 time points at 3, 6,12 and 24 hours after the surgery. Each group was then randomly divided into 4 groups:model control group, low dose emodin group (referred to as low-dose group, 1mg/ml emodin solution was applied), middle dose emodin group (referred to as middle-dose group, 2mg/ml emodin solution was treated) and high-dose emodin group (referred to as high-dose group,4mg/ml emodin solution was used). Emodin-treated groups (low-dose group, middle-dose group and high-dose group) were respectively administrated 1/2 dose of corresponding concentration of sterilized emodin solution (preparation of 4% sucrose fatty acid esters) by intraperitoneal injection in 1 hour before and after modeling (following 1ml/100g of body weight); the model control group was injected with the same volume of 4% sterilized sucrose fatty acid ester solution. The specimens, including serum, pancreatic and lung tissues, were collected from each group at the corresponding time points; the specimens of SO group were collected in 3 hours after modeling. Detection Indicators: the serum amylase and total calcium levels were determined by using an automatic biochemical analyzer; the biopsy of normal pancreatic and lung tissues was performed before HE staining and a pathologist then assigned the pathological scores; left lung wet/dry weight ratio (W/D); MPO content in lung tissues was measured by a spectrophotometer; the apoptosis of pancreatic and lung tissues was detected by using TUNEL assay followed by calculating apoptotic index (AI); the mRNA expression levels of Notch-1, Hes-1 and Hes-5 were determined by RT-PCR; the protein expression levels of Notch-1, Hes-1 and Hes-5 were determined by Immunohistochemistry and Western blotting.Results:(1) Serum amylase and total calcium: the serum amylase levels of model rats showed a significantly increased trend over time with statistically significant difference as compared with SO group at each time point (P<0.01); meanwhile, the serum total calcium of model rats exhibited a decline,especially the serum total calcium was significantly lower than that of SO group after 6h (P<0.01). After various doses of emodin were administrated for interventional treatment, the AMS levels appeared to increase in treatment groups, but the serum total calcium still tended to decline; however, the serum amylase and total calcium levels of treatment groups increased to some extent compared to the model control group within 24 hours. The serum amylase levels of treatment groups were significantly lower than that of model control group (P<0.01),and the high-dose group exhibited a greater reduction than the low-dose group (P<0.05); the serum total calcium levels of treatment groups were significantly higher than that of model control group (P<0.01), and the high-dose group exhibited a higher increment than the low-dose group (P<0.05).(2) Pathological score: the pathological score of pancreas in model group increased at each time point, and all were significantly higher than that of SO group (P<0.01); in the model group, the score at 6h time point was higher than those at 3h and 12h time points whereas the highest score existed at 24h time point. The pathological score of lung tissue in the model group was significantly higher than that of SO group at each time point (P<0.01):the scores showed an increment in the first 12 hours and achieved the peak at 12h time point.After treatment, the pancreatic pathological scores of middle- and high-dose groups were significantly enhanced as compared with the model control group (P<0.01),the improvement was most significant in the high-dose group whereas there was no statistically significant difference between the low-dose group and the model control group (P>0.05); the lung pathological scores were significantly improved in each treatment group as compared with the model control group (P<0.05) while the high-dose group was significantly superior to the low-dose group (P<0.05).(3) Lung tissue MPO: the lung tissue MPO of model rats presented a gradually rising trend at each time point prior to the first 12 hours and the difference was significant as compared with SO group (P<0.01); the MPO level reached the peak at 12h time point which was significantly higher than those at other time points (P<0.01); the MPO level was significantly decreased at 24h time point than prior time points. After treatment, the treatment groups showed a significant reduction in MPO level compared to the model control group(P<0.05), there was no statistical significance between the low-dose group and the middle-dose group (P>0.05), whereas the high-dose group was significantly greater than the low- and middle-dose groups (P<0.01).(4) Lung tissue wet/dry weight ratio (W/D): the lung tissue W/D of model rats exhibited a gradual increment at the prior 12 hours, and the ratio was significantly higher than that of SO group at each time point (P<0.01) while it reached the peak at 12h time point; the lung tissue W/D of the model group significantly reduced with significant difference compared to 6h (P<0.01) and 12h (P<0.01) time points, but still higher than that of the SO group (P<0.01);after treatment, the W/D of the model control group and treatment groups showed an increasing trend in the prior 12 hours and achieved the peak at 12h time point; however, the levels of treatment groups were significantly lower than that of the model group (P<0.01), the high-dose group was significantly superior to the low-dose group (P<0.05).(5) Apoptotic Index (AI): in the model group, the AI of pancreatic cells achieved the peak at 3h, significantly decreased at 6h, recovered at 12h, and then fell down to the lowest at 24h; it was observed that the apoptotic pancreatic cells of model group mainly consist of neutrophils,lymphocytes,pancreatic follicle cells and a small number of vascular endothelial cells. The lung tissue AI value of model group was significantly higher than that of SO group at each time point (P<0.01); at 3h time point, AI reached peak and then showed a decline, but raised again at 24h; it was observed that the apoptotic pancreatic cells mainly include inflammatory cells (e.g. neutrophils and lymphocytes) and a small number of lung alveolar epithelial cells and vascular endothelial cells. In the treatment groups, the AI of pancreatic cells raised to some extent compared to the model control group at various time points,however, there was no statistical significance between the low-dose group and the model control group (P>0.05) while the middle- and high-dose groups were significantly higher than the model group (P<0.05) and the high-dose group exhibited the greatest effect; the lung tissue AIs of treatment groups were significantly higher than that of model control group(P<0.01), moreover, the middle- and high-dose groups were significantly higher than the low-dose group (P<0.05) while the middle- and high-dose groups showed no statistical significance (P>0.05).(6) The mRNA expression levels of Notch-1, Hes-1 and Hes-5 in pancreatic tissue: the Notch-1 mRNA expression of model rats showed a tortuous decline in general and its expression level was significantly lower than that of SO group at each time point (P<0.01),the expression at 6h time point decreased significantly compared to those at 3h (P<0.05) and 12h (P>0.05), the lowest level was observed at 24h time point; the Hes-1 mRNA expression level of model control group was significantly higher than that of SO group at each time point(P<0.05), specifically, the mRNA level reached the peak at 6h, fell down to the lowest at 12h,and recovered again at 24h; in the model group, the mRNA level of Hes-5 reached the peak at 3h which was significantly higher than that of SO group (P<0.05) and that at 24h time point(P<0.05), followed by a decreasing trend. After treatment, the expression level of low-dose group increased significantly than that of model control group (P<0.05),while the increments in the middle- and high-dose group were significantly greater than that of model control group(P<0.05); the Hes-1 mRNA levels of pancreatic tissue varied greatly (data dispersion) in treatment groups, however, the levels increased significantly compared to the model control group in general (P<0.05); in the treatment groups, the Hes-5 mRNA levels of pancreatic tissue increased to some extent than the model control group, although there was no statistical significance between the low-dose group and the model control group,the levels of middle-and high-dose groups significantly increased than the latter (P<0.01).(7) The mRNA expression levels of Notch-1,Hes-1 and Hes-5 in lung tissue: the Notch-1 mRNA expression of model rats was significantly lower than that of SO group at each time point (P<0.05), it fell down to the lowest at 12h and then exhibited an increment at 24h; the Hes-1 mRNA expression of model rats showed a fluctuating trend with a valley at 12h and the level was also significantly lower than that of SO group or that at 24h (P<0.05);the Hes-5 mRNA expression decreased followed by a increment,the level was lowest at 6h and then increased gradually. After treatment, the Notch-1 mRNA expression levels of lung tissues of treatment groups were all higher than that of model control group (P<0.01),specifically, the high-dose group had a greatest effect which was significantly higher than the low- and middle-dose groups (P<0.01); the Hes-1 mRNA expression levels of lung tissues of treatment groups also increased compared to the model control group (P<0.01), specifically,the high-dose group was significantly higher than the low-dose group (P<0.05) whereas there was no statistical significance between the middle- and the high-dose groups (P>0.05); the Hes-5 mRNA expression levels of lung tissues of treatment groups showed an increment compared to the model control group, although the low-dose groups were not significantly different from the model control group (P>0.05), the middle- and high-dose groups are significantly higher than the model control group and low-dose group (P<0.05),and the high-dose group was also higher than the middle-dose group (P<0.01).(8) According to the results of immunohistochemistry, we found that Notch-1 is mainly expressed in cytoplasm,Hes-1 in cytoplasm/nuclear and Hes-5 in cytoplasmic. Notch-1, Hes-1 and Hes-5 are all expressed in neutrophils, lymphocytes and pancreatic acinar cell in pancreatic tissue.Notch-1, Hes-1 and Hes-5 were also expressed in neutrophils,lymphocytes,alveolar epithelial cells and endothelial cells of the lung. The intensity and range of Notch/Hes protein expression tendency are the same trend Consistent with with mRNA expression in pancreatic and lung tissue accordingly.(9) The Notch/Hes protein expression in the pancreatic tissue: the alternation in Notch/Hes protein expression of the pancreatic tissue was generally consistent with its mRNA expression trend, specifically, the protein expression of Notch-1 and Hes-5 was consistent in the model control group while the protein levels of Hes-1 and Hes-5 exhibited a fluctuating trend. After treatment, the Notchl protein expression level of each treatment group increased as compared with that of model control group, and the middle- and high-dose groups showed statistical significance (P<0.01); the high-dose group had a greatest effect which was significantly superior to the low-dose group (P<0.05); the Hes-1 protein level of high-dose group was higher than those of model control group (P<0.01),low-dose group (P<0.01) and middle-dose group (P<0.05), whereas the Hes-1 expression levels showed no significant difference among other groups (P>0.05); the Hes-5 protein levels of middle- and high-dose groups were significantly higher than that of model control group (P<0.05),the high-dose group also showed a higher protein level than the low-dose group (P<0.05), however, there was no significant difference among other groups (P>0.05).(10) The Notch/Hes protein expression in the lung tissue: the alternation in Notch/Hes protein expression of the lung tissue was generally consistent with its mRNA expression trend,specifically,the protein expression of Notch-1 and Hes-5 showed a fluctuating trend in the model control group. After treatment, the Notch-1 protein expression level of each treatment group increased compared to that of model control group (P<0.05), specifically, the high-dose group had the greatest change (P<0.01) while other groups showed no statistical significant difference (P>0.05); the protein level of each treatment group was significantly higher than that of model control group (P<0.05), the high-dose group was significantly superior to the low-dose group (P<0.05),while there was no statistical significance among other groups(P>0.05); the Hes-5 protein levels of middle- and high-dose groups increased compared to that of model control group (P<0.05),the high-dose group was significantly greater than the low-dose group (P<0.05),while other groups had no statistical significance (P>0.05).Conclusion:(1) The animal model of retrograde injection of taurocholic acid via pancreaticobiliary duct reflects the pathological characteristics of SAP lung injury and thereby applies to pathology and pharmacology studies.(2) Apoptosis of pancreatic acini and lung tissues plays an important role in the pathological process of SAP associated with ALI, and the apoptosis is inversely proportional to the extent of tissue injury. The reduction in apoptosis of pancreatic cells is one of important causes of disease progression during SAP; the latency or inhibition of apoptosis in neutrophils makes a great contribution to ALI complication.(3) The Notch/Hes signal can promote cell apoptosis; the Notch-1 signal transduction limitation is a leading factor that causes worsening of pancreatic and lung injury during SAP.The Notch/Hes signal also induces apoptosis of inflammatory cells but inhibits apoptosis of pulmonary vascular endothelial cells, thereby reducing lung MPO level, pulmonary edema and tissue injury (pancreas and lungs).(4) The alternation of Hes-1 and Hes-5 reaches a dynamic equilibrium, thereby amplifying the Notch signal transduction via feedback.(5) Emodin can prevent and cure SAP lung injury through reducing the serum amylase level, increasing the serum calcium level, decreasing the lung MPO level and improving pulmonary edema, thereby leading to a reduction in pathological scores of pancreatic and lung tissues. Emodin exhibits a dose-dependent manner while the high-dose group shows a significant benefit.(6) Emodin not only promotes apoptosis of pancreatic acinar cells,lung alveolar epithelial cells and vascular endothelial cells in SAP lung injury model but also prevents latency (or inhibition) of apoptosis of most inflammatory cells such as neutrophils.(7) Notch/Hes signal pathway is an important target for emodin to promote apoptosis during SAP. Emodin not only induces mRNA expression of Notch-1 but also regulates expression of its downstream genes (Hes-1,5), thus amplifying Notch signaling and enhancing its regulation on apoptosis. |
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