| Excessive inflammatory reaction and resultant multiple organ dysfunction syndrome (MODS) is an important cause of early death due to severe acute pancreatitis (SAP), and control of excessive inflammatory reaction in early stage of disease development is the key to improve SAP survival rate. In case of inflammatory reaction and trauma injury, the organism adopts information exchange between nervous incretion and immunological system to adjust and control immune response inflammatory reflex, maintain internal stability, and avoid further harm to organism.The inflammatory reflex interposed by vagus nerve, i.e. choline energy anti-inflammation passage, is a powerful force that mitigates the impact after fatal injury and infection of organism. The central nervous system receives signal of inflammatory reaction inputted via vagus nerve and outputs adjustment signal via vagus nerve efferent fibers. The vagus nerve signal can mitigate peripheral immunological reaction (including generation of inflammation promotion cytokine, recruitment of WBC, and activation of nucleus agent κb) through a7 nicotine type acetylcholinergic receptor on T-lymphocyte. Activation of this pathway, or stimulation via vagus nerve, or use of a7 cholinergic receptor excitant can prevent whole body inflammatory reaction of a number of models, and mitigate tissue injury during shock and blood poisoning.Our earlier research found increased activity of sympathetic nerve in early stage of SAP, aggravating inflammatory reaction. Use of β receptor blocking agent can mitigate the inflammatory reaction. As an important part of the autonomic nerve system, the vagus nerve works with the sympathetic nerve to jointly maintain nerve immunologic balance. Animal experiment has found that after cutting of vagus nerve, amylase of pancreatitis mice is apparently higher than that of the control group, but adjustment of inflammatory reaction by vagus nerve upon SAP is not clear. Therefore, we assume that SIRS of early stage SAP is related to vagus nerve functions, and if this is true, adjustment of vagal excitation may provide a new way of treatment that controls excessive inflammatory reaction and mitigates functional injury of organs in early stage of SAP.Purposes of the research:1. To verify change of functional status of vagus nerve in early stage of SAP and explore its relation with whole body inflammatory reaction;2. Through blocking/enhancing vagal excitation, verify effect of vagus nerve activity on SAP inflammatory reaction and organ functions, and explore the mechanism.Contents of research:Part Ⅰ:Relationship between SAP rat vagus nerve function and inflammatory reactionPurpose:To verify vagus nerve dysfunction of SAP rat and explore the relation between SAP rat vagus nerve function and inflammatory factor.Method:Take 18 SD male rats and divide them randomly into a Control group (pseudo-operation group), SAP group (regression injection of 3.5% sodium taurocholate into pancreas bile duct,1 ml/kg), and SAP+VGX group (2 hours after left neck vagotomy, regression injection of 3.5% sodium taurocholate into pancreas bile duct, 1ml/kg). After model creation, carry out fluid replacement by tube insertion in femoral vein, to supplement normal saline at 10ml/kg.h. Detect rat HRV Oh, 1h,2h, 4h, and 6h after model stabilization respectively. At 4h after model stabilization, withdraw rat vein blood to detect TNF-α, IL-1β, and IL-10 levels (ELISA). For rats of SAP group,4h after model creation, detect MDA and MPO levels of lung tissue. All measurement data adopt representation of mean value ± standard deviation, and use One-way ANOVA. For one-to-one comparison among a number of groups of data, LSD (SPSS 18.0) method is used and if p<0.05, statistic difference is deemed present.Results:1. As compared with the Control group, Oh, 1h,2h,4h, and 6h after model stabilization respectively, nHF of HRV of groups SAP and SAP+VGX apparently rises. However, this value of group SAP+VGX is lower than that of group SAP, that is to say, for SAP+VGX vs SAP, p<0.05. Value of nLF/nHF also apparently rises after Oh, 1h,2h,4h, and 6h respectively, that is to say, for SAP and SAP+VGX vs Control, p<0.05; and this value of group SAP+VGX is higher than that of group SAP, that is to say, for SAP+VGX vs SAP, p<0.05. As compared with the Control group (127.51±17.28ug/ml),4h after model creation, serum Ach concentration of groups SAP and SAP+VGX rises, that is to say, p<0.05; however, this value of the SAP+VGX group (136.25±13.91ug/ml) is lower than that of the SAP group (165.25±11.93ug/ml), that is to say,* p<0.05 VGX+SAP vs. SAP.2. As compared with the Control group,4h after model creation, lung tissue MDA and MPO levels of groups SAP and SAP+VGX apparently rise, with those of group SAP+VGX higher than those of group SAP. Serum TNF-a level of group SAP, group SAP+VGX, and the Control group is 191.0±33.4pg/ml,272.6±33.5pg/ml, and 34.8±3.9pg/ml respectively, that is to say, serum TNF-α level of groups SAP and SAP+VGX apparently rises (p<0.05), and this level of group SAP+VGX is higher than that of group SAP (p<0.05). Serum IL-1β level of groups SAP and SAP+VGX is 269.7±26.3pg/ml and 334.8±30.6pg/ml respectively, higher (p<0.05) than that of Control group, which is 39.8±6.1 pg/ml; and this level of group SAP+VGX is higher than that of group SAP (p<0.05). Serum IL-10 level of groups SAP and SAP+VGX is 34.1±3.6pg/ml and 30.6±2.2pg/ml respectively, with no apparent statistic difference as compared with that of the Control group (25.2±3.4pg/ml) (p>0.05).3. Analysis of correlation between vagus nerve activity and inflammation mediator levels shows that nLF/nHF and serum TNF-α level have positive correlation, with r=0.837 and/ p<0.05. nLF/nHF and serum IL-1β level have positive correlation, with r=0.827 and p<0.05. Based on normal comparison, change AnHF of vagus nerve has negative correlation with inflammatory factor TNF-a (with r=-0.751 and p<0.05), and negative correlation with increase of IL-1β (with r=-0.870 and p<0.05).Conclusions:Early stage SAP rat is subject to autonomic nerve dysfunction, manifested by increase of sympathetic nerve excitation and vagal excitation, with sympathetic nerve excitation higher than vagal excitation. Blocking of SAP rat vagus nerve activity aggravates inflammatory reaction but does not change anti-inflammation cytokines.Part Ⅱ:Effect of a7nAChR excitant on SAP rat inflammatory reaction and organ functionsPurpose:To observe effect of a7nAChR excitant nicotine on SAP rat inflammatory reaction and organ functions.Method:Take 36 SD male rats and randomly divide them into 3 groups:Control group, SAP group, and SAP+Nico group (each group including 16 rats, half of them used for observation of survival rate). The SAP group refers to establishment of SAP rat model and supplement of normal saline via vein; same as Part I. For the SAP+Nico group, after model creation and stabilization, inject nicotine (100ug/kg.h) into abdominal cavity. Insert tube into femoral artery of all rats to continuously monitor aortic pressure, while monitoring change of heart rate. Withdraw arterial blood from femoral artery for blood gas analysis and kill the rats to take materials (4h after model creation). Through blood gas analysis, dry weight to wet weight ratio of lung, and pathology, assess lung functions. Through serum enzymology and pathology, assess severity of pancreatitis. Through blood biochemical inspection, assess liver and kidney functions. Through serum inflammatory factor, lung tissue enzymology, and activity of MDA and MPO, measure severity of inflammatory reaction.Results:1.4h after injection of nicotine into abdominal cavity, the model was stable. nHF of SAP group rats (66.2±2.9nu) is apparently higher than that of Control group (45.5±1.9nu) (p<0.05). Vagus nerve activity of SAP+Nicotine group rats (72.4±1.6nu) is higher than that of SAP group (p<0.05). Sympathetic nerve/vagus nerve balance (nLF/nHF) of SAP+Nicotine group is better than that of SAP group (1.55±0.11 vs 1.73±0.15, p<0.05). Comparing SAP group with the control group, the change is more apparent (p<0.05).2.4h after injection of nicotine into abdominal cavity, comparing SAP+Nicotine group with SAP group, reduction of TNF-a level has statistic difference (143.4±16.7pg/mL vs 191.1±33.4pg/mL,p<0.05). The IL-1β level of SAP group and that of SAP+Nicotine group are both higher than that of Control group (39.7±6.1pg/mL,p<0.05), and the IL-1β level of SAP+Nicotine group is apparently higher than that of SAP group (269.7±26.3pg/mL vs 181.9±22.6pg/mL), p<0.05. Lung MPO and MDA levels of SAP+Nicotine group and SAP group are both apparently higher than those of the control group (p<0.05). Lung MPO and MDA levels of SAP+Nicotine group are lower than those of the SAP group (p<0.05).3.4h after injection of nicotine into abdominal cavity, lactic acid level of the SAP group is apparently lower than that of the SAP+Nicotine group (3.70±0.32umol/L vs 2.31±0.69 umol/L, p<0.05), but both higher than that of the control group (0.50±0.15umol/L, p<0.05). Lung W/D of the SAP group and the SAP+Nicotine group is higher than that of the control group (3.75±0.25),p<0.05. BE level of the SAP group is apparently lower than that of the control group (0.28±0.16umol/L). Comparing SAP+Nicotine group with SAP group, BE value increases (-1.66±0.55umol/L vs -2.5±0.31 umol/L, p<0.05). Lung tissue pathological grades show that the grade of inflammatory reaction and local necrosis of the SAP+Nicotine group is lower than that of the SAP group (p<0.05). Serum amylase and lipase of both SAP+Nico group and SAP group are higher than those of the control group, but those of the SAP+Nico group are lower than those of the SAP group. Pancreas pathological grades show that structural injury (3.25±0.46) and tissue necrosis (2.12±0.64) of the SAP+Nico group are lower than those (3.50±0.53 and 2.63±0.52) of the SAP group (p<0.05). Optical chalone C level of the SAP+Nicotine group is lower than that of the SAP group (0.63±0.06mg/ml vs 0.48±0.05mg/ml, p<0.05), but not different from that of the Control group (p>0.05). Liver pathology of SAP group and SAP+Nico group shows liver cell edema and inflammatory cell infiltration. Biochemical inspection shows that ALT of the SAP+Nicotine group is lower than that of the SAP group (p<0.05). However, AST detection shows no statistic difference between the SAP group and the SAP+Nico group (p>0.05).4. After injection of nicotine into the abdominal cavity, longest survival of rats of the SAP+Nico group is 72h, higher than that of the SAP group (60h), p<0.05. Conclusions:a7nAChR excitant nicotine can reduce pancreas damage due to SAP, mitigate inflammatory reaction, improve oxygenation and tissue perfusion of SAP rats, and mitigate pulmonary edema of SAP rats.Part Ⅲ The mechanism of control and adjustment of SAP rat inflammatory reaction by vagus nervePurpose:To study the mechanism of control and adjustment of SAP inflammatory reaction by vagal excitation.Method:Experimental animal model creation is the same as Part Ⅰ. Injection of nicotine into abdominal cavity is adopted. Rats are randomly divided into five groups: Control group, SAP group, SAP+Nico group (50ug/kg), SAP+Nico group (100ug/kg), and SAP+Nico group (300ug/kg).4h after creation of model, obtain sample (peripheral blood and pancreas). Use flow cytometer to count CD4+CD25+ and detect albumen expression level of FOXP3 and CTLA-4. Use fluorescent quantitative PCR to detect levels of FOXP3 and CTLA-4 mRNA (relative quantities). Each detection index adopts standard control group 1 as reference and data adopt expression of mean value ± standard deviation. SPSS 13.0 statistic software is used for LSD analysis of multiple groups of data, to detect correlation among research data by rank correlation.Results:1.4h after injection of nicotine into abdominal cavity, peripheral blood T cell CD4+CD25+ expression level is apparently lower in SAP group than in the Control group. CD4+CD25+ expression level of the Nico (100ug/kg) group (0.61±0.04) and the Nico (300ug/kg) group (0.82±0.03) is higher than that of the SAP group (0.45±0.06), with p<0.05, and the trend of increase with increase of nicotine dose.2. Trend of change of FOXP3 and CTLA-4 expression level in these three groups of rats are the same as that of CD4+CD25+.4h after injection of nicotine into abdominal cavity, FOXP3 expression level of the Nico (100ug/kg) group (0.90±0.06) and the Nico (300ug/kg) group (1.18±0.07) is higher than that of the SAP group (0.78±0.03) (p<0.05). CTLA-4 mRNA level of the Nico (100ug/kg) group (0.89±0.03) and the Nico (300ug/kg) group (1.18±0.08) is higher than that of the SAP group (0.70±0.03) (p<0.05). FOXP3 and CTLA-4 expression levels of the Nico (100ug/kg) group are not different from those of the SAP group (p>0.05). FOXP3 and CTLA-4 albumen expression levels have concentration dependence on nicotine dose. FOXP3 and CTLA-4 mRNA expression levels are higher than those of SAP group, p<0.05.3.Correlation analysis found the following:Nicotine concentration has positive correlation with CD4+CD25+ expression FOXP3 mRNA level, and r=0.962 and p< 0.05. CD4+CD25+ expression FOXP3 and CTLA-4 levels increase with increase of FOXP3 mRNA level, and r=0.912 and p<0.05. FOXP3 albumen level has negative correlation with inflammation medium level, and with increase of FOXP3 expression, inflammation medium TNF-a level will decrease, and r=-0.844 and p<0.05. Expression level of the CTLA-4 series has the same trend with FOXP3.Conclusion:Nicotine receptor excitant increases SAP rat peripheral blood CD4+CD25+ level, promoting CD4+CD25+ expression FOXP3 and CTLA-4. Increase of expression level of FOXP3 and CTLA-4 is closely related to reduction of pro-inflammation factor. |
PDF Full Text Request