| Objective:To evaluate the preventive effect of QingXia HuaYu(QXHY)granules on severe acute pancreatitis(SAP)by observing the effect of early treatment with QXHY granules on the incidence of SAP in patients with predicted severe acute pancreatitis(PSAP).And further explore correlation between the preventive effect and the improved gastrointestinal function from both clinical and experimental aspects,so as to provide a new theoretical basis for the clinical advantages of traditional Chinese medicine(TCM)in the treatment of acute pancreatitis(AP).Methods:1.Clinical study of QXHY granules to improve gastrointestinal dysfunction to prevent acute pancreatitis from becoming severePatients with PSAP who were hospitalized from January 2019 to March 2021 in a multicenter were collected and randomly divided into treatment and control groups.The treatment group received QXHY granules orally or by nasojejunal tube for 7 days starting early(within 24 h)on admission on the basis of conventional western medical treatment.The control group was treated with conventional western medical treatment only.Data were collected from patients before treatment,on day 3 and day 7 of treatment,and were followed up for 28 days.(1)To observe the incidence of SAP and the occurrence of organ failure(OF)in PSAP patients to assess its preventive effect on the severity of AP.(2)To evaluate its improvement and treatment effects on TCM syndromes through syndrome scores and syndrome efficacy.(3)To dynamically assess the changes in the criticality of patients by APACHE II,BISAP,PASS and Marshall scores.(4)To assess the improvement of patients’ inflammatory response by C-reactive protein(CRP),serum inflammatory factors(TNF-α,IL-6)levels and systemic inflammatory response syndrome(SIRS)scores.(5)To assess the improvement of gastrointestinal dysfunction by acute gastrointestinal injury(AGI)grading and changes in intra-abdominal pressure(IAP),improvement in signs and symptoms of gastrointestinal dysfunction(abdominal pain,bloating,cessation of defecation,abnormal bowel sounds),initiation time of enteral nutrition,and changes in serum intestinal mucosal injury markers(diamine oxidase,Dlactate,endotoxin)levels.(6)To observe and compare the prognostic differences between the two groups(pancreatic pseudocysts,encapsulated necrosis,occurrence of necrosis in infected pancreatitis,length of stay and mortality).(7)To investigate the correlation between gastrointestinal dysfunction and the progression and prevention of AP readmission by correlation analysis of AGI score with APACHE II,BISAP,PASS,Marshall,SIRS scores and prognostic indicators.(8)To evaluate the predictive value of APACHE II,BISAP,PASS scores and CRP levels for SAP by subject operating characteristic curve(ROC).2.Experimental study of QXHY granules in the treatment of severe acute pancreatitis by improving intestinal mucosal barrier injuryThe SAP model was established by retrograde injection of 2%sodium taurocholate into the biliopancreatic duct in mice.QXHY granules solution was administered for 3 times by gastric gavage 1h before surgery,5h and 11h after surgery,and the following materials were taken 24h after surgery.(1)HE staining was performed to observe the histopathological changes of pancreas and ileum.(2)Transmission electron microscopy was used to observe the structural changes of tight junctions of ileal epithelial cells.(3)ELISA was applied to detect the changes of pancreatic injury markers(serum amylase,pancreatic myeloperoxidase),serum intestinal mucosal injury markers(diamine oxidase,D-lactate,endotoxin)and serum inflammatory factors(TNF-α,IL-6,IL-1β)expression levels.(4)Changes in gene and protein expression of tight junction proteins(Claudin-1,Occludin and ZO-1)in ileal tissue were detected by fluorescence quantitative-PCR(RTPCR)and protein immunoblotting(Western blot).(5)Flow cytometry was employed to detect the expression of group 3 innate lymphoid cell(ILC3)in ileal tissues,and RT-PCR was used to detect the changes in the expression of effector cytokines(IL-17,IL-22)and their key transcription factors(RORyt,AhR)of ILC3 in ileal tissues.Results:1.Clinical study of QXHY granules to improve gastrointestinal dysfunction to prevent acute pancreatitis from becoming severe(1)Subject recruitment and grouping:86 patients who met the inclusion criteria were enrolled in the two centers from January 2019 to March 2021,and 41 and 42 patients in the treatment and control groups,respectively,eventually completed the trial.(2)Effect on preventing AP from becoming severe:After 7 days of treatment,a total of 25(30.12%)PSAP patients developed SAP,including 8 in the treatment group and 17 in the control group,with a significantly lower incidence of SAP in the treatment group compared to the control group(19.51%vs.40.48%,P=0.04).Although the difference in the overall incidence of organ failure(OF)between the two groups was not statistically significant(53.67%vs 59.52%,P=0.59),and the difference in the number of single organ failure/multi-organ failure(SOF/MOF)was not statistically significant(19/3 vs 20/5,P=0.56),more patients in the treatment group had transient OF(<48h)than in the control group(63.63%vs 32.00%,P=0.03),and fewer patients experienced persistent OF(>48h)than the control group(36.36%vs 68.00%,P=0.03).(3)Effect on TCM syndromes:The results of intra-group comparison showed that both treatments were effective in reducing the TCM symptoms scores of PSAP patients(P<0.001 and P=0.01 before and after treatment in the treatment and control group,respectively).Before treatment,the difference in TCM syndrome scores between the two groups of patients was not statistically significant(P=0.51);after treatment,the TCM syndrome points of both groups decreased,but the decreasing trend was more obvious in the treatment group,and the difference between the groups of syndrome points on the 3rd and 7th day of treatment was statistically significant(P3=0.002,P7<0.001).After 7 days of treatment,the total efficiency of the symptoms in the treatment group and the control group was 92.68%and 71.43%,respectively(P=0.02),and the treatment group was better than the control group.(4)Effects on systemic scores:The APACHE Ⅱ,BISAP,PASS and Marshall scores of patients in both groups decreased to different degrees as the treatment progressed,and the values of the above scores of patients in the treatment group were significantly lower than those in the control group on the 3rd and 7th days of treatment(all P<0.05).(5)Effects on inflammatory indexes:The differences in serum CRP,TNF-α,IL-6 levels and SIRS scores between the two groups before treatment were not statistically significant(P>0.05).With the progress of treatment,all the above indicators decreased to different degrees.The differences in CRP levels,SIRS scores on day 3 and day 7 and IL6 levels on day 7 of treatment were statistically significant(all P<0.05).The levels of TNF-α and IL-6 on day 3 and TNF-α on day 7 of the two groups of patients were reduced,but there was no statistically significant difference between the groups(all P>0.05).(6)Effects on gastrointestinal function:①About 96.39%(80/83)of the patients had different degrees of acute gastrointestinal injury(AGI)before treatment,and the AGI grading was mainly grade Ⅰ and Ⅱ.On day 3 of treatment,the number of patients with AGI grade 0 and Ⅰ increased in the treatment group,and by day 7 more than half of the patients(65.85%)had recovered AGI(grade 0),and there were 11 patients(26.83%)and3 patients(7.32%)with AGI grade Ⅰ and Ⅱ,respectively,and no patients with grade Ⅲand Ⅳ.In the control group,the AGI grade changed relatively slowly until the 7th day of treatment,there were still only 11 cases of grade 0(26.19%),17 cases of grade I(40.48%),11 cases of grade Ⅱ(26.19%),2 cases of grade Ⅲ(4.76%),and 1 case of gradeⅣ(2.38%).The differences in the composition of AGI grading on day 3 and day 7 of treatment were statistically significant in both groups(P3=0.019,P7=0.004).②The abdominal pain scores of patients in both groups at the time of admission were about as high as about 8 points,which decreased rapidly after treatment.The interval from admission to significant improvement of abdominal pain(3 points)was about 56 h in the treatment group and about 88 h in the control group.③The time of disappearance of signs and symptoms of gastrointestinal dysfunction and the time of starting enteral nutrition in patients in the treatment group were earlier than those in the control group(all P<0.05).④The levels of IAP,diamine oxidase,D-lactate,and endotoxin were significantly decreased in patients in the treatment group on day 3 and day 7 of treatment compared with those in the control group(all P<0.05).(7)Effect on prognostic indicators:The mean length of stay of patients in the treatment group was 10.80±4.18 days,which was significantly shorter than that of patients in the control group,which was 15.36±6.18 days(P<0.001).During the 28-day follow-up period,abdominal septal compartment syndrome occurred in 3(7.32%)and 10(23.08%)patients in each of the treatment and control groups(P=0.04).The differences in mortality and the incidence of late local complications(pancreatic pseudocyst,encapsulated necrosis,and infectious pancreatitis necrosis)between the two groups were not statistically significant(P>0.05).(8)Correlation analysis between AGI score and severity of disease and clinical prognosis:The AGI score of patients before treatment had positive correlations with progression to SAP,OF duration>48h,APACHE Ⅱ,PASS and BISAP scores to different degrees(r 0.57,0.57,0.25,0.27,0.37,respectively,all P<0.05).And there was a negative correlation with OF duration<48h(r=-0.33,P<0.01).AGI scores on treatment day 7 had different degrees of positive correlation with APACHE Ⅱ,PASS,Marshall,SIRS scores and prolongation of patients’ hospital stay(r=0.32,0.29,0.33,0.33,0.26,respectively,with P<0.05 in all cases).(9)The AUCs of APACHE Ⅱ,PASS,BISAP scores and CRP levels at admission to predict SAP were 0.89,0.80,0.78 and 0.81,respectively,by ROC curve analysis(P<0.01 in all cases).2.Experimental study of QXHY granules to improve intestinal mucosal barrier damage in the treatment of severe acute pancreatitis(1)Effects on pancreatic pathological damage:HE staining results showed that the model group mice showed structural destruction of pancreatic lobules,extensive coagulative necrosis with inflammatory cell infiltration,and increased pathological score,serum amylase and pancreatic myeloperoxidase(MPO)levels compared with the sham-operated group mice(all P<0.001).Compared with mice in the model group,mice in the QXHY group showed significantly less structural destruction of pancreatic lobules,less tissue necrosis and inflammatory cell infiltration,and lower pathological scores,serum amylase and pancreatic MPO levels(all P<0.01).(2)Effect on serum inflammatory factors:ELISA results showed that the levels of serum pro-inflammatory cytokines TNF-α,IL-6 and IL-1β were significantly increased in the model group mice compared with the sham-operated group mice(all P<0.001).The levels of serum TNF-α,IL-6 and IL-1β were reduced in the QXHY group mice,and the differences were statistically significant compared with the model group(all P<0.05).(3)Effects on pathological injury of intestinal mucosa:Light microscopy showed that the ileal mucosa of mice in the model group was congested and edematous,and the villi were shortened,detached and defective with inflammatory cell infiltration,and the pathological score was increased compared with that of mice in the sham-operated group(P<0.001).In the QXHY group,chorionic edema was seen,but other pathological damage was significantly reduced compared with the model group mice,and the pathological score was lower(P<0.01).ELISA results showed that serum diamine oxidase,D-lactate,endotoxin levels were significantly higher in the model group mice compared with the sham-operated group mice(P<0.001).The serum diamine oxidase,Dlactate,endotoxin levels were significantly lower in the QXHY group mice compared with the model group mice(P<0.01,<0.01,<0.001,respectively).(4)Effects on intestinal tight junction morphology and related protein expression:Transmission electron microscopy showed that the tight junction structure of ileal epithelium was reduced and the cell gap was widened in the model group mice,while the tight junction structure was more intact and the cell gap widening was reduced in the QXHY group mice.The RT-PCR and Western blot results showed that the expression levels of tight junction proteins Claudin-1,Occludin and ZO-1 mRNA and protein were significantly lower in the ileal tissues of the model mice compared with those of the shamoperated mice(all P<0.001).Compared with the mice in the model group,Claudin-1,Occludin and ZO-1 mRNA expression were all increased in the ileal tissues of mice in the QXHY group mice(all P<0.001),and Claudin-1,Occludin and ZO-1 protein expression were all up-regulated(P<0.05,<0.01,<0.001,respectively).(5)Effect on intestinal ILC3:Flow cytometry results showed that the mean frequency percentage of ILC3 cells in the intestine of the model group was significantly lower than that in the sham-operation group(P=0.002).The mean frequency percentage of ILC3 cells in the intestine was higher in the QXHY group compared with the model group(P=0.04).RT-PCR results showed that the expression of IL-17 and IL-22 mRNA,the main effector cytokines of ILC3,and its key transcription factors RORyt and AhR mRNA were down-regulated(all P<0.001).Compared with the model group,the intestinal IL-17 and IL-22 mRNA expressions were up-regulated in the QXHY group,and the differences were statistically significant(all P<0.05),and the RORyt and AhR mRNA expressions were up-regulated,and the differences were statistically significant(all P<0.05)。Conclusion:(1)Gastrointestinal dysfunction is a common complication of PSAP patients and is closely related to the severity of the disease and clinical prognosis.(2)Early combined treatment with QXHY granules can shorten the duration of organ failure,reduce the incidence of SAP in PSAP patients,and thus preventing PSAP from becoming seriously ill with SAP.This effect may be related to its effective improvement of patients’gastrointestinal dysfunction,stabilizing the intestinal mucosal barrier,breaking the cascade of inflammation,and inhibiting the systemic inflammatory response.(3)SAP can cause obvious damage to the intestinal tight junctions of mice,thereby destroying the mechanical barrier of the intestinal mucosa,leading to increased intestinal permeability,and causing systemic inflammation and intestinal endotoxemia in SAP mice.(4)QXHY granules have a protective effect on the tight junction damage and intestinal mucosal barrier injury caused by SAP in mice,and this effect may be related to its regulation of the number and function of intestinal ILC3 and up regulation of intestinal tight junction protein expression in SAP mice. |
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