| Objective1.In this study,on the basis of the establishment of a diarrhea model by intravenously administering irinotecan to rats,the effect of a Chinese Herbal Formula,Shengjiang Xiexin Decoction(SXD),on the apoptosis of intestinal cells,the proliferation of intestinal cells,the expression of intestinal stem cells(ISCs),the activity of UGT1A1 in liver andβ-glucuronidase in the intestinal tract were evaluated.The possible mechanisms of this action were explored,all of which will supply scientific basis to the prevention in chemotherapy-induced diarrhea(CID)with TCM.2.The finding will provide experimental evidence to the clinical application of SXD in the prophylaxis of intestinal toxicity induced by irinotecan.MethodsTCM is known with the multi-target effects,the exploration to the inhibitive effect of SXD on the chemotherapy-induced intestinal toxicity in the different targets,is the key point of the illumination of mechanism in preventive effect.According to the metabolism of irinotecan and potential mechanism of enterotoxigenesis,with a diarrhea model of rat induced by irinotecan,the study in vivo was designed to focus on the activity of UGT1A1 in liver,β-glucuronidase in the intestinal tract and the promotion to self-repair ability.1.A diarrhea model was established by intravenously administering irinotecan to rats;2.By the observation of the weight and intake of rats in different groups,the effect of SXD on the general condition of the diarrhea model of rats was verified;and according to the criterion of grade and score of diarrhea,the diarrhea of rats after irinotecan administration were evaluated,the effect of SXD on the symptom of diarrhea;3.By the HE staining,the injury of intestinal mucosa(jejunum,ileum,cecum and colon)caused by irinotecan were observed.Through comparison of the difference in the microscopic pathology of intestinal mucosa in different groups,the effect of SXD on the injury of intestinal mucosa was verified.4.By TUNEL staining and the assay of the activity of caspase-3,the qualitative and quantitative detections of the apoptosis of intestinal cells in rats were implemented respectively.The effect of SXD on the apoptosis of intestinal cells was verified.5.By the immunohistochemical assays,proliferating cell nuclear antigen(PCNA)homing cell adhesion molecule(CD44)were used to label the intestinal proliferative cells and intestinal stem cells(ISCs)respectively,microscopic staining of intestinal epithelium and ISCs were observed.And the integrated option density(IOD)of PCNA and CD44 staining per view was measured using Image-Pro Plus(IPP)software.The expression of PCNA was also quantified by counting the number of positively stained cells per view.Through the difference of quantitative analysis in different groups,the effect of SXD on the proliferation of intestinal cells was verified.6.By real-time PCR,the expression of Lgr5 mRNA in the jejunum was detected as a marker of ISCs in different groups.The effect of SXD on the ISCs of the diarrhea model of rat was verified.7.By the assays of enzymatic activity,P-glucuronidase in the intestinal tract was detected in different groups.The effect of SXD on intestinal environment of the diarrhea model of rat was verified.8.By real-time PCR,the expression of UGT1A1 mRNA in liver was detected in different groups.The effect of SXD on metabolic enzymes in liver was verified.Results1.Though the observation of the general condition,the symptom of diarrhea,pathology of the intestinal mucosa and immunohistochemical assays,verified that the diarrhea model of rat was successes.2.The results from the observation of the general condition had shown,date from Day 7,the cumulative-added of weight was significantly higher in the high-dose and middle-dose SXD groups than it was in the model control group(p<0.05,p<0.01).However,differences between the groups receiving different doses of SXD were not significant.Until Day 9,when the rats in the high-dose SXD group had significantly higher cumulative-added weight than the rats in the low-dose SXD group(p<0.05).The model control and SXD groups had significantly lower intake of food since Day 4(p<0.01)compared with the blank control group.On Day 9,intake of food in the SXD groups was significantly higher than it was in the model control group(p<0.01),but differences between the groups receiving different doses of SXD were not significant.The results from the observation of the diarrhea of the rats had shown,the mean score of diarrhea started to increase in the model control and SXD groups after 24 hours from the last irinotecan injection(Figure 4).After 60 and 72 hours,differences in distribution of diarrhea grade were significant between the middle-dose SXD group and the model control group(p<0.05,p<0.01),and between the high-dose SXD group and the model control group(p<0.01).3.The results from the microscopic pathology of intestinal mucosa had shown,the injuries of intestinal mucosa of rats were observed in model control group,including the architecture of the epithelium is destroyed,epithelial cell arrangement is disordered,and intestinal crypts and villi are reduced.The architecture of the intestinal epithelium in the SXD group rats had more integrity than that of the model control group.The intestinal crypts of the SXD group rats were also better maintained.4.The results from TUNEL staining and the assay of the activity of caspase-3 had shown,the number of positive apoptotic cell in SXD groups was obviously less than model control group.The activity of Caspase3 in the low-,middle-,high-dose SXD group were 0.66±0.10,0.4810.11,0.68±0.19,was obviously lower than model control group(vs 1.00±0.17,p<0.01).5.The results from immunohistochemical assays of PCNA and CD44 had shown,the number of PCNA positive cell in the high-dose SXD group was obviously higher than model control group(531.20±101.64 vs 312.40±43.23,p<0.01).The IOD value of PCNA in the high-dose SXD group was obviously higher than model control group(2494.93±615.00 vs 1026.26±339.00,p<0.05).The IOD value of CD44 in the middle-,high-dose SXD group were 511.35±83.18、613.04±102.42,were obviously higher than model control group(vs 360.99±156.87,p<0.05,p<0.01).6.The results from real-time PCR had shown,compared with the model control group,the expression of Lgr5 mRNA was increased in all-dose SXD groups,however,only in high-dose SXD group did the statistical differences exist in the expression of Lgr5 mRNA(1.75±0.53 vs 0.56±0.54,p<0.01).7.The results from the activity assays of β-glucuronidase had shown,low-and middle-dose SXD treatment had no obvious effect on fecal β-glucuronidase prior to irinotecan administration,while high-dose SXD did have an effect(3.88±1.78 vs 5.92±1.47,p<0.05).After irinotecan administration,fecal β-glucuronidase activity(U)in the low-,middle-,high-dose SXD group was 12.56±1.81,13.55±1.17,11.62±1.78,lower than it was in the model control group prominently(vs 16.09±1.40,p<0.05,p<0.01).There were no significant differences between the SXD groups.8.The results from real-time PCR had shown,compared with the blank control group,the expression of UGT1A1 mRNA in model control group,low-,middle-and high-dose SXD group decreased obviously.Compared with the model control group,the expression of UGT1A1 mRNA in all-dose SXD group had no significant differences.Conclusion1.SXD can alleviate the loss of weight induced by irinotecan,and increase the intake of food after irinotecan administration.2.SXD can lower the grade and score of diarrhea induced by irinotecan.3.SXD can improve the pathological injuries of intestinal mucosa induced by irinotecan.4.SXD can decrease the apoptosis of intestinal cell induced by irinotecan.5.SXD can promote the proliferation of the intestinal mucosal cell after irinotecan administration.6.SXD can promote the expression of ISCs after irinotecan administration.7.SXD can lower the activity of fecal β-glucuronidase after irinotecan administration.8.SXD has no obvious effect on the expression of UGT1A1 mRNA in liver. |
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