| ObjectiveLiver cancer is one of the highest rates of malignancy.Primary liver cancer is the most common type of liver cancer,and its treatment is relatively difficult.Camptothecin is one of the most common drugs for the treatment of liver cancer,and its adverse reactions are manifested in many aspects.The main toxicities and side effects are myelosuppression and intestinal damage.Among them,myelosuppression mainly manifests as a decrease in the number of white blood cells in peripheral blood.Therefore,the clinical application of camptothecin is greatly limited.The cantharidin is a dry body of Insectaceae insects,and cantharidin is derived from the cantharidin of Chinese medicine.However,because of its high toxicity,cantharidin removes 1,2 methyl artificially synthesized cantharidin.Norcantharidin has a significant anti-tumor effect and is a chemotherapeutic drug with an elevated leukocyte effect.This experiment is expected to reduce the side effects of camptothecin myelosuppression by the combination of camptothecin and norcantharidin for clinical treatment provide help.Research purposesTo investigate whether Norcantharidin(NCTD)can relieve myelosuppression caused by camptothecin(CPT)medication.Research methods:Experiment I:Obtaining the median lethal(LD50)dose of camptothecin by experimentFirst,6-week-old Balb/c mice were used as experimental subjects,and the time for intracerebroventricular administration of camptothecin was two weeks.The approximate range of the median lethal dose of camptothecin was obtained according to preliminary experiments,ie,the smallest total lethal dose and the largest Tolerated dose.At the same time,according to the results of the pre-test,we designed five different doses of drugs for intragastric administration,for two consecutive weeks,with the dose and mortality of the corresponding mice,and calculated the results using the method of the probability unit method to obtain the median lethal dose.LD50,and the confidence interval for this value.Experiment 2:Study on the toxic and side effects of short-term camptothecin and norcantharidin1,First,6-week-old Balb/c mice were used as experimental subjects,and were administered intragastrically with CPT LD50.After 3,6,9,12,15,18,21,and 24 hours of administration,the mice took eyeballs and drew about 500?L,mixed upside down and tested for leukocytes using a fully automated hematology analyzer.2,From the results of 1,CPT can reduce the peripheral blood leukocyte levels in mice,and the decrease is most significant at 6 h after administration.Balb/c mice were divided into 6 groups with 8 mice in each group.The rats were administrated with 6 hours of intragastric administration as time points and divided into control group,CPT 1 mg·kg-1 group,and NCTD low dose(10 mg·kg-1)Group,NCTD high dose(20 mg·kg-1)group and CPT1 mg·kg-1+NCTD 10 mg·kg-1 group and CPT 1 mg·kg-1+ NCTD 20 mg·kg-1 group.The condition of leukocytes was detected by an automated hematology analyzer.3,According to the previous grouping,6 hours was selected as the time point for intragastric administration.The mice were divided into 6 groups and the mice were subjected to extraction of bone marrow neutrophils.The cells were counted,and 1×107 cells were added.APC-CD11b and PE-Ly6G(Gr-1)antibodies were added and incubated at room temperature for 30 min in the dark to remove the supernatant.After the antibody was added,500 ?l of PBS buffer was used to resuspend the cells,and the cells were filtered through a 70 ?m strainer.The ratio of neutrophils to bone marrow cells was detected by flow cytometric analysis.4,According to the previous grouping,6h was selected as the time point for intragastric administration,and 6 groups of mice were routinely sacrificed after gavage,and the intestinal tract was removed for histopathological HE staining.According to the staining results,camptothecin was compared.The toxic effects of this drug on the tissue and organs of the cantharidin and combination groups were analyzed and analyzed.Experiment 3:Study on the toxic and side effects of long-term camptothecin and norcantharidinThe control group was set up and balb/c mice were divided into 6 groups of 8 in each group and divided into control group,CPT 1 mg·kg-1 group,NCTD low dose(10 mg·kg-1)group,and high NCTD.Dose(20 mg·kg-1)group and CPT 1 mg·kg-1+NCTD 10 mg·kg-1 group and CPT 1 mg·kg-1+NCTD 20 mg·kg-1 group.One week after the mice were fed adaptively for 14 days,the whole blood analyzer was used to detect leukocyte changes.Six groups of mice were sacrificed according to conventional methods.The intestinal tract was removed for histopathological HE staining.Based on the staining results,the effects of camptothecin and norcantharidin on the toxicity of this organ were analyzed.Research resultExperimental result one From the results of the preliminary experiments,the approximate LD50 range of camptothecin was obtained.The minimum lethal dose in the camptothecin-administered group was 2.0 mg·kg-1,and the LD50 of camptothecin was 1.032 mg·kg-1.95%confidence limit(0.813,1.232).Experimental result two1,The results of leukocyte detection in peripheral blood of mice after administration of camptothecin showed that compared with the control group,the total number of white blood cells at the 6th hour after CPT administration was reduced to a minimum,and the difference was significant(P<0.05).Among them,after 6 hours of camptothecin administration,neutrophils and lymphocytes were significantly decreased(P<0.05),and there was no significant change in the number of eosinophils,basophils,and monocytes.2,From the results of 1,CPT can reduce the peripheral blood leukocyte levels in mice,and the decrease is most significant at 6 h after administration.Therefore,in this experiment,the combination of NCTD and CPT was used to detect the change of white blood cell count in peripheral blood.Compared with the control group,the white blood cells in the CPT group were significantly reduced and significant(P<0.05).Neutrophils were significantly lower(P<0.05).Compared with the CPT group,the number of white blood cells in the NCTD low-dose group and the NCTD high-dose group was significantly increased(P<0.01).Compared with the CPT group,CPT and NCTD low-dose,NCTD high-dose combination group increased the leukocyte effect significantly,the difference was statistically significant(P<0.05).The number of neutrophils was significantly higher(P<0.05),and no significant changes were observed in other cells.3,CD11b+Ly6G+ can be used as a specific marker for the surface of mature neutrophils in peripheral blood and bone marrow.The results of flow cytometry analysis of mouse bone marrow neutrophils showed no significant difference in the ratio of neutrophils among the groups(P>0.05).4,After 6 hours of drug treatment in this experiment,HE staining was used in each group to observe the intestinal pathological changes in each group and to assess whether the combination of CTP and NCTD would increase intestinal toxicity.The results showed that the structure of villous epithelium in the small intestine of each group was clear,there was no defect in villous filling,and no inflammatory cell infiltration was seen.Experimental result threeIn this experiment,the mice were continuously gavaged for 2 weeks and then the white blood cell changes were detected.The experimental grouping was the same as before.Compared with the control group,the number of white blood cells in the CPT alone group was significantly lower(P<0.05).Among them,the number of neutrophils and lymphocytes decreased significantly and was significant(P<0.05).Compared with the CPT group,the white blood cells in the low,high dose,and combination groups of NCTD were significantly higher(P<0.01).The increase in lymphocytes was significant and significant(P<0.01).HE staining showed no infiltration of inflammatory cells in the intestinal tract of each group.ConclusionThe effect of camptothecin on mouse leukocytes has been greatly changed in the experiment results.Norcantharidin can reduce leukopenia caused by camptothecin administration and does not increase its intestinal toxicity.It provides a theoretical reference for the clinical application of camptothecin and reducing its toxicity. |
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