| Objective: To investigate the effects of different doses of Diquat(DQ)poisoning on the inflammatory signal pathway of HMGB1/NF-κB p65 and the balance of M1/M2 macrophages in mouse spleen.Methods: 64 healthy adult male ICR mice were randomly divided into 4 groups with 16 mice in each group: blank control group(control,n=16),12 mg/kg DQ exposure group(12mg/kg DQ,n=16),24 mg/kg DQ exposure group(24mg/kg DQ,n=16)and the48mg/kg DQ exposure group(48mg/kg DQ,n=16).Each group was randomly divided into two subgroups,namely the observation group and the 24-hour sampling group.The control group was given a one-time intraperitoneal injection of isotonic saline with 48 mg/kg/piece for modeling,and the DQ exposure group was given a one-time intraperitoneal injection of1.9 mg/ml DQ at 12 mg/kg/piece,24 mg/kg/piece,and 48 mg/kg/piece,respectively.After successful modeling in the observation group,the behavioral changes of mice were observed each 6 hours and the survival rate was calculated.The observation period was 14 days.The successful modeling after 24 hours,the spleen of the sample group was taken,weighed and recorded,and the spleen index was calculated.Part of the spleen was retained for HE staining and spleen pathological score,part of the spleen was made for transmission electron microscope to observe the ultrastructural characteristics of the spleen.The expression of HMGB1,NF-κB p65,TNF-α,IL-6 and IL-10 in part of spleen was detected by immunohistochemistry,and the protein content of HMGB1 and NF-κB p65 in part of spleen was detected by Western blotting.And part of the spleen was used as spleen single cell suspension for flow cytometry to detect M1 and M2 macrophage subsets.Results:(1)14-day behavior of mice in each group: during the 14-day observation period,there was no poisoning in the control group,and there were symptoms in the DQ exposed group such as listlessness,ptosis,decreased activity,crawling weakness,cyanosis of the oral and nasal mucosa,shortness of breath,reduced food intake,weight loss,slow response to external stimuli and so on appeared in the DQ exposed group.The above symptoms were alleviated and the activity increased after 24 hours of exposure in the 12mg/kg DQ and 24mg/kg DQ groups.(2)The 14-day survival rate of mice in each group: The 14-day survival time of control group,12mg/kg DQ group and 24mg/kg DQ group was 14 days,and the cumulative survival rate was 100%.The 14-day survival time of 48mg/kg DQ group was 4.625±0.42 days,and the cumulative survival rate was 0%,n=8(P < 0.05).(3)Spleen index: compared with control group,the spleen index of DQ group decreased with the increase of poisoning dose,and the spleen index of 24mg/kg DQ group and 48mg/kg DQ group were significantly lower than that of control group(P < 0.05).(4)Pathological score of spleen: there was no pathological manifestation of spleen poisoning by HE staining in control group.Compared with control group,there were pathological manifestations in DQ group,and aggravated with the increase of poisoning dose.The pathological manifestations were obvious in 24mg/kg DQ group and 48mg/kg DQ group,and the pathological score of spleen increased significantly(P < 0.05).(5)Transmission electron microscope of spleen tissue: compared with control group,the tendency of pyknosis and apoptosis of spleen lymphocytes was obvious with the increase of DQ poisoning dose,and the phagocytosis of spleen macrophages was also obvious with the increase of DQ poisoning dose,and phagocytic lymphocytes and fragments could be seen in spleen macrophages of 48mg/kg DQ group.(6)Immunohistochemical results of each index: compared with control group,the expression of HMGB1,NF-κB p65,TNF-α and IL-6 in 12mg/kg DQ group,24mg/kg DQ group and 48mg/kg DQ group increased with the increase of toxic dose(P < 0.05),while the expression of IL-10 decreased with the increase of toxic dose(P < 0.05).(7)HMGB1 and NF-κB p65 protein western blot results:compared with control group,the protein contents of HMGB1 and NF-κB p65 in 12mg/kg DQ group,24mg/kg DQ group and 48mg/kg DQ group increased with the increase of toxic dose(P < 0.05).(8)M1 and M2 macrophage subsets in spleen: compared with control group,the proportion of M1 and M2 macrophage subsets in 12mg/kg DQ group,24mg/kg DQ group and 48mg/kg DQ group decreased significantly with the increase of poisoning dose(P < 0.05).Conclusion: After DQ poisoning,the proportion of M1 and M2 macrophages in spleen decreased,and the ability of immune response decreased,which might be related to DQ activating HMGB1/NF-κB p65 inflammatory signal pathway. |
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