| It’s reported that Linggui Zhugan decoction(LZD)has the effect of inhibiting neuroinflammation and improving neuronal injury.Recently,new research has found that the obstruction of cerebral lymphatic drainage can induce inflammatory response and cause neuronal injury.In this study,the effects of LZD on neuronal injury were studied from the perspective of cerebral lymphatic drainage.Objective:To investigate the protective effect of LZD on neuronal inflammation and neuronal injury via the cerebral lymphatic drainage.Methods:This study consists of two experiments.In experiment 1,C57BL/6J mice were randomly divided into sham groups and operation groups.Each group was divided into 3 groups according to the detection time points,namely,D1 of sham group(D1S),D5 of sham group(D5S),D10 of sham group(D10S),D1 of operation group,D5 of operation group,D10 of operation group.In the operation group,the method of ligating the deep cervical lymph node was used to establish the model of cerebral lymphatic blockade,and three mice in the operation groups were randomly selected for model test by injecting Evans blue into the cisterna magna.On the 1st,5th and 10th day after the operation,hematoxylin-eosin staining and western blot were used to observe the morphological changes of cortex and hippocampus at different time points and the expression of aquaporin 4(AQP4),β-amyloid and tau,and to explore the association between parenchymal lesions and the above proteins at different time points.Based on experiment 1,experiment 2 conducted the intervention study of LZD.In experiment 2,40 C57BL/6J mice were randomly divided into 4 groups:sham group,operation group,LZD high-dose group(12g/kg),and LZD low-dose group(6g/kg),with 10 mice in each group.According to the results of experiment 1,the time point of the most severe brain pathology was set as the intervention duration of LZD.The effects of LZD on the morphology of brain parenchyma were studied by hematoxylin-eosin staining.Enzyme-linked immunosorbent assay was used to investigate the effect of LZD on improving neuronal injury by reducing the level of related inflammatory factors.Results:The model of cerebral lymphatic drainage was successfully established in experiment 1.It was found that on the 5th day after the operation,the phenomena of cortical neuron,which showed nucleus shrinkage,deepened staining and unclear cell structure,were the most significant,and the expression level of cortical AQP4 was the highest at this time point(P<0.05).The differences was also significant between cortical Aβ and tau on day 1(P<0.05).However,there was no significant change in hippocampus morphology on day 1,5 and 10 after the operation,and the expression of AQP4,Aβ and tau in hippocampus had no significant difference at different time points(P>0.05).Based on experiment 1,the intervention time of LZD was selected for 5 days in experiment 2.It was found that LZD in the high-dose group could significantly improve the phenomena of cortical neuron nucleus shrinkage,staining deepening and unclear cell structure,while the low-dose group had no improvement effect.Enzyme-linked immunosorbent assay was used to detect AQP4-related inflammatory factors,and it was found that compared with the sham group,interleukin-1β levels in the operation group were significantly increased(P<0.05),while tumor necrosis factor-α,interleukin-6 and transforming growth factor-β1 levels were not significantly different(P>0.05).LZD significantly reduced interleukin-1β in the high-dose group(P<0.05),while there was no difference in the low-dose group(P>0.05).Conclusion:LZD may reduce AQP4-related interleukin-1β level to improve cortical damage caused by cerebral lymphatic blockade. |
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