| Objective: Through animal experiments,using the CSDH rabbit model and VEGF blocking technology,based on the changes in Ras/MEK/ERK signaling pathway related factors as observation points,Western blot and ELISA methods were used to detect the factors affected by VEGF in the outer membrane of hematoma and the fluid inside the hematoma,further explaining the effect of Peiyuan Huayu Formula on the VEGF and Ras/MEK/ERK signaling pathway related factors in the outer membrane of CSDH hematoma,Exploring the effect of inhibiting VEGF expression on the therapeutic effect of Peiyuan Huayu Formula on CSDH through experiments,and clarifying whether Peiyuan Huayu Formula achieves the therapeutic effect of CSDH by affecting VEGF expression,providing animal experimental basis for the treatment of CSDH with traditional Chinese medicine.Method:1.Clean grade New Zealand rabbits were randomly divided into a blank control group,a sham operation group,a model group,a positive control group(atorvastatin treatment group),a treatment group(Peiyuan Huayu Fang treatment group),and a VEGF inhibition group(VEGF inhibitor bevacizumab pretreatment followed by Peiyuan Huayu Fang treatment).Establish a CSDH model and observe the general situation of the rabbits.After successful modeling,observe the Purdy score of neural function,evaluate the formation of hematoma,and perform craniotomy verification.On the 21 st day after modeling,use CT to identify the CSDH model in the rabbits.Use CT images to measure the volume of intracranial hematoma,and the transverse and sagittal diameters of hematoma section images.2.After the rabbits were killed by air embolism,the hematoma samples were taken out,and the dura mater and brain tissue around the hematoma were separated and cut into small pieces.Western Blot was used to detect the expression of Ras-GAP,MEK,p-MEK,ERK,and p-ERK proteins.3.Separate the hematoma membrane and surrounding tissues,and slowly extract the hematoma fluid using a pipette.Use commercially available enzyme-linked immunosorbent assay(ELISA)kits to measure the levels of VEGF and related HIF-1,PGE2,vWF,ANG-2,NO,MMP-9,and FLT-1.Collect and organize experimental data,summarize and analyze experimental results.Results:(1)Modeling results: On the 21 st day,a total of 112 rabbits survived,of which 36 were in the blank group or sham surgery group without modeling,84 were modeled,and 8 died.There was no statistically significant difference in Purdy scores between the groups;On 21 days of CT scanning,54 rabbits showed subdural formation of equal or low density crescent shadows,all of which were confirmed as CSDH.The success rate of modeling was 64.29%;(2)By measuring the levels of Ras/MEK/ERK pathway related factors such as Ras,MEK,p-MEK,ERK,p-ERK,etc.,the results showed that the levels of Ras,MEK,p-MEK,ERK,p-ERK in the treatment group were lower than the positive control group,far lower than the model group and the VEGF inhibition group,and the difference was statistically significant.This suggests that Peiyuan Huayu Formula can inhibit VEGF expression by affecting the Ras/MEK/ERK pathway;(3)The results of using ELISA to measure the levels of VEGF related factors showed that among the pro-inflammatory factor related indicators HIF-1,PGE2,NO,MMP-9,the levels of HIF-1 and NO in the treatment group were lower than those in the positive control group and the VEGF inhibition group;The PGE2 content in the treatment group was significantly lower than that in the VEGF inhibition group;Compared with the model group,positive control group,and VEGF inhibition group,the MMP-9 content in the treatment group was significantly higher than other groups,and the difference was statistically significant;Among the indicators related to angiogenesis factors such as VEGF,vWF,ANG-2,and FLT-1,the content of VEGF and ANG-2 in the treatment group was lower than that in the positive control group.The vWF content in the treatment group was lower than that in the positive control group and the VEGF inhibition group.The VEGF content in the VEGF inhibition group was significantly lower than that in the model group and the treatment group.The FLT1 content in the VEGF inhibition group decreased significantly compared to the blank control group and sham surgery,and the difference was statistically significant.Conclusion:Peiyuan Huayu Formula can achieve an intervention effect on rabbit CSDH by mediating the Ras/MEK/ERK signaling pathway,and can significantly improve the neurological performance of rabbits.Its mechanism of action may be related to its reduction of Ras GAP,MEK,p-MEK,ERK,p-ERK protein expression.This formula can block the Ras/MEK/ERK signaling pathway and affect the content of VEGF and related factors HIF-1,PGE2,vWF,ANG-2,NO,MMP-9,and FLT-1 in the hematoma fluid of CSDH rabbits.It can significantly exert its inhibitory effect on neovascularization and prevent CSDH recurrence,further elucidating the therapeutic effect of Peiyuan Huayu Formula on CSDH,providing relatively complete theoretical support for Peiyuan Huayu Formula to promote the absorption of hematoma and reduce recurrence. |
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