| Background:Lymphedema is a chronic progressive disease with the characteristics of increased body fluid in the tissue space caused by injury of lymphatic endothelial cells due to various reasons.It is mainly affected by lymphatic stenosis,occlusion and fibrosis caused by radical surgery and radiotherapy,often accompanied by local inflammation.Inflammation is a basic pathological process of defensive response to the stimulation of various damage factors in living tissues with vascular system.It has been found that inflammation plays an important role in lymphedema both clinically and experimentally.At the present stage,despite various treatment methods for lymphedema,there is still a lack of proven and effective drug treatment.Sodium aescinate（SA）is a solution from aescin,and it is widely used in the treatment of brain edema and venous reflux disorders.Aquaporin-4（AQP-4）is an important member of aquaporin family,which is often distributed in a variety of epithelial cells,endothelial cells and other tissue.In recent years,it has been found that AQP-4 can participate in inflammatory response.However,the role of SA and AQP-4in inflammation-related lymphedema and the correlation between SA and AQP-4 expression have not been reported.Objective:To study the role of SA in the prevention and treatment of lymphedema by means of the clinical data and the establishments of cell and mice models.Meanwhile,the expression changes of AQP-4 in endothelial cell inflammatory injury and inflammation-associated lymphedema were further studied,in order to clarify the role and mechanism of SA in the process of lymphedema,and to provide reliable experimental basis for prevention and drug treatment for lymphedema.Materials and methods:1.The clinical data of patients with lymphedema treated in the Department of Lymphatic Vascular Surgery of China-Japan Union Hospital of Jilin University from October 2021 to February 2022 were retrospectively analyzed.The patients treated with SA were included in the treatment group and those treated with other drugs were included in the control group.After treatment for 7 days,the limb circumferences and blood biochemical indexes were detected,and the data were analyzed by software SPSS 22.0.2.Human umbilical vein endothelial cells（HUVEC）were cultured in vitro.The toxicity of 0.4,1.6 and 6.4μg/ml SA to HUVEC was detected by CCK-8,and the inflammatory injury cell model was established by2.5μg/ml LPS for 6h.These cells were divided into 5 groups: the blank group,the model group（2.5μg/ml LPS）,the low dose group（2.5μg/ml LPS + 0.4μg/ml SA）,the middle dose group（2.5μg/ml LPS + 1.6μg/ml SA）and the high dose group（2.5μg/ml LPS + 6.4μg/ml SA）.The expression of IL-6 was detected by ELISA,the m RNA expression of AQP-4 was detected by RT-q PCR,and the expression of AQP-4 protein was detected by Western blot and immunofluorescence.Finally,the data and images were respectively analyzed by software SPSS 22.0 and Image J.3.The adult male mice（C57）were randomly divided into 6 groups:blank group,sham operation group,model group,low dose group（0.25μg/g SA）,middle dose group（1μg/g SA）and high dose group（4μg/g SA）.The inflammation-related lymphedema model was established by ligating the lymphatic vessels of the lower limbs and removing the popliteal and inguinal lymph nodes.The limb circumferences of all mice were measured before operation.After operation,the low,middle and high dose groups were respectively injected with the corresponding dose of SA solution for treatment of lymphedema,while the blank group,sham operation group and model group were given the same dose of normal saline（NS）,with the treatment period of 14 days.The limb circumferences of mice were measured on the 7th and 14 th day after operation,and the proliferation and dilation of lymphatic vessels and the formation of lymphatic collateral circulation in their lower limbs were detected by near infrared imaging.The mice were killed by euthanasia 14 days later,and the expressions of IL-6 and TNF-αin serum of each group were detected by ELISA;the m RNA expressions of AQP-4 in lymphatic tissue of each group were detected by RT-q PCR;the expressions of AQP-4 protein in lymphatic tissue of each group were detected by Western blot.Finally,the data and images were respectively analyzed by software SPSS 22.0 and Image J.Results:1.Compared with the control group,the limb circumferences and the increase of blood biochemical indexes（white blood cell count,neutrophil percentage and CRP）in the treatment group were all significantly lower than those in the control group（P<0.05）.2.The results of CCK-8 detection in vitro showed that there was no significant difference in the percentage of cell activity between the SA groups（treated with 0.4,1.6 and 6.4μg/ml SA for 6 hours,respectively）and the blank group（P>0.05）.3.The results of IL-6 in vitro showed that the relative expressions of IL-6 in the low,middle and high dose groups were all lower than that in the model group（P<0.05）.4.The results of AQP-4 expressions in vitro showed that the relative expressions of the m RNA and the protein of AQP-4 in the low,middle and high dose groups were all higher than that in the model group（P<0.01,P<0.05）,while the result of immunofluorescence was consistent with the results of AQP-4 expressions.5.The measurement results showed that there was no significant change in the limb circumferences of mice between the sham operation group and the blank group（P>0.05）,but that in the model group was significantly longer than that in the sham operation group（P<0.01）.Compared with the model group,the limb circumference of low,middle and high dose groups all decreased significantly in a concentration-dependent manner（P<0.05）.6.The results of near infrared imaging in mice showed that there was no significant change in lymphatic vessels in the blank group and sham operation group on the 7th and 14 th day after operation.While in the model group,there was a slight proliferation of lymphatic vessels on the 7th day after operation,but which increased significantly on the 14 th day after operation,but without obvious lymphatic collateral circulation into the thoracic duct.In the low dose group,the lymphatic vessels proliferation was obvious on the 7th day after operation,with obvious lymphatic collateral circulation into the thoracic duct.On the 14 th day after operation,however,this phenomenon was more obvious than before.In the middle dose group,there was obvious lymphatic proliferation and lymphatic dilatation on the 7th day after operation,but without obvious lymphatic collateral circulation into the thoracic duct.On the 14 th day after operation,however,the lymphatic proliferation and lymphatic dilatation were more obvious than before,with slight lymphatic collateral circulation into the thoracic duct.In the high dose group,obvious lymphatic proliferation and lymphatic dilatation of lymphatic vessels were observed both on the 7th and 14 th day after operation,but without obvious lymphatic collateral circulation into the thoracic duct.7.The results of ELISA detection of serum in mice showed that the expression of IL-6 in the sham operation group increased significantly compared with the blank group（P<0.01）,and that in the model group also increased significantly compared with the sham operation group（P<0.01）.However,compared with the model group,the expressions of IL-6 in the low,middle and high dose groups all decreased significantly in a concentration-dependent manner（P<0.05）.In addition,the expression of TNF-α in the sham operation group had no significant change compared with the blank group（P>0.05）,but that in the model group increased significantly compared with the sham operation group（P<0.01）.And the expressions of TNF-α in the low,middle and high dose groups all decreased significantly in a concentration-dependent manner compared with the model group（P<0.05）.8.The results of RT-q PCR of lymphatic tissue in mice showed that the expression of the m RNA of AQP-4 in the sham operation group decreased significantly compared with the blank group（P<0.01）,and that in the model group decreased significantly compared with the sham operation group（P<0.01）.While the expressions of the m RNA of AQP-4in the low,middle and high dose groups increased significantly in a concentration-dependent manner compared with the model group（P<0.01）.9.The results of Western blot of lymphatic tissue in mice showed that the expression of AQP-4 protein in the sham operation group had no significant change compared with the blank group（P>0.05）,but that in the model group decreased significantly compared with the sham operation group（P<0.01）.And the expressions of AQP-4 protein in the low,middle and high dose groups increased significantly in a concentration-dependent manner compared with the model group（P<0.01）.Conclusion:1.Sodium aescinate can significantly relieve and treat inflammation-associated lymphedema,and even change the disease progression.2.AQP-4 plays an important role in inflammatory injury and inflammation-associated lymphedema,which may be an important target for sodium aescinate to inhibit inflammation-associated lymphedema. |
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