The Roles Of Krüppel-like Factor 2 In Atorvastatin Combined With Low-dose Dexamethasone In The Treatment Of Chronic Subdural Hematoma

Objective:Chronic subdural hematoma(CSDH)is one of the most common diseases in department of neurosurgery,and the annual incidence rate is 20.6/100000.CSDH usually occurs after trauma.CSDH patients may present with mental state changes,focal neurological deficits and / or headache.Most patients recovered well after operation.Although CSDH surgery is considered relatively " straightforward",the elderly is especially prone to show poor surgical results.Moreover,many elderly patients take anticoagulants and antiplatelet drugs,which increase the risk of rebleeding after surgery.Therefore,it is necessary to find other treatments as a supplement or substitute for surgical treatment.Our previous study found that low dose of atorvastatin can reduce the hematoma volume and improve the prognosis of CSDH patients,but there are still nearly 10% patients with no obvious effect.In the further small-scale clinical trial of atorvastatin combined with low-dose dexamethasone in the treatment of 60 cases of chronic subdural hematoma,the researchers found that compared with atorvastatin alone,atorvastatin combined with low-dose dexamethasone can more effectively reduce the hematoma volume and improve the prognosis of CSDH patients.Based on previous studies,we further screened Krüppel like factor 2(KLF-2)in the model of endothelial cell injury induced by hematoma fluid,and its m RNA and protein increased after different treatment methods,especially in the combined treatment group.However,the effect of atorvastatin combined with low-dose dexamethasone in the treatment of CSDH needs further study.Method:In vitro: Monocytes and endothelial cells were cocultured with CSDH patient hematoma samples to mimic the pathological microenvironment of CSDH.After h CMEC/D3 and THP-1 injured by hematoma fluid,we performed PCR and western bolt to test the expression of KLF-2 in different treatment groups.Small RNA was used to knock down KLF-2 in endothelial cells.We evaluated the cellular state by hopping probe ion conductance microscopy(HPICM)scanning.The changes of tight junction associated proteins,endothelial cell infiltration and endothelial inflammation associated adhesion proteins were analyzed by PCR,and western blot.In vivo: We optimized the CSDH modeling method with more stable success rate based on the established spontaneous subdural hemorrhage model by injecting the mixture of b End-3 cells and matrix glue into the subdural space of rats.Firstly,the volume of spontaneous hematoma in CSDH rats at different time points after modeling was counted by MRI.Then,the expression levels of angiogenic factors and relative inflammatory factors in the hematoma cavity at different time points after modeling were detected by PCR and ELISA.With the help of these technologies,we detected the level of KLF-2 m RNA and protein in hematoma capsule of CSDH rats at different time points.Based on the in vitro study and the above related results,we selected the time point of maximum bleeding volume as the end time of treatment to observe the therapeutic effect.The western bolt,PCR and ELISA were performed to evaluate the changes of inflammatory factor pathway and angiogenesis related indexes in CSDH rats after treatment,and the efficacy of different treatment methods was evaluated.Clinical trial: Based on the result of previous basic experiments and clinical trials,we participated in drafting and formulating a multicenter,randomized,double-blind,placebo-controlled RCT to evaluate the clinical efficacy and safety of atorvastatin combined with dexamethasone in the treatment of CSDH.According to the experimental design,the patients were divided into two groups: 1)atorvastatin group(A): taking atorvastatin + dexamethasone placebo;2)atorvastatin + dexamethasone group(A + D): taking atorvastatin + dexamethasone.Combined with the results of pre-experiment and statistical principle,the sample size of each group is estimated to be 120 cases,a total of 240 cases.Block randomization was used.The clinical study included 17 centers.The cases in each center were random Ly divided into groups according to the ratio of 1:1.The DAS for IWRS was used to assign random numbers and dispense drugs,and each center competed for inclusion.Result:After stimulating endothelial cells and monocytes with hematoma fluid from CSDH patients,the expression of KLF-2 m RNA and protein in endothelial cells was decreased.Atorvastatin monotherapy and atorvastatin combined with low-dose dexamethasone can reverse this trend,and the effect of combination therapy is much more significant.KLF-2 can increase the expression of VE-cadherin and ZO-1,with decreasing the expression of VCAM-1,ICAM-1,IL-6 and VEGF and enhancing the expression of IL-10 to rescue the injured cells.These makers play a key role in endothelial inflammation and protect endothelial cells.Moreover,the effect of combination therapy with atorvastatin and low-dose dexamethasone was obviously reduced in endothelial cells with KLF-2 knockdown compared with normal cells.We injected the mixture of b End-3 cells and matrix glue into the subdural space of rats.With the extension of the model time,the hematoma in the model rats gradually increased,peaked on the fifth day(263.8±52.85 μl)and was completely absorbed in two weeks.Notably,KLF-2 expression was significantly decreased with increasing hematoma volume,and then gradually increased in the repair period.The dynamic trends of proinflammatory and proangiogenic factor expression were consistent with the hematoma volume changes,except the IL-10 levels,which showed an opposite trend to that of the hematoma volume during the CSDH modeling period.Both monotherapy and the combined treatment coμld counteract the inflammatory activities,decrease hematoma permeability,and improve hematoma absorption.The combined treatment coμld more effectively increase KLF-2 expression,attenuate the expression of NF-κb and rapidly decrease the volume of hematoma,resμlting in the lowest mortality,than single treatments.In the previous proof of concept(POC)clinical trial,the results showed that compared with atorvastatin alone,atorvastatin combined with low-dose dexamethasone effectively reduced the hematoma volume and improved the neurological prognosis of patients with CSDH.These previous results also support previous studies that prove there is an effective synergistic effect between the two drugs,that is,atorvastatin and dexamethasone can play a role in the immune regulation and vascular repair in the process of CSDH.The previous proof of concept results also provides strong guidance for the later phase III clinical trials,so as to more reliably evaluate the effect of atorvastatin combined with low-dose dexamethasone in the treatment of CSDH.Conclusion Hematoma fluid of CSDH patients can reduce the expression of klf-2 and damage the function of endothelial cells.Atorvastatin combined with low-dose dexamethasone can antagonize the inhibition of KLF-2 induced by hematoma,thus reducing the inflammatory injury and permeability dysfunction of endothelial cells.Atorvastatin combined with low-dose dexamethasone can more effectively improve the local inflammatory injury of hematoma cavity,stabilize the neovascularization,accelerate the absorption of hematoma and improve the prognosis.The clinical application prospect of combined therapy is broad and significant.KLF-2 and NF-κb pathway may play an important role in the process of hematoma formation and prognosis of CSDH,and may become the key factors of drug treatment and prognosis evaluation of CSDH.