| Background:Glioma is the most common primary malignant tumor of the central nervous system.Glioblastoma(GBM)belongs to WHO Ⅳ grade glioma,which is the most malignant,accounting for about 45%of all gliomas.Due to the poor effect of radiotherapy and chemotherapy,obvious adverse reactions,blood-brain barrier obstruction,tumor microenvironment immune-suppression and other factors,the effect of targeting and immunotherapy is limited,the postoperative recurrence rate of GBM patients is high,and the survival time and quality of life are always difficult to improve.The median overall survival time of patients is only 14.6 to 16.7 months.Therefore,it is very important to ensure a good quality of life in the limited survival period of postoperative GBM patients,and it is of great research value to explore methods to improve the efficacy of radiotherapy and chemotherapy and reduce adverse reactions.Shuangshen Sanjie prescription is the clinical experience prescription of Professor Hua Baojin of Guang’ anmen Hospital,China Academy of Chinese Medical Sciences,which is composed of American ginseng,Panax notoginseng and Cordyceps Fungus Powder,with the effect of replenishing qi and activating blood circulation.The clinical observation of small samples in the early outpatient clinic shows that,compared with western medicine alone,Shuangshen Sanjie prescription combined with western medicine has certain advantages in improving the clinical symptoms,improving the quality of life and alleviating adverse reactions of postoperative GBM patients with qi deficiency and blood stasis,but it is lack of detailed clinical data support.Tumor immunosuppressive microenvironment mediated by myeloid derived suppressor cells(MDSCs)is an important factor for the poor effect of conventional therapy on GBM patients.After activation and expansion of MDSCs in peripheral lymphoid organs,MDSCs was recruited into GBM tumor microenvironment(TME).The higher the degree of MDSCs infiltration in TME,the more serious the immunosuppression,and the worse the clinical treatment effect.Reducing the activation and expansion of peripheral MDSCs,thus reducing the degree of MDSCs infiltration in TME is an important method to improve tumor immunosuppressive microenvironment.PTEN/PI3K/Akt signaling pathway is an important pathway to promote the activation and amplification of MDSCs.The previous basic research of our research group showed that Shuangshen Sanjie prescription could regulate the immune-suppressive microenvironment with MDSCs as the core and reduce the content of MDSCs in the peripheral spleen of tumor-bearing mice.Network pharmacological studies also found that Shuangshen Sanjie prescription could regulate PI3K/Akt signaling pathway.We will start with MDSCs to explore the mechanism of Shuangshen Sanjie prescription in the treatment of GBM.Objective:1.Through prospective clinical study,to explore the clinical efficacy and safety of Shuangshen Sanjie prescription in improving the quality of life of postoperative GBM patients,so as to provide clinical evidence and data support for the prevention and treatment of GBM.2.Through basic research,with the help of network pharmacology and modern molecular biology technology,to observe the effect of Shuangshen Sanjie prescription on PTEN/PI3K/Akt pathway in MDSCs of peripheral spleen of U87MG glioma bearing mice.The mechanism of Shuangshen Sanjie prescription will be explored,and the scientific connotation of the method of replenishing qi and activating blood circulation in the prevention and treatment of GBM will be revealed,and the scientific basis for the prevention and treatment of GBM by traditional Chinese medicine(TCM)will be provided from the point of view of inhibiting the activation and expansion of peripheral MDSCs and reducing MDSCs infiltration in TME.Methods:1.A prospective non-randomized controlled study design was used to screen GBM patients who were treated in Guang’ anmen Hospital,China Academy of Chinese Medical Sciences and Beijing Tiantan Hospital,Capital Medical University from September 2020 to November 2021.The baseline data,medication status and examination data of eligible GBM patients were collected,the case report form was filled out,and the Excel database was established.According to the grouping criteria,patients were divided into Shuangshen Sanjie group(hereinafter referred to as Shuangshen group)and control group.Statistical analysis was carried out by using SPSS22.0 software to evaluate the different changes of TCM syndrome score,life quality score,neurological function state,functional state and drug safety between the two groups before and after treatment.2.Basic research:(1)The expression level of MDSCs phenotype CD33 in tumor pathological tissues of patients with WHO Ⅱ-Ⅳ gliomas was detected by Immunohistochemistry(IHC),and the infiltration degree of MDSCs in tumor tissues of patients with different grades of gliomas was observed.(2)By using the research method of network pharmacology,the active components of Shuangshen Sanjie prescription were screened by TCMSP database,the action targets of Shuangshen Sanjie prescription were predicted by SwissTarget-Prediction platform,and the disease targets of GBM were screened comprehensively by GeneCards,TTD and OMIM databases.By using String online platform,the protein interaction network was constructed for the intersecting targets,and the key targets were screened.The visualization network of "Shuangshen Sanjie prescription-active ingredient-GBM-target" was constructed by using Cytoscape3.7.2 software.Through the Metascape online platform for gene ontology enrichment(GO)analysis and KEGG pathway enrichment analysis,we explored the possible regulatory pathway of Shuangshen Sanjie prescription in the treatment of GBM.(3)U87MG cell line was inoculated into the right axilla of BALB/c nude mices to establish the model of U87MG glioma-bearing mices.After 35 days of intervention with pure water,temozolomide(TMZ),Shuangshen Sanjie prescription combined with TMZ,the tumor weights in each group were observed,the tumor volume growth curve and body weight curve were drawn,and the inhibitory effect of Shuangshen Sanjie prescription combined with TMZ on mice with GBM was verified.(4)Establish the model of U87MG glioma-bearing mice and set up the blank group of BALB/c mice without tumor.After 35 days of intervention with pure water,TMZ,Shuangshen Sanjie prescription combined with TMZ,the MDSCs of spleen and transplanted tumor tissue of each group was detected by flow cytometry and immunofluorescence(IF),and the effect of Shuangshen Sanjie prescription combined with TMZ on MDSCs phenotype was observed.(5)Establish the model of U87MG glioma-bearing mice.After 35 days of intervention with pure water,TMZ,Shuangshen Sanjie prescription combined with TMZ,we selected the spleen MDSCs of each group by using magnetic activated cell sorting,furthermore,we used Western Blot to detect the protein expression of PTEN,PI3K,Akt and p-Akt in MDSCs.Results:1.Clinical research:(1)A total of 60 postoperative GBM patients with qi deficiency and blood stasis were enrolled in this study,and 5 patients fell off.28 patients in the Shuangshen group and 27 patients in the control group were treated and followed up.There was no significant difference in age,gender,body mass index(BMI),surgical site,degree of tumor resection,number of tumors,methylation of MGMT promoter and IDH mutation between the two groups.(2)TCM syndrome score:After treatment,the total score of TCM syndrome scale in the Shuangshen group was better than that in the control group,and there was significant difference between the two groups(P<0.05).The improvement of dizziness,headache,nausea and vomiting,fatigue,limb numbness,tremor and insomnia in the Shuangshen group was better than that in the control group.(3)Life quality score:The baseline of the two groups was balanced.After treatment,the total score of life quality in the Shuangshen group was higher than that in the control group(P>0.05).In terms of each module of the brain tumor quality of Life specificity scale(Fact-BrV4.0),the scores of physiological status,social/family situation,emotional status and functional status in the Shuangshen group were significantly higher than those in the control group,and the difference was statistically significant(P<0.05).(4)Neurological functional status score:We used the Neurologic Assessment in Neuro-Oncology(NANO)scale to evaluate the neurological functional status of postoperative GBM patients.After treatment,the neurological function of postoperative GBM patients in the Shuangshen group and the control group did not improve,but the neurological function of the patients in the Shuangshen group remained stable and had no progress,while 2 patients in the control group progressed,and the difference was not statistically significant(P>0.05).(5)Functional status score:We used Karnofsky performance status(KPS)score scale to evaluate the functional status of postoperative GBM patients.There was no significant difference in KPS score between the two groups before treatment.However,KPS score in the Shuangshen group was significantly higher than that in the control group after treatment.(6)Safety index:There was no significant difference in adverse reactions of blood system,digestive system and urinary system between the Shuangshen group and the control group(P>0.05).There was no significant difference in the incidence of adverse reactions between the two groups(P>0.05).2.Basic research:(1)After IHC staining,there were differences in the expression of CD33 in pathological tissues of patients with different grades of gliomas.The expression of CD33 was the lowest in grade WHO Ⅱ gliomas,and the expressions of CD33 in grade WHO Ⅲ and Ⅳ gliomas were significantly higher than that in grade WHO Ⅱ gliomas.At the same time,the expression of CD33 in WHO Ⅳ grade GBM tumor tissue was higher than that in WHO Ⅲ grade tumor tissue(P<0.01).With the increase of the pathological grade of WHO,the degree of MDSCs infiltration in glioma tissues gradually increased.(2)Based on the study of network pharmacology,23 active ingredients were selected from Shuangshen Sanjie prescription.The top 10 active ingredients were papaverine,quercetin,cerevisterol,DFV,arachidonic acid,mandenol,cordycepin,linoleyl acetate,stigma-7-enol and cholesteryl palmitate.There were 158 key targets of Shuangshen Sanjie prescription in the treatment of GBM,among which TP53,SRC,PIK3CA,MAPK3,PIK3R1,MAPK1,AKT1,STAT3,HSP90AA1 and VEGFA were the top 10 core targets.The main enrichment pathways of Shuangshen Sanjie prescription in treating GBM were PI3K/Akt signaling pathway,Sphingolipid signaling pathway,FoxO signaling pathway,Ras signaling pathway,Rap signaling pathway and so on.Combined with the previous research of our group,we proposed that PI3K/Akt signal pathway might be an important pathway for Shuangshen Sanjie prescription to regulate the activation and amplification of peripheral MDSCs in GBM patients.(3)After 35 days of intervention,the average tumor weight of tumor-bearing mice in TMZ group and SSG+TMZ group was significantly lower than that in Model group(P<0.01),and the tumor weight in SSG+TMZ group was the lowest.From the growth curve of transplanted tumor volume of tumor-bearing mice in each group,compared with Model group and TMZ group,SSG+TMZ group had the best inhibitory effect on tumor growth rate and continuous anti-tumor effect.The body weight of tumor-bearing mice in each group showed an increasing trend,and there was no significant difference in body weight of tumor-bearing mice at different time points(P>0.05).(4)The results of flow cytometry showed that the content of MDSCs in spleen of Model group was significantly higher than that of blank group.After 35 days of intervention,the content of MDSCs in spleen of TMZ group and SSG+TMZ group was significantly lower than that of Model group,and the decrease of spleen MDSCs of SSG+TMZ group was greater than that of TMZ group.The IF results showed that the expression of CD 11 b+Ly6C+and CD 11 b+Ly6G+in TMZ group and SSG+TMZ group was lower than that in Model group,and the expression in SSG+TMZ group was lower than that in TMZ group.(5)PTEN/PI3K/Akt signaling pathway is an important pathway to promote the activation and amplification of peripheral MDSCs.After 35 days of intervention,Western Blot results showed that Shuangshen Sanjie prescription combined with TMZ could significantly enhance the expression of PTEN protein and inhibit the expression of PI3K,Akt and p-Akt protein in peripheral spleen MDSCs of tumor-bearing mice compared with Model group and TMZ group.Conclusion:1.Compared with the standard radiotherapy and chemotherapy regimen of western medicine alone,Shuangshen Sanjie prescription combined with standard radiotherapy and chemotherapy of western medicine can effectively improve the clinical symptoms and quality of life of postoperative GBM patients with qi deficiency and blood stasis,stabilize the neurological state and improve their functional state,and it is safe and feasible.2.The combination of Shuangshen Sanjie prescription and TMZ can inhibit the growth of transplanted tumor in U87MG glioma-bearing mice,and the anti-tumor effect is better than that of TMZ alone.The mechanism of inhibiting tumor growth may be related to reducing the proportion of MDSCs in the peripheral spleen of U87MG glioma-bearing mice and reducing the degree of MDSCs infiltration in TME.3.The mechanism of Shuangshen Sanjie prescription in reducing the proportion of MDSCs in peripheral spleen and inhibiting the activation and amplification of MDSCs in U87MG glioma-bearing mice may be related to PTEN/PI3K/Akt signaling pathway. |
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