| Objective:To study the effect of Danggui Buxue Decoction on PI3K/Akt signaling pathway in mice with ovarian cancer by network pharmacology,and to provide effective prescription and experimental basis for the prevention and treatment of ovarian cancer by traditional Chinese medicine.Methods:In this study,network pharmacology and molecular docking were used to analyze the signaling pathway,key components and core targets of Danggui Buxue Decoction in the treatment of ovarian cancer,and to provide theoretical basis for carrying out subsequent experimental research.Ovarian cancer model was prepared by injecting ID8 cell line suspension of 0.2m L(5×10~6/0.2m L)each into the right axilla of C57BL/6mice.After successful modeling,the mice were randomly divided into model group(Model),positive drug control group(DDP),low dose group of Danggui Buxue Decoction(DBD-L),medium dose group of Danggui Buxue Decoction(DBD-M)and high dose group of Danggui Buxue Decoction(DBD-H),8 normal mice were selected as blank control group(Normal).Model group,DBD-L group,DBD-M group,DBD-H group and Normal group were intragastrically administered for 15 days,and DDP group was intraperitoneally injected every five days.The mental status,hair quality,food consumption and water intake of the mice were observed.Tumors and blood were collected on day 16 for subsequent experiments.The tumor volume of mice was observed and the tumor weight was weighed to calculate the tumor inhibition rate.HE staining was used to observe tumor cell morphology;TUNEL assay was used to detect tumor cell apoptosis in ovarian cancer model mice;immunohistochemistry was used to detect the expression of TAMs-M1 characteristic protein CD86 and TAMs-M2 characteristic protein CD163 in tumor tissues;ELISA was used to detect the expression of M1 secreted inflammatory cytokines TNF-αand M2 secreted inflammatory cytokines IL10 in serum of ovarian cancer model mice;Western blot was used to detect the expression of p-PI3K,p-Akt1 and p-Akt2 protein in tumor tissues of ovarian cancer model mice.Results:1.Network pharmacology results showed that Danggui Buxue Decoction contained 25 active components,acting on 707 targets,280 intersection targets were obtained by mapping with 2066 ovarian cancer target images,and its main signaling pathway against ovarian cancer was PI3K/Akt,and the key components were isorhamnetin,jaranol,quercetin,isoflavanone,and the core targets were Akt1,EGFR,VEGFA,TNF,and IL6;molecular docking results showed that the key components isorhamnetin and quercetin had the strongest binding to the core target Akt1,and the number of hydrogen bonds was the highest,indicating that isorhamnetin and quercetin Danggui Buxue Decoction were important components in the treatment of ovarian cancer,and Akt1 was an important target of Danggui Buxue Decoction for the treatment of ovarian cancer.2.By observing the ovarian cancer model mice,it was found that in terms of mental status:the DDP group had a slightly worse mental status than the model group,easily curled up into a litter and close to each other,the range of motion was weak,and it was easy to grasp.The ovarian cancer model mice in the DBD-H group,DBD-M group,and DBD-L group had a good mental status,a strong range of motion,and a high reaction intensity when grasping.In terms of food consumption:the DDP group consumed less food than the ovarian cancer model mice,and the DBD-H,DBD-M,and DBD-L groups consumed more food.In terms of drinking water consumption:the DDP group consumed less drinking water than the ovarian cancer model mice,and the DBD-H,DBD-M,and DBD-L groups consumed more drinking water.In terms of hair quality:DDP group had poorer hair quality,severe hair loss during grasping,and poor gloss than model group;DBD-H group,DBD-M group,and DBD-L group had dense hair quality and slight hair loss during grasping,but better gloss.3.The results showed that the body weight of mice in Normal group increased,while the body weight of mice in DBD-H and DBD-M groups showed a downward trend,but the downward trend was slower than that in DBD-L group,Model group and DDP group.4.The results of tumor inhibition rate showed that(1)the tumor weight of DDP and DBD-M groups was lower than that of Model group;Compared with DDP group,tumor weight in DBD-H group,DBD-M group and DBD-L group increased(2)Tumor inhibition rates in DBD-H group,DBD-M group,DBD-L group and DDP group were 15.75%,45.17%,31.73%and 94%,respectively.5.The results of HE staining showed that the tumor cells in the Model group and the drug intervention group were distributed patchy and arranged closely,the cells were enlarged and varied,the nuclei were enlarged,the nucleoplasmic ratio was abnormal,the mitotic figures were increased,and the atypia was obvious;local necrosis was observed in the cancer tissues in the DDP group and the DBD-H group,and apoptotic changes such as nuclear pyknosis and vacuolization were observed in the tumor cells.6.TUNEL results showed that the apoptosis rate of ovarian cancer cells was significantly increased in the DDP and DBD-H groups compared with the Model group;the apoptosis rate of ovarian cancer cells was significantly decreased in the DBD-L group compared with the DDP group.7.The results showed that(1)p-PI3K expression was down-regulated in the DDP and DBD-H,DBD-M,and DBD-L groups compared with the Model group;p-PI3K expression was down-regulated in the DBD-H,DBD-M,and DBD-L groups compared with the DDP group(2)p-Akt1 expression was down-regulated in the DBD-M and DBD-H groups compared with the Model group;p-Akt1 expression was significantly down-regulated in the DBD-H group compared with the DDP group(3)p-Akt1 expression was up-regulated in the DDP,DBD-L,DBD-M,and DBD-H groups compared with the Model group,and p-Akt2 expression was up-regulated in the DBD-M and DBD-H groups compared with the DDP group.8.ELISA results showed that(1)TNF-αexpression showed an up-regulation trend in the Model group compared with the Normal group,but it was not statistically significant,and TNF-αexpression was up-regulated in the DBD-H and DBD-L groups;TNF-αexpression levels were up-regulated in the DBD-H and DBD-M groups compared with the Model group;TNF-αexpression levels were up-regulated in the DBD-H and DBD-L groups compared with the DDP group(2)IL10 expression levels were up-regulated in the Model and DBD-L groups compared with the Normal group;IL10 expression was significantly down-regulated in the DBD-H,DBD-M,and DDP groups compared with the Model group;DBD-L expression levels were up-regulated compared with the DDP group.9.Immunohistochemical results showed that(1)CD86 expression was up-regulated in the DDP group and significantly up-regulated in the DBD-L and DBD-H groups compared with the Model group;CD86 expression was significantly up-regulated in the DBD-L and DBD-H groups compared with the DDP group(2)CD163 expression was significantly down-regulated in the DDP,DBD-M,and DBD-H groups and down-regulated in the DBD-L group compared with the Model group.Conclusion:1.Danggui Buxue Decoction can improve the quality of life of ovarian cancer model mice,control the weight loss of mice,and inhibit tumor growth.2.Danggui Buxue Decoction can induce the polarization of TAMs to M1 by regulating the phosphorylation level of key proteins in the PI3K/Akt pathway,promote the expression of TAMs-M1-related inflammatory cytokines TNF-αand M1-characteristic protein CD86,and inhibit the expression of TAMs-M2-related inflammatory cytokines IL10 and M2-characteristic protein CD163,which may be one of the mechanisms of Danggui Buxue Decoction against ovarian cancer. |
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