Effects And Mechanism Of Royal Jelly On The Proliferation Of 4T1 Cells In Vitro And In Vivo

Royal jelly(RJ)is a milkly secretion that is used to feed the queen,as well as bee larvae within three days old.It is secreted by the hypopharyngeal glands and maxillary glands of breeding bees.It was repotred that RJ possesses abundant pharmacological properties,including anti-oxidation,immunity improvement,effects of protecting liver and kidney,antibacterial effects and improving sleeping and so on.However,there are few a series of reports about the effects of RJ on breast cancer.It is reported that 10-HDA,a particular substance in RJ,plays an important role in the antitumor process.In this paper,the effects of rape RJ(10-HDA,2.01%)on the proliferation of 4T1 cells were studied in vivo and in vitro,and the effects of camellia RJ(10-HDA,1.62%)on the growth of 4T1 cells in vivo were also studied.Finally,RNA-seq was employed to investigate the effect of RJ on the growth of the tumor in 4T1-bearing mice from gene level.These results provided a systematic research results about effects of RJ on 4T1 cells.The main technical lines and results of our study are as follows:1.The effects of rape RJ on 4T1 cells in vivo and in vitroa)Cell experiments showed that there were no significant effects of royal jelly(after honey bee collecting nectar on rape)or its components on the proliferation of 4T1 cells by MTT method.RJ could promote 4T1 cells to secrete MMP-9 and MMP-2 protease under normal growth conditions.While it had no significant effect on the production of MMP-9 protease secreted by 4T1 cells in the lacking of nutritional conditions.Regardless of whether the media contains fetal bovine serum,the expression trends of MMP-2,MMP-9,MMP-13 and MMP-14 genes were inconsistent.The results can explain that there is an overall regulatory role of RJ on the expression of these genes,therefore RJ had no significant effect on the proliferation of 4T1 cells.b)RJ has no obvious effect on the organ of the normal mice,but it can improve the antioxidant capacity and immunity of serum,and enhance the antioxidant capacity of liver and kidney.The experiments were done by feeding4T1-bearing mice with RJ using prophylactic-therapeutic and therapeutic methods.The results showed that,the weight of tumor was significantly reduced(RJ 0.5,1.5 g/kg)(P<0.05)in groups processed by prophylactic-therapeutic method;the levels of interleukin(IL)-2(RJ 0.5,1.5 g/kg),interferon(IFN)-α,superoxide dismutase(SOD)and total antioxidant capacity(T-AOC)were significantly elevated(P<0.01)in serum,but the concentrations of IL-4(RJ 0.5,1.5 g/kg)and IL-10(RJ 1.0 g/kg)were significantly decreased(P<0.05);the activities of T-AOC and glutathione reductase(GR)were significantly improved in liver(P<0.01);while the activities of T-AOC and GR were notably surged only under the dose of 0.5 g/kg(P<0.01)in kidney.For therapeutic group,the antitumor effect of RJ was not notable;the changes of IL-2,IFN-α,SOD and T-AOC in serum,and the changes of T-AOC and GR in liver were similar to those in a prophylactic-therapeutic method.RJ treatment not only could reduce development of breast tumor,but also could improve the antioxidant capacity of serum,liver,and kidney,especially in a prophylactic-therapeutic method.These results corroborated the efficacy of RJ supplementation in diets.The antioxidant and immunomodulatory activities of RJ would play an important role on anti-tumor growth.c)The mice were injected 4T1 cells with or without RJ though vein in tail.In serum,the level of IL-4 in the groups of RJ50 and RJ 100 was significantly decreased(P<0.05)in relation to CT group,and the concentrations of IFN-α and T-AOC were notably surged(P<0.05).In addition,the level of IFN-y significantly improved in RJ50 group(P<0.05).In kidney,the activity of T-AOC was notably improved only under the dose of 100 mg/mL(P<0.01).The number of pulmonary metastases in mice was significantly reduced when injected RJ(rape)and the mixture of 4T1 cells in the tail vein.The immunity and antioxidant capacity of mice has been improved.2.The effects of camellia RJ on 4T1 cells in vivoCamellia RJ had no obvious effect on the levels of body weight and the tumor growth in 4T1-bearing mice of 4T1 breast cancer cell by prophylactic-therapeutic method.The result showed that RJ(0.5 g/kg)had a significant immunomodulatory effect on 4T1-bearing mice,which was reflected in serum by the augmentation of tumor necrosis factor-alpha(TNF-α),immunoglobulin G(IgG)and the reduction of interleukin-6(IL-6)(P<0.05).Meanwhile,IL-10 production in spleen lymphocytes was also significantly reduced(P<0.001),which also reduced the Th2 response.RJ(1.5 g/kg)also had a significant immunomodulatory effect on 4T1-bearing mice by improving the level of IgG and reducing the concentration of IL-6 in serum(P<0.05).And histopathological examinations with RJ were more effective.3.The effect of RJ on the tumor growth of 4T1-bearing mice by RNA-seqThere are 281 genes differentially expressed in RJ vs CT of which the number of immune related gene is 56.Differentially expressed genes involved a total of 167 pathways.The pathways enriched dramatically in the second and fourth are TNF signaling pathway and Cytokine-cytokine receptor interaction,respectively.Both these pathways are concerned with the immune capacity.Our result also showed that RJ played an important role in the immune regulation of 4T1 breast cancer cells in mice.