The Effect And Mechanism Of Jianwei Shuangshen Granules In Preventing Spontaneous Lung Squamous Cell Carcinoma Mice

Background:Shuangshen Granules is a prescription developed by Professor Bao-jin Hua based on Fei Liu Ping Ointment.It consists of three precious Chinese herbs,namely American ginseng,Panax notoginseng and Cordyceps.It is mostly used in patients with lung cancer after surgery to reduce the recurrence and metastasis.Whether Shuangshen Granules can prevent the occurrence of lung cancer,we still need to find a suitable animal model for scientific research.Professor Dr.Yinling Hu reported spontaneous lung squamous cell carcinoma mouse model for the first time in the world.In this mouse model,spontaneous lung squamous cell carcinoma was caused by knock-in of IKK? kinase inactivation,which usually takes 4-6 months to form tumors.The model could better simulate the markers of human lung squamous cell carcinoma.There existed inflammatory microenvironment,the activation of oncogenes,the infiltration of a large number of inflammatory cells and cytokines,the activation of the NF-?B signaling pathway,and increased expression of various growth factors in the lung tissue.This will provide a better animal model for long-term intervention of Chinese herbs.In previous study,Dr.Rui Liu used this mouse model to observe the effect and mechanism of Shuangshen Granules in Dr.Yinling Hu's laboratory.After 5 months of intervention,it could effectively reduce skin ulcers,reduce the proportion of macrophages and increase the proportion of neutrophils,which suggested that Shuangshen Granules had a certain preventive effect.Considering the insufficient resources of Cordyceps sinensis and the high price,we only used American ginseng and Panax notoginseng trying to observe whether Jianwei Shuangshen Granules could reduce the incidence of lung tumor and the skin phenotype in this model.In addition,multiple immune cells,NF-?B signaling pathways in the inflammation microenvironment and peripheral blood cytokines were examined to explore possible mechanism of Jianwei Shuangshen Granules.Objective:To observe the effect and mechanism of Jianwei Shuangshen Granules in preventing spontaneous lung squamous cell carcinoma miceMethod:Twenty-four KAKA mice were randomly divided into a model group(KAKA group)and a Jianwei Shuangshen granules group(KAKA+XS group).Each group contains 12 mice.Another 5 wild-type mice were used as a blank control group(WT group).After weaning,the mice in e WT group and KAKA group ate normal diet,and the mice in KAKA+XS group ate normal diet containing 0.788mg/g/d of Jianwei Shuangshen Granules.After 5 months of intervention with Jianwei Shuangshen Granules,these aspects of mice including the mental state,skin hair condition,body weight,lung weight,spleen weight,index of lung penetration,index of spleen penetration were observed.The incidence of lung squamous cell carcinoma of mice was calculated.Inflammation of other organs was observed using by H&E staining.The proportion of CD4+T cells,CD8-T cells,NK cells,macrophages and neutrophils in lung and spleen tissue were detected by flow cytometry.The expression of IL-2,IL-6,IL-10,TNF-? and IFN-? in peripheral blood serum was detected by cytometric bead array.Western Blot was used to detect the expression of IKK?,p-I?B?,I?B?,COX-2 and VEGF in lung tissue.RT-qPCR was used to detect the expression of CHUK.Trim29,CDK1,and IGF1 mRNAResult:1.Mental and skin and hair condition of mice:After 5 months of intervention,the mice in WT group was in good mental state,and the mice in KAKA group had eyelid edema and poor mental state.The mice in KAKA+XS group also had eyelid edema.but the mental condition was better than that of the mice in KAKA group.The hair of mice in the WT group was smooth and shiny.The hair in the KAKA group was rough and dull at the age of 9 weeks,and ulcers appeared on the skin of neck and back.The hair in the KAKA+XS group was also rough and dull at the age of 10 weeks and ulcers also appeared on the skin of neck and back,but compared with the mice in the KAKA group,the ulcer area was significantly reduced,and the ulcer phenotype appeared later than that in the KAKA group.There was a statistically significant difference between the two groups(P<0.05).2.Body weight,lung weight,lung coefficient,spleen weight,and spleen coefficient:(1)After 5 months of intervention,compared with the WT group,the body weight of mice in KAKA group was reduced,and the difference was statistically significant(P<0.05);There was no significant difference in the body weight of mice between the KAKA+XS group and the KAKA group(P>0.05).(2)After 5 months of intervention,compared with the WT group,the lung weight,lung coefficient,spleen weight and spleen coefficient were significant increased in the mice in KAKA group(P<0.05);Compared with the KAKA group,the KAKA+XS group had no significant differences in lung weight,lung coefficient,spleen weight,and spleen coefficient(P>0.05).3.Tumor incidence rate in mice:After 5 months of intervention,4 mice in the KAKA group developed spontaneous lung squamous cell carcinoma(4/8),while no mice in the KAKA+XS group developed spontaneous lung squamous cell carcinoma(0/9),there was a statistical difference between the two groups(P<0.05),4.HE staining of organs:(1)The alveolar structure in the WT group was clearly visible,and there was no obvious congestive edema and inflammatory cell infiltration.In the KAKA+XS group,the alveolar structure was damaged,congested and edema,and there was a large number of inflammatory cell infiltration.There were signs of lung squamous cell carcinoma "keratinized foci" and the cells were heterotypic.In the KAKA+XS group,the alveolar structure was also damaged,congested and edema,and inflammatory cell infiltration was observed.Compared with the KAKA group,the degree of inflammation in the lung tissue was not significantly reduced.(2)In the WT group,normal skin tissue was stained with HE.In the KAKA and KAKA+XS group,the cuticle of the epidermis and spinous cortex were significantly thickened and the cells were edema and a large number of lymphocytes were seen.Compared with the KAKA group,the degree of KAKA+XS group was significantly reduced.(3)The central vein was visible in the WT group,and the hepatic lobules were intact.The interlobular veins,arteries,and bile ducts were visible in the manifold area.In the KAKA group and KAKA+XS group,fibrous tissue proliferation and lymphocyte infiltration near the central vein were seen.No significant difference was seen between the two group.(4)In the WT group,normal kidney tissue was stained with HE.Glomerular capillary hyperemia and renal interstitial hyperemia were seen in both KAKA group and KAKA+XS group.There was no significant difference between the two groups.(5)In the WT group,normal pancreas tissue was stained with HE.Pancreatic acinar tissue atrophy,interstitial diffuse fibrous tissue proliferation,and a large number of lymphocyte infiltration were seen in the KAKA and KAKA+XS group.Compared with the KAKA group,the degree of inflammation in KAKA+XS group was significantly reduced.5.Effect of Jianwei Shuangshen Granules on lung immune cells:(1)At the age of 6 months,there was no statistical difference in the proportion of CD4+T and CD8+T cells in the lung tissue of mice in the WT group and KAKA group(P>0.05);Compared with the KAKA group,the proportion of CD4+T and CD8+T cells in the lung tissue of mice in the KAKA+XS group was significantly increased(P<0.05).(2)Compared with the WT group,the proportion of NK cells in lung tissue of mice in KAKA group was significantly increased(P<0.05);compared with the KAKA group,the proportion of NK cells in lung tissue of mice in KAKA+XS group was significantly increased(P<0.05).(3)Compared with the WT group,the proportion of neutrophils in lung tissue of mice in KAKA group was significantly increased(P<0.05);the difference between KAKA+XS group and KAKA group was not statistically significant(P>0.05).(4)Compared with the WT group,the proportion of macrophages in lung tissue of mice in the KAKA group was significantly increased,(P<0.05);compared with the KAKA group,the proportion of KAKA+XS group was significant reduced(P<0.05).6.Effect of Jianwei Shuangshen Granules on immune cells of spleen tissue:(1)After 5 months of intervention,compared with WT group,the proportion of CD4+T and CD8+T cells in spleen tissue of KAKA group mice was significantly increased(P<0.05).There was no significant difference in the proportion of CD4+T and CD8+T cells between KAKA and KAKA+XS groups(P>0.05).(2)Compared with WT group,the proportion of NK cells in spleen tissue of mice in KAKA group significant increased(P<0.05);compared with the KAKA group,the proportion of NK cells in KAKA+XS group significant increased(P<0.05).(4)There was no significant difference in the proportion of neutrophils and macrophages in the spleen tissue between the WT and KAKA group(P>0.05).There was no significant difference in the proportion of neutrophils and macrophages in the spleen tissue between the KAKA and KAKA+XS group(P>0.05).7.Effect of Jianwei Shuangshen Granules on peripheral blood inflammatory factors in mice:After 5 months of intervention,compared with WT group,serum levels of IL-2,IL-6,and IL-10 in the KAKA group were all increased,and the differences were statistically significant(P<0.05).There was no significant difference in the expression levels of TNF-? and IFN-y(P>0.05).Compared with the KAKA group,the levels of IL-2,IL-6,IL-10 and TNF-? in peripheral blood serum of mice in the KAKA+XS group were not significantly different,but the expression levels of IFN-?were increased,and the difference was statistically significant(P<0.05).8.Effect of Jianwei Shuangshen Granules on the related proteins in the lung tissue of mice:compared with the KAKA group,the expression of IKKa in the lung tissue of mice in the KAKA+XS group increased,the expression of p-IkBa decreased,the expression of IkBa increased,the expression of COX-2 and VEGF decreased,and the differences were statistically significant(P<0.05).9.Effect of Jianwei Shuangshen Granules on the expression of the related genes in the lung tissue of mice:compared with the WT group,CHUK mRNA expression in the lung tissue of the mice in KAKA group decreased,and the expression of Trim29 mRNA,CDK1 mRNA and IGF1 mRNA increased and the differences were statistically significant(P<0.05).Compared with the KAKA group,CHUK mRNA expression in the lung tissue of mice in KAKA+XS mice increased,and the expression of Trim29 mRNA,CDK1 mRNA and IGF1 mRNA decreased,and the differences were statistically significant(P<0.05).Conclusion:1.Jianwei Shuangshen Granules can improve the mental state,skin hair and ulcers of KAKA mice to a certain extent,prolong the time of skin phenotypes,reduce the incidence of spontaneous lung squamous cell carcinoma,and reduce skin and pancreatic inflammation.2.Jianwei Shuangshen Granules may reduce the incidence of spontaneous lung squamous cell carcinoma by increasing the proportion of CD4+T cells,CD8+T cells,NK cells,and reducing macrophages.3.Jianwei Shuangshen Granules may reduce the incidence of spontaneous lung squamous cell carcinoma by increasing the level of peripheral blood serum IFN-y to enhance the killing function of immune cells.4.Jianwei Shuangshen Granules may play a role in inhibiting angiogenesis of lung squamous cell carcinoma by increasing IKK?,I?B?,decreasing p-I?B?,COX-2,and VEGF and inhibiting the NF-?B signaling pathway.5.Jianwei Shuangshen Granules may reduce the incidence of spontaneous lung squamous cell carcinoma by reducing the expression of Trim29,CDK1,IGF1 mRNA,and inhibiting the excessive proliferation of cells.