| Objective: In this study,Osteosarcoma K7 model mice were the object of observation.To observe the comparison of the general situation,iconography and the difference of immunological effect of the combination of acupuncture point and acupoint in the intervention of osteosarcoma model mice and acupuncture intervention effect of intestinal flora consistent with the holistic view of traditional Chinese medicine.To establish non-invasive in vivo imaging monitoring.To explore the effect law of the compatibility of acupoints and single acupoint and its possible immunological mechanism,in order to provide evidence for the scientific application of acupoints combination.Methods:1.There were 60 female BALB/C mice,building osteosarcoma K7 subcutaneous tumor-burdened model mice,and were randomly divided into the control group,the model group,the "Baihui"(DU20)group,the "Shenshu"(BL23)group,the "Zusanli"(ST36)group,and acupoints combination group.The control group made no model and no treatment,model control group only build model and no treatment,the "Baihui"(DU20)group made model then acupunct point "Baihui"(DU20),the "Shenshu"(BL23)group made model then acupunct point "Shenshu"(BL23),the "Zusanli"(ST36)group made model then acupunct point "Zusanli"(ST36),acupoints combination group made model then acupunct acupoints compatibility("Baihui"(DU20),Shenshu,(BL23),and "Zusanli"(ST36)).2.The changes of hair color,mental state and body weight in different groups of mice were observed and recorded.3.The application of multimode optical In Vivo imaging(In Vivo Xtreme technology)and vernier caliper were used to observe the change of mice tumor size,and changes in tumor photon volume.4.Flow cytometry was used to detect the changes of immune-related indicators,such as the immune-related indicators changes of T cells and NK cells.5.Enzyme Linked Immunosorbent Assay(ELISA)was used to observe the changes inimmune-related indicators of the serum cytokine IL-6,IFN-? and spleen tissues TNF-?immune factors.6.Intestinal flora was used to detect the changes of intestinal microorganisms after acupuncture intervention in different groups of mice.Results:1.There was no difference in body weight before acupuncture treatment;After acupuncture treatment,mice in the model group lost significant weight after day 12,the weight of mice in the control group tended to increase over time,the weight of mice in the treatment group was relatively stable.On days 8 and 10 of acupuncture treatment,compare to the control group,the body weight were decreased in the model group,the "Baihui"(DU20)group,the "Shenshu"(BL23)group,the "Zusanli"(ST36)group,and acupoints combination group,and there were differences in these groups(P<0.05).On day12 of acupuncture treatment,compare to the control group,the body weight were significantly decreased in the model group,the "Baihui"(DU20)group,the "Shenshu"(BL23)group,the "Zusanli"(ST36)group,and acupoints combination group,and there were significant differences in these groups(P<0.01);compare to the model group,the body weight was increased in the "Baihui"(DU20)group,and there were differences between the two groups(P<0.05),the body weight were significantly increased in the "Shenshu"(BL23)group,the "Zusanli"(ST36)group,and acupoints combination group,and there were significant differences in these groups(P<0.01).On day 14 of acupuncture treatment,compare to the control group,the body weight were significantly decreased in the model group,the "Baihui"(DU20)group,the "Shenshu"(BL23)group,the "Zusanli"(ST36)group,and acupoints combination group,and there were significant differences in these groups(P<0.01);compare to the model group,the body weight were significantly increased in the "Baihui"(DU20)group,the "Shenshu"(BL23)group,the "Zusanli"(ST36)group,and acupoints combination group,and there were significant differences in these groups(P<0.01).2.There was no difference in tumor size and volume before acupuncture treatment;Onday 8 of acupuncture treatment,compare to the model group,the tumor volume in the acupoints combination group was smaller,and there were differences between the two groups(P<0.05);compare to the acupoints combination group,the tumor volume were bigger in the "Baihui"(DU20)group,the "Shenshu"(BL23)group,the "Zusanli"(ST36)group,and there were differences in these groups(P<0.05).On day 10 of acupuncture treatment,compare to the model group,the tumor volume were smaller in the "Zusanli"(ST36)group and acupoints combination group,and there were differences in these groups(P<0.05);compare to the acupoints combination group,the tumor volume were bigger in the "Baihui"(DU20)group,the "Shenshu"(BL23)group,the "Zusanli"(ST36)group,and there were differences in these groups(P<0.05).On day 12 of acupuncture treatment,compare to the model group,the tumor volume were much smaller in the "Baihui"(DU20)group,the "Shenshu"(BL23)group,the "Zusanli"(ST36)group and acupoints combination group,and there were significant differences in these groups(P<0.01);compare to the acupoints combination group,the tumor volume were much bigger in the "Baihui"(DU20)group,the "Shenshu"(BL23)group,the "Zusanli"(ST36)group,and there were significant differences in these groups(P<0.01).On day 14 of acupuncture treatment,compare to the model group,the tumor volume were much smaller in the "Baihui"(DU20)group,the "Shenshu"(BL23)group,the "Zusanli"(ST36)group,and acupoints combination group,and there were significant differences in these groups(P<0.01);compare to the "Shenshu"(BL23)group,the tumor volume were bigger in the "Baihui"(DU20)group and the "Zusanli"(ST36)group,and there were differences in these groups(P<0.05);compare to the acupoints combination group,the tumor volume were much bigger in the "Baihui"(DU20)group,the "Shenshu"(BL23)group and the "Zusanli"(ST36)group,and there were significant differences in these groups(P<0.01).3.In vivo imaging results show,there was no difference at baseline;After one week of acupuncture treatment,from the imaging picture,compare to other groups,the "Shenshu"(BL23)group and acupoints combination group had smaller tumors.After two week of acupuncture treatment,from the imaging picture,the tumors in the model groupwere significantly larger,compare to the "Shenshu"(BL23)group and acupoints combination group,the tumors in the model group,the "Baihui"(DU20)group,the "Zusanli"(ST36)group,and acupoints combination group were much larger.4.In vivo imaging photon volume statistics show that,in the previous two phases,there was no difference among groups.The amount of photons collected at the end of the treatment was shown,compare to the model group,the quantitative value was decreased in the "Shenshu"(BL23)group,and there were differences between the two groups(P<0.05),the quantitative value was significantly decreased in the acupoints combination group,and there were significant differences between the two groups(P<0.01);compare to the "Shenshu"(BL23)group,the quantitative value was increased in the "Baihui"(DU20)group and the "Zusanli"(ST36)group,and there were differences in these groups(P<0.05);compare to the acupoints combination group,the quantitative value was increased in the "Shenshu"(BL23)group,and there were differences between the two groups(P<0.05),the quantitative value were significantly increased in the "Baihui"(DU20)group and the "Zusanli"(ST36)group,and there were significant differences in these groups(P<0.01).5.After the treatment,tumor tissue images and weight were compared,compare to the model group,the tumor tissue weight were much less in the "Shenshu"(BL23)group,the "Zusanli"(ST36)group and the acupoints combination group,and there were significant differences in these groups(P<0.01);compare to the "Shenshu"(BL23)group,the tumor tissue weight was heavy in the "Baihui"(DU20)group,and there were differences between the two groups(P<0.05);compare to the acupoints combination group,the tumor tissue weight were much heavier in the "Baihui"(DU20)group,the "Shenshu"(BL23)group and the "Zusanli"(ST36)group,and there were significant differences in these groups(P<0.01).The comparison of tumor weight difference was basically consistent with the tumor volume measured by vernier caliper.6.In the change of Helper T cells,compare to the control group,the quantitative value were significantly decreased in the model group,the "Baihui"(DU20)group,the "Shenshu"(BL23)group,the "Zusanli"(ST36)group,and the acupoints combination group,and there were significant differences in these groups(P<0.01);compare to the model group,the quantitative value was increased in the "Shenshu"(BL23)group,and there were differences between the two groups(P<0.05),the quantitative value was significantly increased in the acupoints combination group,and there were significantly differences between the two groups(P<0.01);compare to the "Shenshu"(BL23)group,the quantitative value were decreased in the "Baihui"(DU20)group and the "Zusanli"(ST36)group,and there were differences in these groups(P<0.05);compare to the acupoints combination group,the quantitative value were decreased in the "Baihui"(DU20)group and the "Zusanli"(ST36)group,and there were differences in these groups(P<0.05).In the change of Toxic T cells,compare to the control group,the quantitative value was significantly increased in the model group,the "Baihui"(DU20)group,the "Shenshu"(BL23)group,the "Zusanli"(ST36)group,and the acupoints combination group,and there were significant differences in these groups(P<0.01);compare to the model group,the quantitative value was significantly decreased in the "Baihui"(DU20)group,the "Shenshu"(BL23)group,the "Zusanli"(ST36)group and the acupoints combination group,and there were significant differences in these groups(P<0.01);compare to the "Shenshu"(BL23)group,the quantitative value were increased in the "Baihui"(DU20)group and the "Zusanli"(ST36)group,and there were differences in these groups(P<0.05);compare to the acupoints combination group,the quantitative value were significantly increased in the "Baihui"(DU20)group,the "Shenshu"(BL23)group and the "Zusanli"(ST36)group,and there were significant differences in these groups(P<0.01).In the change of NK cell,compare to the control group,the quantitative value were significantly increased in the model group,the "Baihui"(DU20)group,the "Shenshu"(BL23)group,the "Zusanli"(ST36)group,and the acupoints combination group,and there were significant differences in these groups(P<0.01);compare to the model group,the quantitative value was decreased in the "Shenshu"(BL23)group,and there were differences between the two groups(P<0.05),the quantitative value was significantly decreased in the acupoints combination group,and there were significant differences between the two groups(P<0.01);compare to the "Shenshu"(BL23)group,thequantitative value were increased in the "Baihui"(DU20)group and the "Zusanli"(ST36)group,and there were differences in these groups(P<0.05);compare to the acupoints combination group,the quantitative value were increased in the "Baihui"(DU20)group and the "Zusanli"(ST36)group,and there were differences in these groups(P<0.05).7.In the change of TNF-?,compare to the control group,the quantitative value were significantly increased in the model group,the "Baihui"(DU20)group,the "Shenshu"(BL23)group,the "Zusanli"(ST36)group,and the acupoints combination group,and there were significant differences in these groups(P<0.01);compare to the model group,the quantitative value were significantly decreased in the "Baihui"(DU20)group,the "Shenshu"(BL23)group,the "Zusanli"(ST36)group and the acupoints combination group,and there were significant differences in these groups(P<0.01);compare to the "Shenshu"(BL23)group,the quantitative value were increased in the "Baihui"(DU20)group and the "Zusanli"(ST36)group,and there were differences in these groups(P<0.05);compare to the acupoints combination group,the quantitative value were significantly increased in the "Baihui"(DU20)group,the "Shenshu"(BL23)group and the "Zusanli"(ST36)group,and there were significant differences in these groups(P<0.01).In the change of IL-6,compare to the control group,the quantitative value were significantly increased in the model group,the "Baihui"(DU20)group,the "Shenshu"(BL23)group,the "Zusanli"(ST36)group,and the acupoints combination group,and there were significant differences in these groups(P<0.01);compare to the model group,the quantitative value was decreased in the "Shenshu"(BL23)group,and there were differences between the two groups(P<0.05),the quantitative value was significantly decreased in the acupoints combination group,and there were significant differences between the two groups(P<0.01);compare to the "Shenshu"(BL23)group,the quantitative value were increased in the "Baihui"(DU20)group and the "Zusanli"(ST36)group,and there were differences in these groups(P<0.05);compare to the acupoints combination group,the quantitative value were significantly increased in the "Baihui"(DU20)group and the "Zusanli"(ST36)group,and there were significant differences in these groups(P<0.01),the quantitative value wasincreased in the "Shenshu"(BL23)group,and there was differences between the two groups(P<0.05).In the change of IFN-?,compare to the control group,the quantitative value were significantly increased in the model group,the "Baihui"(DU20)group,the "Shenshu"(BL23)group,the "Zusanli"(ST36)group,and the acupoints combination group,and there were significant differences in these groups(P<0.01);compare to the model group,the quantitative value was decreased in the acupoints combination group,and there was differences between the two groups(P<0.05);compare to the "Shenshu"(BL23)group,the quantitative value was increased in the "Baihui"(DU20)group,and there was differences between the two groups(P<0.05);compare to the acupoints combination group,the quantitative value were increased in the "Baihui"(DU20)group and the "Zusanli"(ST36)group,and there were differences in these groups(P<0.05).8.In the change of intestinal flora,At the phylum level,the significant changes of model group were mainly observed in Bacteroidetes,Firmicutes and Candidatus Saccharibacteria compared to control group.Acupuncture treatment works on delaying the decrease of Bacteroidetes and the increase of Firmicutes and Candidatus Saccharibacteria.Apparently,Alloprevotella(genus,P<0.05,by ANOVA)was the most abundant differential microbiota in the treatment group.The relative abundances of Clostridiales(order,P<0.05,by ANOVA),Unassigned(genus,P<0.05,by ANOVA),Lachnospiraceae(family,P<0.05,by ANOVA),Lactobacillus(genus,P<0.05,by ANOVA),Eisenbergiella(genus,P<0.05,by ANOVA),Firmicutes(phylum,P<0.05,by ANOVA),and Odoribacter(genus,P<0.05,by ANOVA)were significantly higher in the model group,while the relative abundances of Bacteroides(genus,P<0.05,by ANOVA),Unassigned(genus,P<0.05,by ANOVA),Bacteroidales(order,P<0.05,by ANOVA),Prevotellaceae(family,P<0.05,by ANOVA),and Prevotella(genus,P<0.05,by ANOVA)were significantly higher in the model group.Conclusion:1.In this study,in vivo imaging monitoring for acupuncture intervention of subcutaneous tumor bearing osteosarcoma was successfully established;2.The combination of acupoints and single acupuncture point had good intervention effect on various indexes of tumor model mice;3.The combination of acupoints is more effective than single point;Acupuncture at single point in the "Shenshu"(BL23)had the best effect;4.Acupuncture intervention was used to adjust the immune and intestinal microbiological indicators,the effect of tumor intervention can be achieved by regulating the immune homeostasis and intestinal homeostasis.5.Acupuncture can delay tumor growth in mice with osteosarcoma,regulates changes in immune responses and gut microbes in mice,it suggests that acupuncture provides the possibility for the holistic and systematic treatment of tumor. |
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