| Objective:As one of the major public health problems in the world,malignant tumors will greatly harm human health and will become the number one killer of humanity in the new century.Chemotherapy,as an effective method for tumor treatment,not only kills cancer cells effectively,but also has multiple adverse consequences.One of them is bone marrow suppression,which is mainly manifested in patients with peripheral blood white blood cells,red blood cells,platelets and even hemoglobin.In severe cases,it can cause all blood cells to decrease,hinder the treatment of the disease,and affect the basic life of patients.In this study,the effective extract of Schisandra chinensis Schisandrin A（JY-1）was used as a research object to study the protective effect of chemotherapeutic drugs（cyclophosphamide（CTX）,cisplatin（DDP））on bone marrow suppression,and to explore the improvement effect of JY-1 on bone marrow suppression caused by chemotherapy.The intestinal microecology is the entry point,and the possible mechanism of its resistance to bone marrow suppression is discussed in depth.Methods:1.JY-1 resistance to CTX,DDP induced bone marrow suppression animal experiments:Kunming mice were randomly divided into CON group,CTX&DDP group,JY1 group,CTX group,DDP group,JY1（CTX）group,JY1（DDP）group,each7 groups,7 groups in total.Among them,JY-1 group,JY1（CTX）group and JY1（DDP）group were given 60 mg/kg JY-1 by intragastric administration for 14 days.CTX&DDP group and JY1 group were intraperitoneally injected with 3 mg/kg DDP and 60mg/kg CTX;CTX group Intraperitoneal injection of 60 mg/kg CTX with the JY1（CTX）group;intraperitoneal injection of 3 mg/kg DDP in the DDP group and JY1（DDP）group for three consecutive days,taking materials every other day,through mice food intake,body weight changes and epididymal fat HE stain To evaluate the improvement of JY-1 on chemotherapy-induced malnutrition.Through blood test,bone marrow smear,femoral HE staining,bone marrow cell apoptosis,and cycle detection,the improvement effect of JY-1 on CTX&DDP,CTX,DDP induced bone marrow suppression was evaluated.2.JY1 resistance to bone marrow cytotoxicity test caused by chemotherapy drugs:Aseptically take mouse bone marrow,suspend in 96-well plate,add CTX and DDP respectively to make the final concentration of the two drugs 5μmol/L,10μmol/L,50μmol/L,100μmol/L to determine the concentration that causes the highest rate of inhibition of bone marrow cell activity.Bone marrow cells added with chemotherapy drugs were treated with JY-1 of 0.1μmol/L,1μmol/L,and 5μmol/L,respectively,and cell viability was measured by CCK-8.After clarifying the concentration and JY-1concentration at which the activity suppression rate of bone marrow cells is the highest,bone marrow cells are treated at this concentration to detect the apoptosis rate.3.Hematopoietic function and intestinal wall integrity testing after intestinal flora transplantation:experimental grouping:Kunming mice were randomly divided into CON group,CTX&DDP group,FMT-CM group,FMT-MC group,JY1 group,FMT-JY1 group.The hematopoietic function of mice was evaluated by detecting blood routine,bone marrow smear,HE staining of femur,apoptosis of bone marrow cells,and cycle.The integrity of the mouse intestinal barrier was evaluated by immunohistochemical detection of the mouse small intestine tight junction protein Claudin5,immunofluorescence detection of ZO1 expression and WB detection of the expression of inflammatory factors IL-1β,TNF-α,iNOS protein.4.JY1 improved immune system disorders caused by chemotherapy drugs through intestinal flora:aseptically remove feces in the rectum of mice,and perform 16srRNA sequencing of mouse intestinal microorganisms in Guangzhou Gidio to detect intestines of each group Road flora.After the peripheral blood of the mouse was lysed by erythrocytes,the immune status of the mice was evaluated by detecting the proportion of CD4 and CD8 cells in the blood cells of the mouse.5.Detection of serum inflammatory factor content and in vitro experiments of serum conditioned culture:ELISA kits were used to detect IL-1β,TNF-αinflammatory factors and promoting factors in the serum of CON,CTX&DDP,JY1,FMT-JY1 four groups of mice.Erythropoietin（EPO）content.Bone marrow cells were added to mouse serum from each experimental treatment group（CON,CTX&DDP,JY1,FMT-JY1）to detect the apoptosis,cycle and red blood cell colony forming ability of bone marrow cells.6.Bone marrow cells treated by IL-1β,TNF-αtreatment in vitro:Bone marrow cells cultured in a 96-well plate were treated with IL-1β,TNF-αwith different density,and CCK-8 reagent was added to determine cell viability.20ng/ml,50 ng/ml IL-1β,TNF-αwere used to treat suspended bone marrow cells and bone marrow cells in erythrocyte colony forming medium.Suspended cells were used for apoptosis and cycle detection,and red blood cell colony formation culture was used to observe Effect of inflammatory factors on colony forming ability.7.Statistical analysis method:The experimental data are expressed as mean±standard deviation,analyzed by 16.0 SPSS software,compared between two groups by t test,and comparison between multiple groups by single factor analysis of variance.#P<0.05,##P<0.01,###P<0.001,####P<0.0001,compared with the Control group;*P<0.05,**P<0.01,***P<0.001,****P<0.0001,compared with the MOD group;ns,P>0.05.Results:1.JY-1 can resiste malnutrition and bone marrow suppression induced by CTX and DDP:After CTX&DDP chemotherapy treatment,mice showed significant food intake and weight loss（P<0.05）,and epididymal fat atrophy significantly,and after modeling JY-1 administration can effectively improve the above-mentioned malnutrition caused by CTX&DDP（P<0.05）.At the same time,it can reduce blood image,decrease bone marrow nucleated cells,increase bone marrow cell apoptosis rate,and increase S-phase cells,all have effects（P<0.05）.Using CTX and DDP alone can also cause adverse reactions such as decreased blood image（P<0.05）and reduced bone marrow nucleated cells（P<0.05）in mice.The recovery of blood and bone marrow was observed when JY-1 was administered（P<0.05）.2.Effect of JY-1 on chemotherapy-induced bone marrow suppression in vitro:CTX has no obvious cytotoxicity（P>0.05）in the treatment of bone marrow cells in vitro.DDP showed significant bone marrow cytotoxicity at a concentration of 10μmol/L（P<0.05）,10μmol/L DDP bone marrow cells were treated with 1μmol/L JY-1,and the bone marrow cell apoptosis in the blank control group,DDP group,and JY1-DDP group was detected respectively.The results showed that JY1 was effective for chemotherapy drugs.There was no direct effect of the bone marrow cell damage（P>0.05）.4.Effect of two-way flora transplantation on hematopoietic function and intestinal wall integrity in mice:16srRNA sequencing results showed that compared with the blank group,the level of intestinal flora was significantly reduced in Bacteroides（P<0.05）Clostridia increased significantly（P<0.05）,and after transplantation of the flora,it can be seen that the flora of the FMT-MC group is closer to the model group,and the FMT-CM group is closer to the blank group.By examining the peripheral blood routine,it was found that the intestinal flora of chemotherapy mice will affect the white blood cells,red blood cells,platelets,lymphocytes and neutrophils of normal mice.No effect（P>0.05）,but the reduction of red blood cells caused by chemotherapy（P<0.05）is improved.Bone marrow smears and femoral HE staining also show the same rules.It can be seen that the intestinal flora affects the hematopoietic function of mice after chemotherapy.It plays a vital role.The expressions of Claudin5 and ZO1 in the model group were significantly reduced（P<0.05）.The two-way flora transplantation results also showed the same trend,indicating that passing the intestinal flora would not only affect the hematopoietic function of mice,but also affect the intestinal barrier.Play a role.5.JY1 improved myelosuppression caused by chemotherapy drugs through intestinal flora:PCOA analysis at the level of OTU classification,UPGMA cluster tree,species composition pie chart,and various levels of flora and species stacking charts all indicate the intestinal tract of mice after chemotherapy The flora is imbalanced,and the JY1group and the FMT-JY1 group have a flora structure closer to the blank group,which directly indicates that JY-1 does play a role in resisting bone marrow suppression through the intestinal flora.In the microbial composition,there are many different bacteria in the JY1 group and the model group.The most prominent are the thick wall bacteria,Fusobacterium and Bacteroides.JY-1 may play a role by adjusting these types of flora.Correlation analysis of all bacteria at the species level with white blood cells,red blood cells,platelets,lymphocytes,monocytes,neutrophils,and CD4/CD8revealed that Lactobacillus＿murinus was significantly positively correlated with lymphocytes and red blood cells（P<0.05）,it can be seen that the hematopoietic function can be improved by increasing the abundance of Lactobacillus＿murinus.By examining the expression of Claudin5 and Claudin ZO1 in the small intestine of mice,it was found that after JY-1 administration and colony transplantation,the damaged small intestinal barrier of mice was significantly repaired and the intestinal inflammation was reduced.The expression of factors IL-1β,TNF-α,and INOS（P<0.05）shows that JY-1 can improve the intestinal flora imbalance caused by chemotherapy and protect the damaged intestinal barrier through the intestinal flora.6.Effect of JY-1 on serum inflammatory factors and serum conditioned culture on bone marrow cells:JY-1 can effectively reduce the serum IL-1βand TNF-αinflammatory factors in mice（P<0.05）.The conditioned culture results showed that the mouse serum of the model group induced apoptosis of bone marrow cells in vitro（P<0.05）,and the serum of the two groups of mice,JY1 and FMT-JY1,could reduce the rate of apoptosis of bone marrow cells compared with the model group（P<0.05）,the cycle and erythrocyte colony formation results showed the same trend of results as in vivo experiments.7.Effects of IL-1βand TNF-αon bone marrow cells:Both IL-1βand TNF-αinduce apoptosis of bone marrow cells（P<0.05）,but IL-1βhas no significant effect on bone marrow cell cycle（P>0.05）,TNF-αcan lead to the increase of bone marrow cells in the S phase（P<0.05）,showing the same experimental result trend as in vivo experiments,and inhibit the formation of bone marrow red blood cell colonies（P<0.05）.It can be seen that in addition to chemotherapy itself,it can cause apoptosis of bone marrow cells and affect the proliferation and differentiation of erythroid progenitor cells.It can also destroy the intestinal mucosa through the intestinal flora,release harmful factors such as TNF-αinto the blood,and increase bone marrow suppression.Conclusion:1.JY-1 has anti-myelosuppressive effect caused by cisplatin,cyclophosphamide and their combination.2.JY-1 does not work by direct means,but by regulating the intestinal flora,resisting the small intestinal barrier damage caused by chemotherapy and the abnormal expression of small intestinal inflammatory factors,thereby inhibiting the inflammatory factors TNF-α,IL-1βRelease into the blood to achieve resistance to bone marrow suppression. |
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