| Background:Gastric cancer(GC)is the highest incidence digestive system cancer in China.In recent years,chemotherapy is still one of the main treatment for patients with advanced stage.Clinically,it is found that the curative effect of patients will gradually inefficacy after the second and third chemotherapy cycles.The reason is that at this time,the chemoresistance of cancer cells to chemotherapy is increasing,which will eventually lead to chemoresistance,recurrence and metastasis.Studies have shown that after entering the tumor microenvironment(TME),chemotherapeutic drugs will not only kill cancer cells,but also damage surrounding stromal cells,such as cancer associated fibroblasts(CAFs),which will enter the senescence state,At the same time,it produces senescence associated secretory phenotype(SASP)to enhance the stemness of cancer cells and inhibit the function of immune cells in TME,so as to cause chemoresistance and eventually lead to chemoresistance.CAFs are the most stromal cells in TME,and their sources are mostly induced and transformed by normal fibroblasts,while there is too much TGF-β activating its SMAD pathway is an important mechanism to complete transformation.Compared with normal fibroblasts,CAFs have stronger secretory function and release more cancer promoting factors.When they are damaged by chemotherapy,the secretory spectrum of CAFs in senescence state changes,and the activated nuclear transcription factors κB(NF-κB)Under the regulation of p38MAPK pathway,SASP containing IL-6,IL-8 and WNT16B was produced.Wnt16B is activation of Wnt/β-catenin pathway can improve the stemness level of cancer cells and express the stemness phenotype.CD44 is one of the recognized cancer stem cell markers.Gastric cancer cells with high expression of CD44 have stronger tumorigenicity and migration ability,and CD44 is Wnt/β-catenin downstream target genes of catenin pathway.Similarly,ALDH1A3,a member of the aldehyde dehydrogenase family(ALDH),is a newly discovered stem cell marker in gastric cancer.It is highly expressed in gastric cancer cells resistant to 5fluorouracil(5-FU),the most commonly used chemotherapeutic drug in gastric cancer,and is also related to the activation of Wnt pathway.Stemness cancer cells have stronger invasive ability,but also strengthen immune evasion.They highly express human leukocyte Antigen-E(HLA-E)on the surface,and inhibiting the killing function of NK cells may be the key reason for their escape from clearance,continuous proliferation,and finally aggravate chemoresistance.Fuzheng Jiedu formula is an effective prescription for the treatment of gastric cancer in the oncology department of Guang’anmen Hospital,Chinese Academy of traditional Chinese medicine.Previous studies have shown that the combination of chemotherapy with Fuzheng Jiedu formula can improve the chemotherapy effect of patients.Based on the basic research,it is proved that Fuzheng Jiedu formula can inhibit the proliferation of cancer cells and down regulate the serum TGF-βlevel in of cancer bearing mice,inhibit cancer angiogenesis.Our recent studies have shown that DNA fragments produced by chemotherapy damage in cells activate cyclic guanosine monophosphate adenosine synthase(cGAS),which activates NF-κB.Release of from SASP activated cancer cells by Wnt/β-catenin pathway,enhance the intracellular level of cancer cells.The accumulation of catenin promotes the stemness of cancer cells.But does Fuzheng Jiedu formula regulate TGF-β/The formation of CAFs mediated by SMAD,interfering with the senescence and senescence secretion of CAFs,reducing the stemness phenotype of cancer cells and improving the killing ability of NK cells are not completely clear.Therefore,this study uses a combination of in vivo and in vitro experiments to verify the role of Fuzheng Jiedu formula in the formation of CAFs,and then the senescence regulation of CAFs,Then explore whether to interfere with the secretion of senescence CAFs and its effect on the Stemnessing of gastric cancer cells.In addition,observe the synergistic effect on NK cells killing Stemness cancer cells.From the perspective of "cancer cell-stromal cell-immune cell" interaction in TME,Further explore the influence of "CAFs formation-senescence secretion after chemotherapy-cancer cell stemnessing-inhibiting NK to avoid clearance" on the occurrence and development of chemoresistance and the intervention effect of Fuzheng Jiedu formula,so as to help solve the clinical bottleneck of chemoresistance of gastric cancer.Purpose:1.The CAFs model and the senescence CAFs secretion model were constructed.At the same time,the senescence conditioned medium(CM)was used to induce the stemness of cancer cells and the co culture system of cancer cells/NK cells.The intervention of Fuzheng Jiedu formula containing serum on TGF-β/SMADmediated CAFs formation pathway,p38MAPK mediated senescence secretion pathway,cancer cell stem phenotype formation pathway and NK cell disability formation pathway by observed in vitro.2.Construct the senescence CAFs/cancer cell mixed cancer bearing immune deficiency mouse model,observe the Fuzheng Jiedu formula to improve the therapeutic effect of chemotherapy in vivo,and verify the way to improve chemoresistance by regulating "CAFs formation-senescence secretion after chemotherapy-cancer cell stemness-inhibiting NK and avoiding clearance".Method:1.Construction of CAFs model and exploration of intervention mechanism of Fuzheng Jiedu formulaTGF-β1 was used in this experiment human embryonic lung fibroblasts(MRC-5)were induced as CAFs in vitro.CCK-8 was used to detect their proliferation ability,and cellular immunofluorescence was used to detect CAFs markers α-SMA and FAP,and the secretion of IL-6 and SDF-1α、TGF-β1 by CAFs were detected by ELISA.RTPCR was used to detect the effect of Fuzheng Jiedu formula on the SMAD mRNA of CAFs formation pathway,and Western blot was used to detect the SMAD2/3 pathway and FAP,α-SMA of Fuzheng Jiedu formula on CAFs.At the same time,the effect of Fuzheng Jiedu formula on the expression of was evaluated by regulating TGF-β/SMAD pathway in the formation of CAFs.2.Construction of senescence CAFs model and reach on the intervention mechanism of Fuzheng Jiedu formulaThis part of the experiment compares the senescent models of CAFs induced by 5-FU and hydrogen peroxide β-galactosidase staining confirmed that the model was successfully constructed,and CCK-8 method and annexin-V/PI double staining method were used to evaluate the stability of the model.After the senescent model induced by hydrogen peroxide was determined,Fuzheng Jiedu formula containing serum was used for intervention The proportion of senescent cells was detected by βgalactosidase staining,the expression of HLA-E in senescent cells was detected by flow cytometry,the expression of cyclin and p38 MAPK pathway were detected by RT-PCR and Western blot to evaluate the effect of Fuzheng Jiedu formula on the senescent of CAFs.3.Study on the intervention mechanism of Fuzheng Jiedu formula on the stemness of carcinogenic cells caused by senescence CAFs-CM.In this part of the experiment,CAFs senescent model was used to prepare conditioned medium(CM)to culture gastric cancer cells(MKN-45),and the proliferation ability of MKN-45(CM-MKN-45)was measured by CCK-8 method.The malignant behavior of CM-MKN-45 was evaluated by scratch test,plate clone formation test and Transwell invasion test.The expressions of SOX2,LGR-5,CD44 and ALDH1A3 related to stemness were detected by RT-PCR and Western blot,At the same time,Fuzheng Jiedu formula containing serum was used to intervene to evaluate the role of Fuzheng Jiedu recipe in inhibiting the formation of cancer cell stemness.4.Study on the mechanism of Fuzheng Jiedu formula improving the killing ability of NK cells to Stemness cancer cells induced by senescence CAFs-CMThis part of the experiment takes effector cells(NK-92 cells)and target cells(senescent CM-MKN-45)as the research objects.Firstly,the optimal intervention concentration and intervention effect of Fuzheng Jiedu formula containing serum on NK-92 are determined by CCK-8 method,and the optimal effect target ratio of the two cells is selected.Then,the killing activity of NK-92 cells is determined by lactate dehydrogenase(LDH)method,and the IFN-y of NK-92 cells is detected by ELISA method flow cytometry was used to detect the expression of CD 107a in NK-92 cells to observe the degranulation ability.In addition,the Transwell chamber co culture system was established to observe the effects of senescent CM-MKN-45 on the recognition receptor and functional receptor between senescent CM-MKN-45 and NK cells.The expression of surface activated MICA/B and inhibitory HLA-E receptor of senescent CM-MKN-45 and the expression of functional receptors NKG2A and NKG2D of NK92 cells were detected by flow cytometry.At the same time,the drug containing serum of Fuzheng Jiedu recipe was used to intervene to evaluate the role of Fuzheng Jiedu recipe in enhancing the killing of stemness gastric cancer cells by NK cells.5.Study on the intervention effect of Fuzheng Jiedu formula on senescence CAFs gastric cancer cell mixed inoculation mouse model and the regulation mechanism of NK function.This part of the experiment is to construct the cancer bearing mouse model by inoculating senescent CAFs and MKN-45 into CB17-SCID mice with only NK cells and monocytes in the immune system.The control group,simple Fuzheng Jiedu formula group(traditional Chinese medicine group),chemotherapy group and Fuzheng Jiedu formula+chemotherapy group(traditional Chinese medicine+chemotherapy group)are set to observe the effect of Fuzheng Jiedu formula on improving chemoresistance,and the tumor volume,tumor weight and spleen weight are measured,Tumor tissue immunohistochemical staining,ELIS A test of blood inflammatory factors,flow cytometry to detect the proportion of NK cells,and Western blot to detect the stemness index to verify the relevant mechanism.Results:1.Construction of CAFs model and exploration of intervention mechanism of Fuzheng Jiedu formula10ng/mL TGF-β1 intervene MRC-5 for 24 hours can induce the formation of CAFs,while Fuzheng Jiedu recipe can inhibit the formation of CAFs in TME and reduce its characteristic markers expression of α-SMA and FAP down regulated their characteristic TGF-β1,IL-6 and SDF-1α level of cancer promoting factor,its mechanism and inhibition of TGF-β1/SMAD2/3 pathway activation is related.2.Construction of senescence CAFs model and Study on the intervention mechanism of Fuzheng Jiedu formula60μM hydrogen peroxide intervention for 24 hours can build CAFs senescent model,while Fuzheng Jiedu recipe can inhibit the senescent of CAFs in TME.The mechanism is related to down regulating the expression of cell cycle p16 and p21.At the same time,Fuzheng Jiedu recipe inhibits the expression of p38MAPK pathway,and further prevents the occurrence of senescent,senescent related secretion and the formation of senescent microenvironment.3.Explore the intervention mechanism of Fuzheng Jiedu formula on the stemness of carcinogenic cells caused by senescence CAFs CM.Senescent CAFs-CM can promote the stemness of MKN-45 cells,show stronger ability of proliferation,migration and invasion,and express higher levels of stemness markers.Fuzheng Jiedu recipe can inhibit the senescent in the TME,CAFs-CM induced cancer cells to dry,and significantly reduce the proliferation,migration,invasion and other malignant behaviors of dry cancer cells.Its mechanism is related to the inhibition of Wnt/β-catenin,the most common downstream of is related to CD44,gastric cancer specific ALDH1A3 and 5-FU specific LGR-5 stemness expression.At the same time,it also reduces the expression of SOX2,a primary stemness marker outside this pathway,which shows that Fuzheng Jiedu recipe can reduce the stemness expression of cancer cells induced by various factors and prevent the formation of chemoresistance.4.Study on the mechanism of Fuzheng Jiedu formula improving the killing ability of NK cells to stemness cancer cells induced by senescence CAFs-CMNK-92 cells can kill MKN-45 gastric cancer cells,and the best effect target ratio is 5:1.The serum containing Fuzheng Jiedu formula can improve the killing efficiency of NK-92 cells,and the best intervention concentration is 10%;Senescent CAFs-CM environment can inhibit NK cells,and Fuzheng Jiedu formula can restore the killing ability of NK cells;NK cells can kill MKN-45 induced by senescent CAFs-CM.Fuzheng Jiedu recipe can comprehensively improve the direct cytotoxicity,anti-tumor factor secretion and degranulation killing ability of NK cells;Under the induction of senescent CAFs-CM,MKN-45 will highly express HLA-E binding NK cell inhibitory receptor NKG2A.Fuzheng Jiedu formula can inhibit this process and up regulate the killing of cancer cell MICA/B binding NK cell activating receptor NKG2D.5.Study on the intervention effect of Fuzheng Jiedu formula on senescence CAFs gastric cancer cell mixed inoculation mouse model and the regulation mechanism of NK function.The mixed subcutaneous inoculation of senescent CAFs and MKN-45 can form 100%tumor in CB17-SCID mice;Fuzheng Jiedu formula combined with chemotherapy can reduce the tumor weight of mixed tumor bearing mice and significantly reduce the tumor volume of mice.Compared with the chemotherapy group,it has a higher tumor inhibition rate.At the same time,it reduces the expression of two cancer cell stemness markers expression of ALDH1A3 and CD44.α-SMA inhibited the formation of CAFs.In addition,it also improved the proportion and function of NK cells to a certain extent,which confirmed that Fuzheng Jiedu formula could inhibit the stemness of cancer cells induced by CAFs in chemotherapy,enhance the function of NK cells and reduce the formation of chemoresistance.Conclusion:1.Fuzheng Jiedu formula has good antitumor and chemotherapy synergistic effects in vitro and in vivo,which can reduce the formation of CAFs and the degree of cancer cell stemness in tumor tissue,improve the proportion and function of NK cells,and reduce the occurrence of chemoresistance in some aspects.2.Fuzheng Jiedu formula can inhibit TGF-β/SMAD pathway mediates the formation of CAFs,regulates cell cycle and senescent related pathway p38 MAPK,inhibits CAFs,down regulates the dry formation of MKN-45 promoted by senescent CAFs-CM,and improves NK cytotoxicity,degranulation ability and IFN-y release level,regulate cancer cells and NK cells to receive ligands,and enhance the killing ability of dry MKN-45.3.The above comprehensive description shows that Fuzheng Jiedu formula can regulate the interaction of"cancer cells-stromal cells-immune cells" in TME,inhibit the pathway of "CAFs formation-senescent secretion after chemotherapy-cancer cell drying-inhibiting NK to avoid clearance",and effectively reduce the occurrence of chemoresistance. |
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