| In recent years,tumor radiotherapy has developed rapidly.Radiotherapy can effectively kill tumor cells to inhibit the growth of tumor cells and improve the survival of patients.Even about 40%of tumor patients can be completely cured by this.Clinical studies have shown that the level of high mobility group protein B1（HMGB1）in tumor patients is significantly increased after radiotherapy,and high level of HMGB1 is significantly related to poor prognosis of patients.Tumor cells were damaged and died and released a large amount of HMGB1 in tumor microenvironment after radiotherapy.As one of the important component in tumor microenvironment,HMGB1 can significantly promote the proliferation,invasion and metastasis of tumor cells.And there is no clinically effective means to control the recurrence and metastasis of tumor cells.HMGB1 is a highly conserved non-histone nucleoprotein with a variety of receptor binding regions on its molecule,which can interact with heparin,advanced glycation end products（RAGE）,toll-like receptor 4（TLR4）and others to play different roles.TLR4 is the main receptor for the innate immune system to recognize pathogenic microorganisms and plays a key role in the innate immune response.Studies have shown that the binding of HMGB1 to TLR4 on the tumor cell membrane can promote the proliferation of tumor cells,as well as metastasis and angiogenesis around tumor tissues.HMGB1 can activate dendritic cells（DCs）in the tumor microenvironment,and induce the secretion of interferon-γ by promoting the activation of CD8+T cells mediated by DCs.HMGB1 directly activate the Thl polarization of T cells and enhance T cell infiltration.Studies have found that low-dose radiotherapy induces the conversion of macrophages to the anti-tumor M1 type,while HMGB1 promotes the conversion of macrophages to the tumor-promoting M2 type.MMP-9 enters the blood vessel by degrading and adjusting the cell matrix into the blood vessel.HMGB1 activates the MAPK/MyD88 and p38/Nrf2 signaling pathways downstream of TLR4,which can up-regulate the transcription and translation of MMPs,thereby promoting tumor development.Transforming growth factor-β（TGF-β）is responsible for initiating the epithelial-mesenchymal transition process of cells.Studies have reported that HMGB1 activates the signal pathway mediated by TLR4,induces the release of pro-inflammatory cytokines IL-1b and IL-6 and recruits myeloid-derived suppressor cells.Myeloid-derived cells inhibit the release of TGF-β1,increasing the invasiveness of tumor cells.Studies have found that heparin has a high affinity with HMGB 1.After binding to the 6-12th amino acid sequence of the N-terminal of HMGB 1,heparin can change the two-dimensional structure of HMGB1,thereby,interferes the binding of HMGB1 with RAGE.Therefore,it can be inferred that the binding of heparin to HMGB 1 can also interfere and inhibit the binding of the latter to TLR4 and the mediated tumor growth and invasion effects.This may help cope with tumor recurrence and metastasis after radiotherapy.But the strong anticoagulant activity of heparin also determines the risk of bleeding in its application.Therefore,when developing HMGB1 inhibitors,heparin needs to be derivatized to reduce its anticoagulant activity while retaining its high affinity activity with HMGB1.In the early stage of this research group,heparin was treated by N-desulfation/peracetylation derivatization to obtain N-desulfated acetylated heparin derivative（NDSAH）with low anticoagulant activity.Studies have found that NDSAH protects both vascular endothelial cells and intestinal epithelial cells glycocalyx by inhibiting the up-regulation of tumor necrosis factor-α（TNF-α）,IL-6 and matrix metalloproteinas-9（MMP-9）,thereby improving the survival rate of septic mice.In this project,heparin sodium was derivatized to prepare NDSAH,and its structure was confirmed by nuclear magnetic resonance and its anticoagulant activity was investigated.At the molecular level,the affinity of heparin sodium,NDSAH,HS,three low molecular weight heparins（nadroparin calcium,enoxaparin sodium,dalteparin sodium）,dipotassium glycyrrhizinate（HMGB1 inhibitor）and TLR4 to HMGB1 were investigated by surface plasmon resonance（SPR）technology.And the binding ability of above analytes to HMGB1 compete with TLR4 was tested.Three molecules with high affinity to HMGB1,heparin sodium,NDSAH and HS,were screened out.At the cellular level,the effect of radiotherapy on the proliferation and release of HMGB1 in 4T1 tumor cells were examined,and then the ability of heparin sodium,NDSAH and HS to antagonize the HMGB1 on promoting the proliferation and metastasis of 4T1 tumor cells were investigated.At the animal level,radiotherapy and NDSAH or other analytes were combined in 4T1 tumor-bearing mice,and the tumor tissue weight,the levels of HMGB1,MMP-9 and TGF-β in tumor tissues and plasma,the number of IFN-y+CD8+T cells,M1 and M2 macrophages in tumor microenvironment were investigated to explore the effect of NDSAH combined with radiotherapy to antagonize HMGB1 to promote tumor cell proliferation and migration.The main results and conclusions of this research are:1.Preparation of NDSAH and detection of its anticoagulant potency1.1 Preparation of NDSAH and confirmation of its structureIn this project,the N-sulfate group of heparin sodium was selectively desorbed in the reaction of pyridine and DMSO/MeOH,and N-acetylation of N-desulfated heparin derivative was carried out in the presence of NaHCO3/MeOH and acetic anhydride,finally,NDSAH was obtained,the NMR results were in line with expectations.1.2 Anticoagulant potency test of NDSAHThe anticoagulant titer of NDSAH and HS was detected by goat plasma method,and the results were:NDSAH 0.39IU/mg,HS 1.94IU/mg.The anticoagulant potency of NDSAH is much lower than that of heparin sodium（200IU/mg）,which indicates that NDSAH is a non-anticoagulant heparin derivative and has no risk of bleeding.2.The affinity of NDSAH and other analytes with HMGB1 and the ability to antagonize the binding of HMGB1/TLR42.1 Study on the affinity of NDSAH and other analytes with HMGB1The SPR was used to investigate the affinity of heparin derivatives,HS and dipotassium glycyrrhizinate with HMGB1.The results showed that the affinity of the above analytes with HMGB1 is in the order of:heparin sodium>NDSAH>HS>dipotassium glycyrrhizinate>TLR4>dalteparin sodium>enoxaparin sodium>nadroparin calcium,which indicates that the molecular weight and the degree of sulfation and N-acetylation of heparin derivatives all affect there affinity with HMGB1.2.2 Investigation on the antagonistic effect of NDSAH and others on the affinity binding of HMGB1 and TLR4SPR was used to detect the ability of heparin derivatives,HS and dipotassium glycyrrhizinate to antagonize the binding of HMGB1/TLR4 under Dual injection mode.The results showed that heparin sodium,NDSAH and HS can antagonize the combination of TLR4 with HMGB1,while the three low molecular weight heparins--dalteparin sodium,nadroparin calcium and enoxaparin sodium cannot.This indicates that the ability of heparin sodium and others to compete with TLR4 for binding to HMGB1 depends on there affinity with HMGB1.Heparin derivatives whose affinity with HMGB1 is higher than that of HMGB1 with TLR4 can antagonize the binding of HMGB1/TLR4,but not vice versa.3-Effects of NDSAH,heparin sodium and HS on the proliferation and Migration of 4T1 tumor cells treated with HMGB13.1 The effect of X-ray on the proliferation of 4T1 cells4T1 cells were irradiated with 0Gy,6Gy or 12Gy X-rays respectively,and the cell proliferation rate was measured with CCK8 at 24h,48h and 72h after irradiation.The results showed that the cell proliferation rate of 4T1 cells after 24h,48h and 72h exposed to 6Gy or 12Gy X-rays were significantly decreased than that of the 0Gy group（**P<0.01,***P<0.001）.Compared with the cell proliferation rates at 24h after irradiation,the cell proliferation rates at 48h were further reduced（6Gy,#P<0.05;12Gy,###P<0.001）.Compared with the cell proliferation rates at 24h and 48h after irradiation,the cell proliferation rates at 72h continued to decrease（24h and 48h,###P<0.001）.The above results indicate that radiotherapy affects the proliferation of 4T1 cells.With the passage of time after irradiation,the cell proliferation ability continues to decrease,and the greater the dose of X-rays,the lower the potential for cell proliferation,which indicating that the effects of X-rays on cell proliferation have a dose dependent.3.2 Determination of HMGB1 levels in 4T1 cells culture supernatant after irradiationELISA was used to investigate the levels of HMGB1 in the cell culture supernatant after different does of irradiation applyed to 4T1 cells.The results showed that,compared with the 0Gy group,after 6Gy or 12Gy X-rays irradiated the cells for 24h,48h and 72h,the levels of HMGB1 in the cell supernatant were significantly increased（24h,**P<0.01,***P<0.001;48h,#P<0.05,*P<0.05;72h,**P<0.01,***P<0.001）.And at 24h and 72h after irradiation,the concentrations of HMGB1 in the cell supernatant after 12Gy irradiation were significantly greater than that in the cell supernatant after 6Gy irradiation（24h,#P<0.05;72h,###P<0.001）.Compared with 24h after irradiation,there were no significant differences in the amounts of HMGB1 released from 4T1 cells at 48h and 72h after each dose of irradiation（P>0.05）.The above results indicate that X-rays affect the levels of HMGB1 released by 4T1 cells in a dose-dependent manner.As the dose of X-rays increases,the levels of HMGB1 released by cells increases.After each dose of X-rays irradiated 4T1 cells for 24 hours,the concentrations of HMGB1 released by 4T1 cells no longer increased.3.3 Effects of different concentrations of HMGB1 on the proliferation of 4T1 cellsAfter 4T1 cells were treated with 125ng/mL,250ng/mL and 500ng/mL HMGB1,CCK8 was used to detect the cell proliferation at 24h and 48h.The results showed that at 24h,the cell proliferation rates of 250ng/mL HMGB1 group and 500ng/mL HMGB1 group were significantly increased compared with the blank group（**P<0.01,***P<0.001）,and the cell proliferation rate of the 500ng/mL HMGB1 group was greater than that of the 250ng/mL HMGB1 group.At 48h,compared with the blank group,different concentrations of HMGB 1 significantly promoted the proliferation of 4T1 cells（***P<0.001）.At 24h and 48h,there were no significant differences in the proliferation ability of 4T1 cells between the different groups（P>0.05）.The above experimental results show that HMGB1 can promote the proliferation of 4T1 cells.When the concentration of HMGB 1 is 500ng/mL and the treated time is 48h,the proliferation effect of HMGB 1 on cells is highest.3.4 Effects of NDSAH,heparin sodium and HS on the proliferation of 4T1 cellsAfter treated with 62.5μg/mL,125μg/mL,250μg/mL and 500μg/mL NDSAH,heparin sodium or HS for 24h,CCK8 was used to detected the proliferation of 4T1 cells.The results showed that,compared with the blank group,different concentrations of NDSAH,heparin sodium or HS significantly inhibited the proliferation of 4T1 cells（NDSAH,**P<0.01,***P<0.001;heparin sodium,**P<0.01,***P<0.001;HS,***P<0.001）.However,the inhibitory effects of NDSAH,heparin sodium or HS on the proliferation of 4T1 cells are not concentration-dependent.3.5 Study on the inhibitory effect of NDSAH and other analytes on the proliferation of 4T1 cells induced by HMGB1After different concentrations of NDSAH,heparin sodium or HS treated with 4T1 cells,CCK8 was used to detect the proliferation of 4T1 cells.The results showed that,compared with the blank group,the cell proliferation rate of the HMGB1 group was significantly increased（##P<0.01）.Compared with the HMGB1 group,after 4T1 cells were treated with different concentrations（62.5μg/mL,250μg/mL and 500μg/mL）of NDSAH,heparin sodium or HS,the cell proliferation rates were significantly reduced（NDSAH,***P<0.001;heparin sodium,***P<0.001;HS,***P<0.001,**P<0.01）.And they inhibit the proliferation-promoting effect of HMGB1 in a concentration-dependent manner.The ability of NDSAH,heparin sodium and HS to antagonize the pro-proliferation effect of HMGB 1 is:heparin sodium>NDSAH≈HS.Compared with the HMGB1 group,the cell proliferation rates of the glycyrrhizic acid group（HMGB1 inhibitor）and the TLR4-IN-C34 group（TLR4 inhibitor）were significantly reduced（***P<0.001）.The above results indicate that HMGB1 can promote the proliferation of 4T1 cells,and NDSAH,heparin sodium and HS all antagonize the proliferation effect of HMGB1 in a concentration-dependent manner,which may be related to the HMGB1/TLR4 signaling pathway.3.6 Study on the inhibitory effects of NDSAH and other analytes on the migration of 4T1 cells promoted by HMGB1Wound healing experiment was used to observe the inhibitory effects of different concentrations（62.5μg/mL,250μg/mL and 500μg/mL）of NDSAH,heparin sodium and HS on the effect of HMGB1 on promoting cell migration at different times（24h and 48h）.The results showed that,compared with the blank group,the wound healing rates of cells in the HMGB1 group were significantly increased（24h and 48h,###P<0.001）.Compared with the HMGB1 group,the wound healing rates of 4T1 cells at different concentrations（62.5μg/mL,250μg/mL and 500μg/mL）of heparin sodium or HS were significantly reduced after 24h and 48h（*P<0.05,**P<0.01,***P<0.001）.Compared with the HMGB1 group,the wound healing rates of 500μg/mL NDSAH group were significantly reduced（24h and 48h,*P<0.05）,and 62.5μg/mL and 250μg/mL NDSAH could not inhibit the cell migration promotion effect of HMGB1.The ability of NDSAH,heparin sodium and HS to inhibit HMGB1 on promoting cell migration is as follows:heparin ≈HS>NDSAH.Compared with the HMGB1 group,the wound healing rates of cells in the glycyrrhizic acid group and TLR4-IN-C34 group were significantly decreased（*P<0.05）.The above results indicate that HMGB1 can promote the migration of 4T1 cells.And NDSAH,heparin sodium and HS can inhibit the pro-migration effect of HMGB1.This inhibitory effect may be achieved by targeting the HMGB1/TLR4 signaling pathway.4.Effects of NDSAH and other analytes on tumor microenvironment and tumor growth in 4T1 tumor-bearing mice after radiotherapy4.1 Detection of HMGB1,MMP-9 and TGF-β in plasma in 4T1 tumor-bearing mice after NDSAH or other analytes combined with radiotherapyIn this experiment,the ELISA method was used for detection.The results showed that,compared with the normal saline group,the levels of HMGB1,MMP-9 and TGF-β in plasma in the X-ray group were significantly increased（HMGB1,MMP-9,###P<0.001;TGF-β,#P<0.05）.Compared with the X-ray group,combined radiotherapy with NDSAH,heparin sodium or HS can reduce the levels of HMGB1（***P<0.001,***P<0.01,***P<0.001）,MMP-9（***P<0.001）and TGF-β（*P<0.05,**P<0.01）in plasma.In addition,compared with radiotherapy alone,glycyrrhizic acid or TLR4-IN-C34 combined with radiotherapy can significantly decreased the levels of HMGB1（***P<0.001）,MMP-9（***P<0.001）and TGF-β（*P<0.05,**P<0.01）in plasma.The above results indicate that radiotherapy can promote the release of HMGB1,MMP-9 and TGF-β.NDSAH,heparin sodium and HS can reduce the levels of MMP-9 and TGF-β in plasma after radiotherapy by inhibiting the release of HMGB1.4.2 Detection of HMGB1,MMP-9 and TGF-β in tumor tissues of 4T1 tumor-bearing mice after NDSAH or other analytes combined with radiotherapyIn this experiment,the immunohistochemical was used for detection.The results showed that,compared with the normal saline group,the levels of HMGB1（##P<0.01）,MMP-9（#P<0.05）and TGF-β（#P<0.05）in the tumor tissues of the mice were increased after radiotherapy.Compared with the X-ray group,after combined radiotherapy with NDSAH,heparin sodium,HS,glycyrrhizic acid or TLR4-IN-C34,the levels of HMGB1（**P<0.01,*P<0.05）,MMP-9（***P<0.001;**P<0.01）and TGF-β（*P<0.05,**P<0.01）were significantly reduced.The above results indicate that NDSAH,heparin sodium and HS can reduce the levels of HMGB1,and further reduce the levels of MMP-9 and TGF-β in tumor tissues after radiotherapy.4.3 Detection of some immune-related cells in tumor tissues of 4T1 tumor-bearing mice after NDSAH or other analytes combined with radiotherapyIn this experiment,the immunofluorescence double-labeling method was used for detection.The results showed that,compared with the normal saline group,there were a large number of IFN-γ+CD8+T cells in the tumor tissue after radiotherapy.Compared with the X-ray group,the number of IFN-y+CD8+T cells in the tumor tissue of mice were decreased after NDSAH,heparin sodium,HS,TLR4-IN-C34 or glycyrrhizic acid combined with radiotherapy.The detection of macrophages showed that,compared with the normal saline group,the number of M1 macrophages in the tumor tissue of the X-ray group was increased,and the number of M2 macrophages was decreased.Compared with the X-ray group,the number of M1 macrophages in mouse tumor tissues was increased after NDSAH,heparin sodium,HS,glycyrrhizic acid or TLR4-IN-C34 combined with radiotherapy,and the number of M2 macrophages was decreased.The above results indicate that NDSAH and other analytes can inhibit the infiltration of IFN-γ+CD8+T cells into tumor microenvironment after radiotherapy,and promote the differentiation of macrophages into M1 macrophages with anti-tumor activity.4.4 Effects of NDSAH and others on the weight of tumor tissue in 4T1 tumor-bearing mice after radiotherapyThe tumor tissue was dissected and weighed after NDSAH,heparin sodium or HS combined with radiotherapy.The results showed that,compared with the normal saline group,the tumor weight of the mice in the X-ray group was significantly reduced（*P<0.05）.Compared with the X-ray group,the tumor tissue weight of the mice in the other combination treatment groups were significantly reduced（#P<0.05）except for glycyrrhizic acid（P>0.05）.The results indicate that radiotherapy can inhibit the proliferation of tumor cells.NDSAH,heparin sodium and HS can further inhibit the growth of tumor tissue after radiotherapy,which may be related to NDSAH’s antagonism of the combination of HMGB1 and TLR4.5.ConclusionThe non-anticoagulant heparin derivative NDSAH can reduce the release level of HMGB1 after radiotherapy,and antagonizes the HMGB1/TLR4 signaling pathway.So it can reduce the levels of MMP-9,TGF-β and promote the differentiation of macrophages into M1 type.Thereby,NDSAH inhibits the effect of HMGB1 on the proliferation and migration of tumor cells after radiotherapy.NDSAH may reduce the risk of tumor cell recurrence and metastasis after radiotherapy,providing a new option for combination therapy of tumor radiotherapy. |
PDF Full Text Request