Study On The Negative Regulatory Effect And Mechanism Of Biodegradable Magnesium Metal On Non-small Cell Lung Adenocarcinoma

Objective:Recently,the incidence rate and mortality of lung cancer are on the rise,becoming a major public health problem threatening human life and health.The most common histopathological classification of lung cancer is non-small cell lung adenocarcinoma.In recent years,minimally invasive interventional therapy for non-small cell lung cancer has developed a variety of treatment methods,such as perfusion chemotherapy,chemoembolization,ablation therapy,radioactive particle implantation technology,but there are still many problems,and scientific researchers need to innovate materials,technologies and methods to improve the accuracy and effectiveness of treatment.Relevant research shows that magnesium metal and magnesium base alloy materials have the function of selectively killing tumor cells and inhibiting tumor growth.In addition,their good biodegradability and biocompatibility make magnesium metal materials become potential biodegradable metal materials in the field of biomedicine and interventional therapy.As one of the important trace elements in human body,magnesium plays an important role in maintaining human health and normal life activities.It participates in energy metabolism and protein synthesis in the body,can maintain the stability of cell membrane,participate in DNA replication and play the role of antioxidation.Magnesium and magnesium base alloy materials show cytotoxicity to a variety of tumor cells,including osteosarcoma,colorectal cancer,gallbladder cancer,etc.,and have the ability to kill tumor,prevent recurrence,inhibit invasion and induce tumor cell apoptosis.Hedgehog signal pathway is generally activated and over-expressed in non-small cell lung adenocarcinoma.The up-regulation of the expression of this pathway can promote the transcription of cell cycle regulatory factors,accelerate mitosis,and significantly induce tumor cell proliferation;In addition,the abnormal activation of Hedgehog signaling pathway also involved in tumor cell invasion,migration,inhibition of apoptosis autophagy,and radio-chemotherapy resistance.From the point of pain and difficulty of clinical interventional treatment of lung cancer,this topic studies the inhibitory function and mechanism of biodegradable magnesium metal on non-small cell lung adenocarcinoma.The effect of biodegradable magnesium metal degradation products on the biological behavior of tumor cells（proliferation,migration,invasion,apoptosis and cell cycle）of non-small cell lung adenocarcinoma cells was proved by experiments,and the relationship between the degradation products and the Hedgehog signal pathway was explored,so as to explore the tumor treatment effect and mechanism of biodegradable magnesium metal on non-small cell lung adenocarcinoma.The research results of this project will provide theoretical basis and scientific basis for the application of biodegradable magnesium metal in the clinical interventional treatment of lung cancer.Methods:In this study,firstly,according to the national standard ISO10993-12,the magnesium powder was extracted with serum free medium or normal saline at the concentration of 0.2g/m L,and the p H value of the extraction solution was measured with a p H meter during the extraction process until the p H value remained stable,and then the culture medium containing magnesium extraction solution with saturated Mg²⁺concentration and normal saline were obtained,The concentration of Mg²⁺in the culture medium containing magnesium extract and the physiological saline containing magnesium extract was determined with the magnesium detection kit.Then dilute the culture medium containing magnesium extract and physiological saline 3 times,10 times,30 times and 100 times respectively,and measure the p H value of the culture medium containing magnesium extract and physiological saline with different Mg2+concentrations.Observe the proliferation of A549 cell line,H1975 cell line and HBE cell line through CCK-8 experiment,and select the concentration that has the strongest inhibitory effect on non-small cell lung adenocarcinoma and has less influence on normal tissue cells as the follow-up experiment concentration.In the cell experiment,CCK-8 experiment was used to study the inhibitory effect of magnesium-containing extract medium on the proliferation of non-small cell lung adenocarcinoma cells after 24h,48h and 72h of treatment.The neutral p H magnesium-containing extract medium and alkaline p H magnesium-containing extract medium were added respectively to study the effect of Mg2+and OH-on cell proliferation.In addition,the distribution of tumor cell cycle was detected by flow cytometry to study the inhibition of biodegradable magnesium on tumor cell cycle progression.Next,the invasion ability of tumor cells was studied by coating the Transwell cell with matrix glue in advance,and the culture medium and alkaline and neutral p H value magnesium extract medium were added to the lower cell respectively to study the inhibition effect of biodegradable magnesium metal on the invasion of non-small cell lung adenocarcinoma cells;At the same time,the scratch distance before and after drug treatment was compared through scratch test to study the inhibition of migration of non-small cell lung adenocarcinoma cells.Then,flow cytometry was used to observe the proportion of apoptotic cells and study the effect of biodegradable magnesium metal on apoptosis of non-small cell lung adenocarcinoma cells.For the research on the mechanism of action,firstly,the probe was loaded by the reactive oxygen detection kit,and the fluorescence intensity of the probe was detected by flow cytometry to determine the content of reactive oxygen species in tumor cells;Then the expression of Hedgehog signal pathway in tumor cells was studied by q RT-PCR,Western blot,immunohistochemistry,immunofluorescence and other technical means to verify the possible mechanism of the inhibition of biodegradable magnesium on non-small cell lung adenocarcinoma.Finally,the activation of the Hedgehog signal pathway was stimulated by adding pathway agonists.The expression of the Hedgehog signal pathway in tumor cells was studied by q RT-PCR and Western blot.The proliferation,cycle,invasion and migration of cells inhibited by the culture medium containing magnesium extract were reactivated and promoted by CCK-8experiment,cell cycle experiment,Transwell invasion experiment,scratch experiment,cell apoptosis experiment and reactive oxygen detection,The proportion of tumor cell apoptosis and the content of reactive oxygen species in cells were observed.The biodegradable magnesium metal can play a negative role in regulating non-small cell lung adenocarcinoma through the Hedgehog signal pathway.In animal experiments,after successful subcutaneous tumor implantation in nude mice,physiological saline containing magnesium extract was injected for 14 days.By comparing the size of tumor volume and tumor growth rate during treatment and at the end of treatment,the inhibitory effect of biodegradable magnesium metal on non-small cell lung adenocarcinoma in vivo environment was studied.Results:According to the national standard ISO10993-12,the biodegradable magnesium metal was extracted in serum-free medium and physiological saline for 72h to obtain the saturated Mg²⁺concentration of the magnesium-containing extract medium and the magnesium-containing extract physiological saline.The measured Mg²⁺concentration in the magnesium-containing extract medium was 0.72mmol/L,and the p H value was 12.22,and the Mg²⁺concentration in the magnesium-containing extract physiological saline was0.72mmol/L,and the p H value was 12.23.Then,dilute the Mg-containing extract medium with saturated Mg²⁺concentration by 3 times,10 times,30 times and 100 times respectively,and the p H values of the Mg-containing extract medium with different Mg²⁺concentration gradients are 10.45,8.91,7.82 and 7.34 respectively.Because there are two components of Mg²⁺and OH^-in the culture medium containing magnesium extract that may inhibit non-small cell lung adenocarcinoma,dilute hydrochloric acid titration is used to prepare the culture medium containing magnesium extract with different Mg2+concentrations at p H 7for experimental research.Through the CCK-8 experiment,observe the absorbance value of the culture medium containing magnesium extract with different concentrations of Mg²⁺with alkaline and neutral p H after treating non-small cell lung adenocarcinoma,and select the treatment concentration with the highest inhibition rate of tumor cells as0.72mmol/L.At this concentration,the inhibition rate of the culture medium containing magnesium extract on non-small cell lung adenocarcinoma is close to the half lethal dose,and then select this concentration as the experimental concentration.In the proliferation experiment,it was observed that the biodegradable magnesium metal and its degradation products Mg²⁺and OH^-had an inhibitory effect on the proliferation of non-small cell lung adenocarcinoma tumor cells,and the longer the duration of the culture medium containing magnesium extract,the higher the inhibition rate of tumor cells.The results of cell cycle experiments show that biodegradable magnesium metal and its degradation products Mg²⁺and OH^-have inhibitory effects on the cell cycle process of non-small cell lung adenocarcinoma,can block the tumor cells from G0/G1 phase to S phase,and also have certain inhibitory effects on the process from S phase to G2/M phase,thus affecting the cell division of tumor cells and inhibiting the cell proliferation of non-small cell lung adenocarcinoma.In the Transwell invasion test and scratch test,it was observed that the number of invasion and migration distance of tumor cells were significantly reduced after the treatment of the culture medium containing magnesium extract,indicating that the biodegradable magnesium metal and its degradation products Mg²⁺and OH^-had an inhibitory effect on the invasion and migration of non-small cell lung adenocarcinoma cells.The results of cell apoptosis experiment showed that biodegradable magnesium metal and its degradation products Mg²⁺and OH^-could promote the apoptosis of tumor cells and kill tumor cells.In animal experiments,after 14 days of treatment,compared with the nude mice injected with normal saline,the tumor volume of the nude mice injected with normal saline containing magnesium extract was significantly smaller,and during the treatment,the size and growth rate of the subcutaneous tumor of the nude mice injected with normal saline containing magnesium extract were lower than those of the nude mice injected with normal saline,indicating that the occurrence of non-small cell lung adenocarcinoma caused by biodegradable magnesium metal in vivo environment Development has an inhibitory effect.The experiment further studied the possible mechanism of the negative regulation of biodegradable magnesium metal on non-small cell lung adenocarcinoma,and found that the culture medium containing magnesium extract could inhibit the content of reactive oxygen species in tumor cells;At the same time,under both in vivo and in vitro conditions,the culture medium containing magnesium extract and normal saline can down-regulate the expression of proteins such as the Hedgehog signal pathway SHH,Ptch1,Smo,Gli1 in non-small cell lung adenocarcinoma cells;After the addition of exogenous recombinant SHH protein,the Hedgehog signal pathway in tumor cells was activated again,the expression of related m RNA and protein was up-regulated,the cell proliferation,cell cycle,cell invasion and migration ability inhibited by the magnesium extract medium was reactivated and promoted,the number of cell apoptosis was reduced,and the content of reactive oxygen species in cells was increased.It shows that biodegradable magnesium metal and its degradation products Mg²⁺and OH^-can achieve negative regulation of biological behavior of non-small cell lung adenocarcinoma by preventing oxidative stress damage in tumor cells,down-regulating the expression of Hedgehog signal pathway in tumor cells.Conclusion:1.Biodegradable magnesium metal has a negative regulatory effect on the biological behavior of non-small cell lung adenocarcinoma,which can effectively inhibit the occurrence and development of tumor cells by inhibiting the cell proliferation,cell cycle progression,cell invasion and migration,and promoting tumor cell apoptosis.2.Biodegradable magnesium metal can inhibit the content of reactive oxygen species in non-small cell lung adenocarcinoma cells,prevent oxidative stress damage,and down-regulate the expression of Hedgehog signal pathway in tumor cells,thus realizing the negative regulation of non-small cell lung adenocarcinoma.3.Biodegradable magnesium metal not only inhibits non-small cell lung adenocarcinoma,but also has little effect on normal tissues and cells,and has good biological safety.