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Objective To investigate the efficacy,safety,and associated mechanisms of injected ozonated saline in the treatment of VX2 tumors.Methods A total of 90 rabbits bearing VX2 tumors on their left hind legs were randomly divided into three groups.The control group (A) received normal saline,while groups B and C received 20 μg/mL and 40 μg/mL O3/O2 ozonated saline,respectively.Rabbits were anesthetized and 2 mL of blood was drawn directly from the heart to measure serum concentrations of interleukin (IL-6) and tumor necrosis factor (TNF-α).The skin covering the VX2 tumor was cut in each rabbit and the maximum and vertical diameters of the tumors were measured under direct visualization.Several milliliters of saline,saline pre-treated with 20 μg/mL O3/O2,or saline pre-treated with 40 μg/mL O3/O2 were directly injected into the tumors of groups A,B,and C,respectively (injection volume (milliliter) =1/2 volume of the tumor,V =1/2ab2).On days 4,8 and 12 following treatment,10 rabbits were randomly selected from each group for blood sample collection,and serum IL-6 and TNF-α were measured.The tumor growth rate was calculated by measuring the maximum and vertical diameters of the VX2 tumors under direct visualization.All selected rabbits were euthanized and the tumors,livers,and lungs were removed for pathological examination.The tumor necrosis rate was calculated by cutting the tumors into half along the longitudinal axis and measuring the maximum diameters of the intratumoral necrotic regions.Results The average tumor volume in the three groups increased to different degrees at each time point;however,the average tumor growth rates in groups B and C were substantially lower than that in group A,exhibiting a statistically significant difference.The difference in the tumor growth rate between group B and group C was not statistically significant.The serum concentrations of IL-6 and TNF-α increased in the three groups at each time point,with larger increases occurring in groups B and C;however,the greater increases did not reach statistical significance.Although the diameters of the necrotic areas were larger in both groups B and C than that in group A,significant differences in necrotic area diameters were only found when comparing groups A and C on days 4 and 12 following treatment.Conclusion Direct injection of different concentrations of ozonated saline into VX2 tumors significantly increased intratumoral necrosis and reduced the tumor growth rate.The associated mechanism may be partially mediated by IL-6 and TNF-α,as the serum concentrations of these molecules increased after the treatment.