Effects Of Pinus Massoniana Needle Extract On Cytotoxicity Of 16HBE Induced By Cadmium Chloride

Backgrouds:Cadmium（Cd）and its compounds are important industrial toxicants and environmental pollutants.They are classified as Class I carcinogens by the International Agency for Research on Cancer（IARC）and are used by the American Toxic Committee（ATSDR）and the US Environmental Protection Agency（EPA）.)Listed as the 7th toxic substance that threatened human health.Inhalation of cadmium into the body through inhalation of cadmium polluted air or ingestion of cadmium-contaminated water sources and foods can easily lead to kidney damage,liver damage,respiratory diseases,nervous system disorders,skeletal system damage,and even related tumors.However,the specific molecular mechanism of cadmium-induced cell and organ damage has not yet been fully elucidated,and there is also lack of ideal and safe prevention and control measures.For the mechanism study of cadmium poisoning,the relationship between cadmium toxicity and oxidative damage is the focus of most researchers and has made some progress.Studies have shown that cadmium can indirectly cause oxidative stress caused by the imbalance of redox state of cells through a variety of ways and approaches.Our previous studies showed that pine needles from Pinus massoniana have good anti-oxidant efficacy and were granted national invention patent（Patent No.ZL.201410568707.2）.This study intends to create an interventional model of human bronchial epithelial cells（16HBE）in vitro with different cadmium toxicities under different treatment conditions of Pinus massoniana pine needles.A new way of thinking is to investigate the effect of Pinus massoniana pine needle extract on the cytotoxicity of 16HBE induced by cadmium chloride.Objectives:2.1 Preparation of Pinus massoniana pine needle extract using ethanol extraction method,testing its total antioxidant activity to ensure that the pine needle extract used in subsequent experiments has good antioxidant activity.CCK8 cell activity assay Determined the cytotoxicity of Pinus massoniana pine needle extract and IC₅₀ of Cd Cl₂ to determine the experimental concentration of pine needle extract and Cd Cl₂in the prevention and antagonism of pine needle extract on Cd-induced 16HBE cytotoxicity.2.2 Establish 16HBE cell cadmium toxicity Pinus massoniana pine needle extract prevention model,through 16HBE cell survival rate,oxidative stress related enzymes MDA,LDH,SOD content,apoptosis rate,apoptosis-related genes Bax,Bcl-2,c-myc The detection of m RNA expression levels of DNA repair genes h OGG1 and h MSH2was conducted to investigate the preventive effect of Pinus massoniana pine needle extract on cadmium induced 16HBE cytotoxicity.2.3 Establish 16HBE cell cadmium toxicity Pinus massoniana pine needle extract antagonistic model,through 16HBE cell survival rate,oxidative stress related enzymes MDA,LDH,SOD content,apoptosis rate,apoptosis-related genes Bax,Bcl-2,c-myc The expression levels of DNA repair genes h OGG1 and h MSH2 were examined to investigate the antagonism of Pinus massoniana pine needle extract against cadmium induced 16HBE cytotoxicity.Methods:3.1 Based on the country’s authorized patent formula,the anti-oxidant active substances in pine needles of Pinus massoniana were extracted at a temperature of70.1°C,44%ethanol at a feed-liquid ratio of 1:37 and a time of 72 minutes under light conditions.Petroleum ether,ethyl acetate and n-butanol were used to extract the Pinus massoniana pine needle extract.The total antioxidant activity was measured by T-AOC kit.CCK8 cell activity assay was used to determine the effect of Pinus massoniana pine needle extract and Cd Cl₂ on the proliferation of 16HBE cells.The cytotoxicity of Pinus massoniana pine needle extract and IC₅₀ of Cd Cl₂ were determined.3.2 Preventive Models Five experimental groups were set up as control group,Cd Cl₂ alone treatment group,1ug/ml PMNE group,100ug/ml PMNE group,and500ug/ml PMNE group.Each PMNE group was pretreated with 16HBE cells for 6hours.25μmol/L Cd Cl₂ cultured cells to 24h,48h,72h.The antagonistic model was set up in five experimental groups:control group,25μmol/L Cd Cl₂ alone treatment group,1ug/ml PMNE combined with 25μmol/L Cd Cl₂ group,100ug/ml PMNE combined with 25μmol/L Cd Cl₂ group,500ug/m combined with 25μmol/L In the Cd Cl₂ PMNE group,the cells were cultured for 24 hours,48 hours,and 72 hours.3.3 The survival rate of 16HBE cells in each experimental group was detected by CCK8 method,malondialdehyde（MDA）content in 16HBE cells was detected by TBA method,and the content of superoxide dismutase（SOD）in 16HBE cells was detected by WST-1 method.Lactate dehydrogenase（LDH）content in liquid,Annexin V-FITC/PI method was used to detect the apoptosis rate of 16HBE cells,q PCR was used to detect apoptosis-related genes Bax,Bcl-2,c-myc,and DNA repair genes h OGG1,h MSH2 m RNA To investigate the prevention and antagonism of the cytotoxicity of 16HBE induced by cadmium in Pinus massoniana pine needle extract.Results:4.1 The T-AOC kit detected total antioxidant capacity of pine needles of Pinus massoniana was 176.3 U/ml.The results of CCK8 cell activity assay showed that the survival rate of 16HBE cells at 24h,48h,and 72h in the concentrations of Pinus massoniana pine needle extract groups was not statistically different from that of the control group（P>0.05）,indicating that the pine needle extracts at each concentration did not cause 16HBE cell death.The IC₅₀ of 24 h Cd Cl₂ was 26.48μmol/L,the IC₅₀of 48 h Cd Cl₂was 24.20μmol/L,and the IC₅₀ of 72 h Cd Cl₂ was 15.59μmol/L.4.2 The preventive experiment of cadmium-induced 16HBE cells extracted from Pinus massoniana pine needles showed that the cell survival rate of 16HBE cells treated with pine needles at 24h,48h,and 72h was higher than that treated with cadmium chloride alone（P<0.01）.The contents of kinases LDH and MDA decreased（P<0.05）,SOD levels of antioxidative enzymes increased（P<0.05）,and the relative expression levels of apoptosis-related genes Bax and c-myc decreased（P<0.05）.Anti-apoptotic gene Bcl-2,The relative expression levels of DNA repair genes h OGG1 and h MSH2 were increased（P<0.05）,and the apoptosis rate was decreased（P<0.05）.4.3 Antagonistic test of Pinus massoniana pine needle extract against cadmium16HBE cells showed that LDH and MDA content of oxidative kinases were decreased in 16HBE cells treated with pine needles combined with cadmium chloride at 24h,48h,and 72h compared with 16HBE cells treated with cadmium chloride alone（P<0.05）,SOD content of antioxidative enzymes increased（P<0.05）,relative expression levels of apoptosis-related genes Bax and c-myc decreased（P<0.05）,anti-apoptosis gene Bcl-2,DNA repair gene h OGG1 The relative expression of h MSH2 gene was increased（P<0.05）,and the apoptosis rate was decreased（P<0.05）.4.4 Prevention and Antagonistic Experiments The concentration of 16HBE cells in the pine needle treated group at each time point was higher than that in the antagonistic experiment corresponding concentration group（P<0.05）.The concentration of pine needles at the 24h and 72h concentrations was the best at all time points.The apoptosis rate of group 16HBE cells was lower than that of the corresponding concentration treatment group（P<0.05）.In the 48h prophylactic experiment,the apoptosis rate of the high-concentration pine needle treated group was lower than that of the corresponding concentration treatment group（P<0.01）.Conclusions:The antagonistic and preventive effects of Pinus massoniana pine needle extracts on cadmium cytotoxicity showed that the survival rate of Cdxin 16HBE cells increased,the apoptosis rate decreased,the content of oxidative kinases LDH and MDA decreased,and the content of antioxidant enzymes SOD increased.The expression of apoptosis-related genes Bax and c-myc was down-regulated and the expression of anti-apoptosis gene Bcl-2 and DNA repair genes h OGG1 and h MSH2were up-regulated,and preventive effect can more effectively improve the survival rate of 16HBE cells suggesting that this pine needle extract has certain antagonistic and preventive effects on Cd-induced 16HBE cytotoxicity.The mechanism may be related to the antioxidation function of Pinus massoniana pine needle extract,inhibition of cell apoptosis and repair of cell damage.