| Chloroquine is a basic public health system essential drug widely used in the prevention and treatment of malaria and the treatment of autoimmune diseases.Chloroquine also has a certain antiviral effect and is also a commonly used autophagy inhibitor.Recently,it has attracted much attention because of its controversial role in the treatment of new coronary pneumonia virus.However,relevant research on chloroquine in aquatic organisms is still very limited.At present,studies on the effects and mechanisms of chloroquine on different organs of fish have not been carried out.This situation poses a huge obstacle to a comprehensive understanding of the autophagic response mechanism of fish to chloroquine and the potential environmental threat of chloroquine in water.In this study,the adult Danio rerio is used as the object to explore the effect of different concentrations of chloroquine on the behavior and survival rate of Danio rerio,and the 96-hour LC50 is determined.By comparing the effects of different concentrations of chloroquine on the submicroscopic structure of gill,spleen,testis and ovarian tissue,and by a comparative study of the effects of LC3/BECN1 and Cx43 on three concentrations of chloroquine in 13 tissues of Danio rerio included gill,liver,spleen,stomach,intestine,kidney,heart,muscle,skin,eye,brain,testis and ovary.Among them,it also includes the fastest response time,the peak expression time and the increase or decrease of expression.Reveal the response of different tissues to gradient concentration of chloroquine,and obtain the optimal target tissue and the corresponding optimal detection time.Therefore,the mechanism of autophagy response of Danio rerio to chloroquine is elucidated from the behavioral,survival,histological and molecular biology levels.This study provides important basic data for further research on the impact of chloroquine on fish,and provides a scientific basis for clarifying the potential environmental impact of chloroquine in water.The main findings are as follows:1.The effect of different concentrations of chloroquine on the survival rate of Danio rerio and the 96 h LC50Through the high(2.5mg/mL),medium(1.0mg/mL)and low(0.1mg/mL)chloroquine treatment experiments,three conclusions are found: There is a strong correlation between Danio rerio mortality and chloroquine concentration.As the concentration of chloroquine increases and the processing time elapses,the time for the first occurrence of Danio rerio deaths and the time to reach the death peak gradually advance.The chlorquine’s 96-hour lethal concentration(LC50)for adult Danio rerio is 1.12 mg/mL by the Kou method.This result is basically consistent with the verification result(1.0 mg/mL)of the probabilistic unit method.Histological studies have shown that after treatment with different concentrations of chloroquine,all tissues show significant damage.Among them,gill mainly show epithelial edema and tissue vacuolation,and the degree of change is positively correlated with the concentration of chloroquine.Follicular cell margins in the spleen and ovary show varying degrees of tissue vacuolation,and increase number of apoptotic bodies and autophagolysosomes.The spermatocytes and Sertoli cells in the testis have increased vesicles,autophagolysosomes and apoptotic bodies.And in the low concentration group,the nucleus and cytoplasm of spermatocytes show severe disintegration and autolysis,reflecting the strong interference of chloroquine on the spermatogenesis process.The number of autophagosomes(autophagolysosomes)and apoptotic bodies present in these organs increased significantly,indicating that chloroquine in water strongly is interfered with the autophagy system.The triggering response of chloroquine to the apoptotic system also corresponds to the massive death of Danio rerio.2.Response of autophagy marker gene LC3/BECN1 of various Danio rerio tissues to different concentrations of chloroquineThe expression of LC3 in the skin is the highest in the control group.In the high and medium concentration groups,the expression of LC3 in gills,liver,spleen,stomach,intestine,kidney,heart,muscle,eye,testis,and ovary are significantly increased.The expression of LC3 on the skin is significantly reduced.Compared with the high-concentration group,the fastest response time and peak time of LC3 in each organ in the medium-concentration group has different degrees of lag(no change in the brain).This result indicates that there is a clear dose-dependent response of LC3 to chloroquine in each tissue.High,medium,and low concentrations of chloroquine all cause a significant increase in LC3 expression in the ovary,gills,and spleen.And the higher the concentration,the greater the increase of LC3.Among them,the fastest response time of gills is concentrated in 0.5h-1h.The fastest response time and peak appearance time of the spleen lag behind the gills relatively.The fastest response time of ovary is concentrated in 2h-4h,and the peak is later than gill and spleen.The changes of LC3 in testes in high and medium concentration groups are similar to gills,spleen and ovary.There is a significant increase,and the increase factor is positively correlated with the concentration of chloroquine.In the control group,the expression of BECN1 is highest in the skin.In the high and medium concentration groups,BECN1 in gills,liver,spleen,stomach,kidney,heart,muscle,eye,brain,and ovary all increase significantly.Among them,the rise of gills and spleen is the most significant.The BECN1 of stomach,kidney,heart and ovary show a consistent pattern of change.BECN1 in the testis,skin,and liver decrease significantly.Similar to the results of LC3,the fastest response time and peak value of BECN1 in the organizations of the middle concentration group lag behind the high concentration group to varying degrees.This result shows a clear dose dependence.In the low-concentration group,the fastest response time of gills and ovaries is concentrated at 0.5-1h.The spleen shows extremely high sensitivity to the high and medium concentration groups.In the high-concentration group,the splenic BECN1 increase is higher,and the fastest response and peak appeared earlier.However,the spleen do not change significantly in the low-concentration group,proving that the lowest threshold of the spleen’s response concentration to chloroquine is in the range of 0.1-1.0 mg/mL.Testis BECN1 shows very high sensitivity to chloroquine.In the testis,the change trend in the three concentration groups is very consistent,all of which show a significant decrease at 0.5h.Then it remains at a low level until the end of the experiment,and the moment when the lowest value appeared is advanced as the concentration decreased.3.Response of autophagy-related gene Cx43 of various Danio rerio tissues to different concentrations of chloroquineThe expression of Cx43 in the eye is the highest in the control group.Comparing the three groups with high,medium and low concentrations,it can be found that with the decrease of the concentration of chloroquine,the fastest response time and peak value of Cx43 in each organization have different degrees of lag(except for the spleen of medium concentration).This result indicates that there is a clear dose-dependent response of Cx43 to chloroquine.In addition to the medium concentration of intestine,Cx43 expression in other tissues increases significantly at high and medium concentrations.Among them,the increase of Cx43 in spleen,testis and ovary is the most significant.But gills is always among the fastest responding organizations(0.5h-1h).At the same time,the overall change trend of these tissues is more regular,and the increase in the highest expression level is positively correlated with the concentration of chloroquine.That is,the higher the concentration,the greater the increase in expression.The Cx43 in the low-concentration group shows periodic fluctuations,and the fluctuation period is extended with time.Through comprehensive analysis of anatomy,histology,and results in LC3/BECN1 and Cx43,we believe that gills,spleen,testis and ovary are ideal target tissues for studying the effects of different concentrations of chloroquine on LC3/BECN1 and Cx43 in Danio rerio.At the same time,we also obtain the best detection period to study the effect of different concentrations of chloroquine on the expression of LC3/BECN1 and Cx43.It provides important scientific basis and useful exploration for future research in related fields. |
