The Intervention Effect Of Riboflavin On Testicular Pyroptosis Induced By Fluoride

Preliminary laboratory studies have shown that fluorosis can cause testicular toxicity,destroy the morphological structure of the testis,interfere with the internal homeostasis of the testis,cause symptoms such as oxidative stress,cell death and inflammatory damage,and affect male reproductive function.As a highly pro-inflammatory way of cell death,it has not been reported whether pyroptosis is involved in fluoride-induced testicular inflammatory injury.Riboflavin has good anti-inflammatory and antioxidant properties,and the effect of riboflavin on fluoride-induced testicular inflammatory injury is not clear.Therefore,the fluorosis models of ICR mice and IL-17 A knockout mice were established in this paper.Hematoxylin-eosin staining(HE staining),qRT-PCR,Western blot,ELISA and immunofluorescence staining were used to detect the pathology of testicular tissue and the classical pathway of pyroptosis in each model.The purpose of this study was to explore whether the testicular inflammatory injury caused by fluorosis is related to pyroptosis,and whether riboflavin can alleviate the testicular inflammatory injury induced by fluorosis,so as to provide a theoretical basis for the study of the mechanism of reproductive toxicity caused by fluorosis.At the same time,it also provides practical reference for the potential clinical application of riboflavin in the treatment of fluorosis.(1)The results of fluorosis model of ICR mouse : 32 healthy ICR mice were randomly divided into four groups: the control group,the Na F group,the Ribo group and the Na F + Ribo group,with 8 mice in each group.The control group was fed with distilled water.The results of bone fluoride showed that fluoride could increase bone fluoride content and riboflavin could decrease bone fluoride content.The results of HE and semen quality test showed that fluoride could damage testicular tissue structure,reduce spermatogenic cells and spermatozoa in mice,while riboflavin could alleviate these injuries to some extent.Furthermore,qRT-PCR,ELISA,immunofluorescence and immunoblotting were used to detect testicular pyroptosis related gene proteins NLRP3,Caspase-1,GSDMD,IL-1β and IL-18,.It was found that fluoride could increase the expression of GSDMD,NLRP3,Caspase-1 and the content of IL-18 and IL-1β in testis,while riboflavin could reduce the protein expression of GSDMD,NLRP3 and Caspase-1.(2)The results of fluorosis model of IL-17 knockout mice: 32 healthy IL-17A-/-mice and 32 WT mice(both C57 mice)were divided into four groups: the control group: 0.9% normal saline group,the Na F group: 24mg/kg sodium fluoride group,the Ribo group: 5mg/kg riboflavin sodium phosphate group,the Na F+Ribo group: 24mg/kg sodium fluoride + 5mg/kg riboflavin sodium phosphate group,with 8 mice in each group.The above conditions were given by intragastric administration and fed for 13 weeks.The results of bone fluoride and dental fluoride showed that fluoride could increase the content of bone fluoride and dental fluoride,while oral administration of riboflavin sodium phosphate could not reduce the content of bone fluoride and dental fluoride.The results of HE and semen quality test showed that fluoride could damage the structure of testicular tissue,disordered arrangement of cells and loose structure in mice,and sodium riboflavin phosphate could alleviate these injuries to some extent,but IL-17 A knockout could not improve the morphological and structural damage of testis caused by fluoride.Furthermore,ELISA and Western blotting were used to detect testicular pyroptosis related gene proteins NLRP3,Caspase-1,GSDMD,IL-1β and IL-18,.It was found that fluoride could increase the expression of GSDMD,NLRP3,Caspase-1 and the content of IL-18 and IL-1β in testis,while riboflavin sodium phosphate could reduce the expression of GSDMD,NLRP3,Caspase-1 and the content of IL-18 and IL-1β.Moreover,the expression of GSDMD and NLRP3 and the content of IL-18 and IL-1β decreased in the testis of IL-17A-/-miceIn summary,the conclusions of this experiment are as follows: Fluorosis can cause pyroptosis,and ultimately lead to damage to the morphology and structure of testicular tissue,decreased sperm quality,and damage to reproductive function,and riboflavin can alleviate testicular pyroptosis caused by fluorosis by inhibiting NLRP3/Caspase1/GSDMD signal pathway.IL-17 A knockout mice could reduce the expression of pyroptosis-related proteins GSDMD,NLRP3,IL-18 and IL-1β in testes.Riboflavin addition and IL-17 A knockout had the same effect on mice as riboflavin addition or IL-17 A knockout alone,and riboflavin addition was better than IL-17 A knockout in alleviating testicular morphological and structural damage.