Protective Effect Of Lycium Barbarum Polysaccharides And Sodium Selenite On Cadmium Chloride Induced Testicular Toxicity In Rats

Objective This study was conducted to investigate whether cadmium chloride（CdCl₂）could cause male reproductive toxicity by causing oxidative damage,disturbance of energy metabolism and imbalance of iron homeostasis in rat testes,and the possible mechanisms by which sodium selenite（Na₂SeO₃）and Lycium Barbarum Polysaccharides（LBP）could intervene to antagonize the testicular toxicity of cadmium（Cadmium,Cd）.Methods（1）One hundred and forty Sprague-Dawley（SD）male rats were randomly divided into five groups according to their body weight（weighing about 180～200 g）,i.e.,saline（NS）control group and CdCl₂-dosed group（0.5 mg/kg,1.0 mg/kg,1.5 mg/kg,2.0 mg/kg）,and the rats were continuously injected intraperitoneally with poison for three months.The food intake and body weight changes were recorded.After 1,4,8 and 12 weeks of CdCl₂ poisoning,the rats were killed and testicular tissues were collected,and the testicular organ coefficients were calculated.The histopathologic changes of rat testis were observed by HE staining.Cadmium content in testicular tissues was determined by inductively coupled plasma mass spectrometry（ICP-MS）.Testicular marker enzymes and oxidative stress-related enzyme activities were measured,with testicular marker enzymes including acid phosphatase（ACP）,alkaline phosphatase（AKP）,lactate dehydrogenase（LDH）and glutathione transpeptidase（γ-GT）,and oxidative stress-related enzymes including glutathione peroxidase（GSH-Px）,superoxide dismutase（SOD）and lipid peroxidation malondialdehyde（MDA）.（2）The testicular tissues of rats in the high dose CdCl₂-stained and NS control groups were selected for high-throughput gene sequencing,and the differentially expressed gene profiles between the CdCl₂-exposured and NS control groups were screened and characterized for bioinformatics analysis.Subsequently,the activities of enzymes related to energy metabolism,including succinate dehydrogenase（SDH）,malate dehydrogenase（MDH）,adenine riboside triphosphate（ATP）and coenzyme I（NAD+）,were detected.Energy metabolism and iron homeostasis related proteins and gene expression levels in rat testis were detected by WB and RT-qPCR.Including cis-aconitase（ACO2）,glutathione peroxidase 1（GPX1）,MDH,SDH,iron regulatory protein 1（IRP-1）,ferritin receptor（TFR）,glutathione peroxidase 4（GPX4）.（3）Forty male SD rats were randomly divided into five groups according to body weight（about160～180 g）.Namely,NS control group,CdCl₂ group（1.5mg/kg）,LBP intervention group（1.5mg/kg CdCl₂+20.0mg/kg LBP）,Na₂SeO₃ intervention group（1.5mg/kg CdCl₂+0.3mg/kg Na₂SeO₃）,Na₂SeO₃and LBP combined intervention group（1.5mg/kg CdCl₂+20.0mg/kg LBP+0.3mg/kg Na₂SeO₃）,8 rats in each group.The control group was given intraperitoneal injection of normal saline and gavage of normal saline 1 hour（h）later.The CdCl₂ group was administered with CdCl₂intraperitoneally and gavaged with saline after 1h.The LBP and Na₂SeO₃ groups were treated with intraperitoneal injection of CdCl₂ and orally gavage 1 hour later,once a day for 35 consecutive days.The rats were killed and testicular tissues were collected to calculate the testicular organ coefficients.The structural changes of rat testicular tissues were observed by HE staining.The expression levels of proteins and genes related to energy metabolism and iron homeostasis in testis tissues were measured by WB technique and RT-qPCR.Results（1）Compared with NS control group,the increase trend of body weight and food utilization of male rats in 0.5,1.0,1.5 and 2.0mg/kg CdCl₂exposed groups slowed down after 8 weeks（P<0.05）.The testis organ coefficient,sperm density and viability of rats showed that compared with NS control group,testis organ coefficient,sperm viability and viability of rats decreased with the increase of CdCl₂ exposure concentration and time（P<0.05）.HE staining showed that the structure of testis cells in CdCl₂ group was irregular,inflammatory cell infiltration increased,spermatogenic cells gradually disappeared.（2）Compared with NS control group,with the increase of CdCl₂ exposure concentration and time,the content of Cd in testicular tissue of male rats in 0.5,1.0,1.5 and 2.0 mg/kg exposure groups was significantly higher than that in the control group.After 4,8 and 12 weeks of exposure,the serum testosterone level of rats in 1.5mg/kg CdCl₂ exposed group decreased gradually（P<0.05）.The results showed that,compared with NS control group,the activities of ACP,AKP,LDH andγ-GT in testis tissue of rats in 1.5mg/kg or above group were decreased after 4 weeks of CdCl₂exposure（P<0.05）.（3）High-throughput sequencing of rat testis tissues showed that in the testis tissues of rats exposed to the CdCl₂ and NS control group,there were 3200 m RNA expressions with a 5-fold difference（Fold change>5）.Among them,958 kinds of m RNA were up-regulated in the testis tissues of rats in the CdCl₂-exposured group,and 2242 kinds of m RNA were down-regulated in the testis of rats in the CdCl₂-poisond group.Among these 3200 differentially expressed m RNAs,the highest up-regulated ploidy was Spp1（Fold change=17.51）and the highest down-regulated ploidy was Ccnk（Fold change=-16.80）.The results of differential gene functional enrichment analysis and signal pathway analysis showed that the up-regulated first 28 genes were related to cell inflammation and immunity,and mainly involved in the regulation of"positive regulation of monocytes,chemotaxis of cells and T-cell homeostasis".Among them,the functional enrichment of the first 28 down-regulated genes is related to peroxidase activity,iron regulation and cell energy metabolism,and is mainly involved in the regulation of"response to oxidative stress,glutathione peroxidase GPX4 and glutathione metabolism".（4）The results of oxidative stress enzyme activity assay showed that the activity of GSH-Px and SOD in the testis tissue of CdCl₂-contaminated rats decreased（P<0.05）and the activity of MDA increased（P<0.05）compared with the NS control group.The results of energy metabolism-related enzyme activities showed that compared with the NS control group,the level of ATP in the rats in the 1.5 mg/kg CdCl₂-infected group decreased gradually with the prolongation of the poisoning time（P<0.05）.The activities of SDH and MDH decreased with the prolongation of the CdCl₂-infected time（P<0.05）,while the activities of NADH/NAD+increased gradually（P<0.05）.The iron content in the testicular tissues of rats at 0.5,1.0,1.5 and 2.0 mg/kg increased with the increase in the concentration of CdCl₂,and also showed a trend of gradual increase with the extension of exposure time（P<0.05）.Compared with that of the NS control group,the expression of ACO2,GPX1,MDH,SDH,IRP-1,TFR and GPX4 in testis tissues of rats in the CdCl₂-infected group were decreased（P<0.05）,and showed a time-dependent effect with the prolongation of the contamination time（P<0.05）.（5）Na₂SeO₃ and LBP interventions in CdCl₂ poisoned rats revealed that compared with the CdCl₂poisoned group,the body weight of rats in the CdCl₂+LBP,CdCl₂+Na₂SeO₃ and CdCl₂+LBP+Na₂SeO₃ groups showed an increasing trend（P<0.05）,and the testis organ coefficient of rats increased（P<0.05）.Changes of sperm viability and density of rats showed that compared with the CdCl₂ infected group,the sperm density and viability of rats in the CdCl₂+LBP,CdCl₂+Na₂SeO₃ and CdCl₂+LBP+Na₂SeO₃ groups were increased（P<0.05）.HE staining results showed that the spermatogenic cells in the testes of rats in the CdCl₂+Na₂SeO₃ group were reduced,and there were obvious cavitation and interstitial changes.In the CdCl₂+LBP group,the testicular spermatogonia of rats were disordered,with significantly fewer cells and significantly larger interstitial gaps.in the CdCl₂+LBP+Na₂SeO₃ group,the testicular vas deferens germ cells of rats were more regular in shape,neatly arranged at the top,and the spermatogonia in the lumen of testicular tissue tubules were significantly increased.The results of the detection of serum testosterone,testicular tissue iron and cadmium in rats suggested that compared with the CdCl₂group,the serum testosterone content of rats in the CdCl₂+LBP+Na₂SeO₃ group increased（P<0.05）,and the mass fraction of cadmium and iron in rat testicular tissues tended to decrease（P<0.05）.（6）Compared with the CdCl₂ group,the SOD activity and GSH-Px activity of testis in CdCl₂+LBP,CdCl₂+Na₂SeO₃ and CdCl₂+LBP+Na₂SeO₃ groups were increased（P<0.05）,while MDA activity was decreased（P<0.05）.Compared with CdCl₂+LBP+Na₂SeO₃ group,The activities of SOD and GSH-Px in testis of CdCl₂+LBP and CdCl₂+Na₂SeO₃ groups were decreased（P<0.05）,while the content of MDA was increased（P<0.05）.Compared with CdCl₂ group,the activities of SDH and MDH in CdCl₂+LBP and CdCl₂+LBP+Na₂SeO₃ combined intervention group were increased（P<0.05）.Compared with CdCl₂+LBP+Na₂SeO₃ intervention group,SDH in CdCl₂+Na₂SeO₃ intervention group had a decreasing trend（P<0.05）.Expression of genes and proteins related to energy metabolism and iron homeostasis in rat testis:Compared with CdCl₂group,the gene expressions of ACO2,MDH,SDH,GPX-1,GPX4 and TFR in CdCl₂+LBP+Na₂SeO₃ group were increased（P<0.05）,and the gene expressions of MDH,SDH,GPX-1 and TFR in CdCl₂+LBP group were increased（P<0.05）.The expressions of MDH and TFR genes in CdCl₂+Na₂SeO₃ group were increased（P<0.05）,while the expression of GPX4 was decreased（P<0.05）.Conclusion（1）CdCl₂ produces reproductive toxic effects on rat testis,and the model of testicular toxicity injury in rats with subchronic exposure to CdCl₂ can provide a basis for further studies on CdCl₂damage to rat testis.（2）CdCl₂ staining caused oxidative damage to rat testis and changes in the activity of enzymes,genes and protein expression levels related to energy metabolism and iron homeostasis in rat testis.（3）Combined intervention of LBP and Na₂SeO₃ could reduce CdCl₂-induced oxidative stress damage in testis.It enhanced the activity and expression levels of key indicators of energy metabolism and iron homeostasis in testis tissues and antagonized the toxic damage of CdCl₂ on testis.