Cadmium Induced Hepatocyte Microtubule System Damage And Protective Effect Of Honokiol

Cadmium is a kind of toxic heavy metal which is harmful to animal and human health.Liver is the central organ of protein synthesis,transport,detoxification and metabolism in animal body,and also the main target organ of Cd accumulation and injury.Microtubule system consists of microtubule network,motor proteins and microtubule-associated proteins attached to microtubules,which are responsible for a wide range of material transport,organelle movement and fission activities in liver cells.In addition to the microtubule system,the normal function of hepatocytes is closely related to the endosomal-autophagosome-lysosome system and the gap junction.Honokiol is a new lignan plant extract with biological activity,targeting a variety of intracellular signaling molecules,with pharmacological properties of antioxidation,anti-inflammation,anti-cancer,neuroprotective,anti-anxiety,antidepression and anti-spasmodic.In this study,BRL 3A cell line was used as the object to investigate the effect of Cd-induced microtubule system damage on endosomalautophagosome-lysosome system and the internalization degradation of gap junction protein.Secondly,the protective effects of honokiol on Cd-induced microtubule injury of chicken hepatocytes and BRL 3A cell injury were investigated in Sanhuang broilers and BRL 3A cells.The main research contents are as follows:1 Effect of Cd-induced microtubule damage on endosomal-autophagosomelysosome system in BRL 3A cellsThe motor proteins kinesin-1 and PIKfyve kinases on microtubules regulate the membrane fission activity of endosome-lysosome.In this study,BRL 3A cells were selected as the object of study.By setting the concentration gradient group of Cd,the groups treated separately and in combination with Cd and nocadazole(microtubule inhibitor),the groups treated separately and in combination with Cd and YM201636(PIKfyve inhibitor),and the NC and sikif5b treatment groups,to study the effect of Cd damage to microtubules on endosome-autophagosome-lysosome and the role of PIKfyve protein in it.The test results showed that:①Cd damaged cellular microtubule network and caused void region,and down-regulated the expression of KIF5B(kinesin-1 subunit protein)and acetylated-α-tubulin.10 μM Cd downregulated the colocalization level of microtubules and microtubule-associated proteins(MAP4 and acetylated-α-tubulin).Cd down-regulated lysosomal marker LAMP2 and up-regulated endosomal marker proteins EEA1,RAB5A and RAB7A.②The concentration gradient of Cd down-regulated the fluorescence intensity and protein level of PIKfyve.Cd and YM201636 alone or in combination increased cell acidity.Cd-damaged microtubules induced late endosome and lysosome vacuolation,but Cd did not directly inhibit PIKfyve protein function.③The protein levels and morphological changes of PIKfyve,EEA1,RAB7A and LAMP2 after nocodazole and Cd alone or in combination were consistent with the results of Cd alone,indicating that the downregulation of PIKfyve protein levels caused by Cd damage to microtubules was the main cause of endosomal/lysosomal vacuolation.④ Cd concentration gradient down-regulated the colocalization of KIF5B and microtubules;After the knockdown of kif5b gene,the volume of endosome and lysosome increased abnormally,and the changes of PIKfyve protein and endosomal/lysosomal protein were consistent with the results of Cd alone,which further indicated that Cd damage microtubule and down-regulation of microtubule-related protein kinesin-1 protein could indirectly lead to the decrease of PIKfyve protein level.Finally,endosomelysosome vacuolation was induced.⑤Nocodazole,YM201636 and kif5b knockdown could block autophagic flux in BRL 3A cells that stably express GFP/RFP-LC3.This study showed that Cd-induced microtubule system damage led to endosomal/lysosomal vacuolation and autophagic flux arrest in the core of BRL 3A cells,and kinesin-1 and PIKfyve protein plays an important role in this process.2 Effect of Cd-induced microtubule damage on the internalization and degradation of Cx43 protein in BRL 3A cellsThe decrease of gap junction between hepatocytes is a cancerous cellular behavior.To investigate the effects of Cd-damaged microtubule system on the internalization and degradation of Cx43,cells were exposed to Cd concentration gradient for 12 h and treated with nocodazole,TP A(promoting the internalization and degradation of Cx43)alone or in combination with Cd for 12 h.The results showed that:① The concentration gradient of Cd damaged microtubules and led to the decrease of Cx43 colocalization levels on microtubules.The levels of Cx43 and PCx43(Ser373)protein were significantly decreased and increased under 10 μM Cd treatment,respectively.② Nocodazole and 10 μM Cd alone or in combination promoted the decrease of Cx43 protein.Increased level of P-Cx43(Ser373)protein.③The results of immunofluorescence and transmission electron microscopy showed that 10 μM Cd could induce the production of AGJ.④Cx43 could be degraded by endosomal lysosome pathway after treatment with 10 μM Cd for 12 h.⑤ Compared with Cd group,the co-treatment of TPA and Cd reduced the Cd-induced cell death of BRL 3A,decreased Cx43 protein level and inhibited the increase of Cd-induced apoptosis protein level.Compared with Cd group,the combined treatment of Cd and TPA reduced the nuclear deformation and damage,indicating that although the internalization of Cx43 was not beneficial to long-term cell survival,acute and shortterm internalization of Cx43 could temporarily protect cells and reduce the toxicity of Cd.This study showed that Cd-induced microtubule damage in BRL 3A cells promoted the internalization and degradation of Cx43 by inducing AGJ generation.3 Protective effects of HNK on Cd-induced microtubule injury in liver cellsIn order to study the protective effect of HNK on Cd-induced hepatocyte injury,female Sanhuang broiler and BRL 3A cell line were selected as the study subjects to explore the protective effect of HNK on Cd-induced hepatocyte injury.In vivo test:Cd and HNK were added into the diet to construct the Cd exposure model and the Cd poisoning-HNK detoxification model.Control,Cd(70 mg/kg CdCl2),(100,200 and 400 mg/kg)HNK group and Cd+(100,200 and 400 mg/kg)HNK group were set up in 8 groups.After 4 weeks of feeding,the results showed that:①Cd inhibited the body weight and liver weight of chickens.Compared with Control group,HNK increased the body weight of chickens.Compared with Cd group,HNK alleviated the effects of Cd-induced liver weight loss,and 400 mg/kg HNK had the best protective effect.② HE staining showed that HNK alleviated the effect of Cd on liver cell pathological injury in chickens.HNK alleviated the increase of ALT,AST and LDH induced by Cd.TUNEL staining showed that HNK alleviated liver apoptosis caused by Cd.③ Compared with Control group,the levels of Mn,Cu and Zn in group Cd increased,while the contents of Fe and Se decreased,and the content of Cd increased significantly.HNK reduced the increase of Cu and Zn levels caused by Cd,but had no significant effect on the increase of Mn content.To some extent,HNK increased the levels of Fe and Se elements,and reduced the accumulation of Cd in chicken liver tissue.④Compared with Cd group,HNK alleviated the decrease of T-AOC,GSH-Px and CAT enzyme activities of Cd,and down-regulated the increase of GSH and MDA contents.⑤Compared with Control group,HNK alleviated the effect of Cd on the decreased levels of GPxs family genes,SIRT3,SOD2 and GJB1 genes.HNK alleviates the effect of Cd on liver ultrastructural damage and vacuolation in hepatocytes.⑥ Tissue immunofluorescence staining showed that HNK alleviated the effects of Cd on the microtubule system of chicken hepatocytes and the decrease of Cx32 internalization,and protected the damage of Cd-induced hepatic gap junction structure.In vitro results showed that 10 μM HNK reduced cell damage,ROS level,mitochondrial membrane potential,and ATP content on BRL 3A induced by Cd.These results indicate that HNK can protect the liver cell damage caused by Cd and relieve the liver cell vacuolation and gap junction protein degradation induced by Cd.In conclusion,the damage of Cd to the microtubule system of hepatocytes can lead to the abnormal enlargement of endosome-lysosome and the block of autophagic flux,and promote the internalization and degradation of gap junction proteins.HNK protects Cd-induced liver damage in chickens,alleviates the abnormal enlargement of endosome-lysosome and the block of autophagic flux caused by Cd on chicken liver microtubule damage,restores the structure of gap junction protein,and it has certain protective effect on Cd-induced liver cell damage in vivo and in vitro.