Mercuric Chloride Induces Sequential Activation Of Ferroptosis And Necroptosis In Chicken Embryo Kidney Cells By Triggering Ferritinophagy

Mercury is a ubiquitous highly toxic heavy metal contaminant,and mainly exists in three forms,including monomeric mercury,inorganic and organic mercury.Mercury chloride（HgCl₂）is a widely worried inorganic mercury which is difficult to degrade and tends to accumulate in the kidney.Programmed cell death（PCD）is a form of cell death controlled and regulated by genes.In which,as the first revealed PCD mode,apoptosis has long been considered as the main mechanism of organ toxicity caused by heavy metals.However,some new PCD modes have been revealed successively,including ferroptosis and necroptosis,also play a non-negligible role in the toxic effects caused by various heavy metals.It suggested that the toxicity of heavy metals are possibly mediated by several PCDs together.What’s notable is that most of the studies on the potential mechanisms of HgCl₂-induced nephrotoxicity have been limited to a single mode of PCD such as apoptosis,and whether and how ferroptosis and necroptosis are involved in HgCl₂-induced kidney injury have not been revealed.In this study,we established a primary chicken embryonic kidney cell injury model of HgCl₂ exposure,taking iron disorder triggered by ferritinophagy as the entry point,aiming to deeply explore the interaction between ferroptosis and necroptosis in chicken kidney injury caused by HgCl₂.The chicken embryonic kidney（CEK）cells were cultured in vitro and treated with different concentrations（0,10,15,20 and 25μM）HgCl₂ for 12 h or 24 h.The cell viability were measured to determine the optimal staining concentration of HgCl₂.We detected the apoptosis rate by apoptosis-specific probe（YO-PRO）and the expression of apoptosis marker proteins（Bcl-2,Bax）,combined with the effect of apoptosis-specific inhibitor（Z-VAD-FMK,z VAD）on cell viability.The results showed that apoptosis was not the main responsible for the cytotoxicity of HgCl₂ in CEK cells.Notably,a time-dependent increase in the rate of PI-positive cells was observed in HgCl₂-exposed CEK cells,and the rate of LDH release also increased in a time-dependent manner,suggesting that HgCl₂ might lead to necrosis in CEK cells.Since the role of necroptosis in kidney injury has been widely reported,the expression of necroptosis in CEK cells was explored primarily.We detect the expression of necroptosis marker proteins（RIPK1,RIPK3,MLKL and p-MLKL）.Combined with the effect of necroptosis specific inhibitor（Necrostatin-1,Nec-1）on cell survival and LDH release rate.The results showed that necroptosis was activated at 24 h after HgCl₂ exposure,but did not occur at 12 h,suggesting that there might be other forms of PCD involved in HgCl₂-induced necrosis of CEK cells.Since the cell morphology of ferroptosis also exhibited necrotic features,we investigated ferroptosis next.We detected the expression of ferroptosis marker protein GPX4.Combined with the effect of ferroptosis specific inhibitor（Ferrostatin-1,Fer-1）on cell viability and LDH release rate.The results showed that ferroptosis was activated 12 h after HgCl₂ exposure.while necroptosis was activated after 24 h.Since ROS is a common trigger of ferroptosis and necroptosis,we speculated that ROS might play a key role in the sequential activation of ferroptosis and necroptosis.We assessed ROS level in CEK cells with a ROS probe（DCFA-DA）,and evaluated the effect of ROS scavenger（N-acetylcysteine,NAC）on cell viability.The results showed that ROS was the initiating factor for HgCl₂-induced ferroptosis and necroptosis in CEK cells.Given that free iron catalyzes the generation of ROS from hydrogen peroxide via the Fenton reaction,which is a key process in ferroptosis,we examined the iron content and the level of labile iron pool（LIP）in CEK cells.Combined with the effects of iron chelator（Deferoxamine,DFO）on ROS,cell viability,LIP level,and PCDs marker proteins were examined.The results indicate that HgCl₂ triggers the sequential activation of both ferroptosis and necroptosis by accumulating excess ROS in an iron-dependent manner.Due to the close association of LIP with iron metabolism,we further investigated the iron metabolism in CEK cells.We detected the expression of iron metabolism marker proteins（FBXL5,IREB2,TFRC,FPN1,FTH and FTL）.The results showed that HgCl₂ exposure led to iron overload in CEK cells and reduced intracellular iron storage capacity.Ferritinophagy is an important pathway for the selective degradation of ferritin,which also plays an important role in iron metabolism.In view of normal cellular iron uptake and excretion,the ferritinophagy pathway was further explored.We detected the expression of ferritinophagy marker proteins（NCOA4,ATG5,ATG7 and LC3）,and the co-localization of LC3 and FTH.The results showed that continuous exposure to HgCl₂activated ferritinophagy in CEK cells.Finally,the effects of ferritinophagy inhibitor（Chloroquine,CQ）on cell viability,ferritinophagy marker proteins,ferroptosis and necroptosis marker proteins were examined.The results showed that HgCl₂ exposure mediated the sequential activation of necroptosis and ferroptosis through ferritinophagy.In conclusion,our results demonstrate that HgCl₂ sequentially activates ferroptosis and necroptosis by triggering NCOA4-mediated ferritinophagy,which disrupts iron homeostasis in CEK cells,elevates LIP,and leads to a subsequent massive accumulation of ROS.These findings provide a new understanding of the nephrotoxic mechanism of HgCl₂,and provides a novel theoretical basis for the mechanism of different types of PCD modalities in heavy metal-induced kidney injury.