Frequent environmental and occupational exposure to Pb has been well-established to induce organ toxicity and subsequent adverse pathological consequences. Many of these diverse toxic effects are manifested at both cellular and molecular levels and share common mechanisms of action across various tissues and organs. Excessive Pb exposure can cause histological alterations of chicken kidneys [23] and renal damage of rats [24]. Pb treatment caused tubular degeneration, cell swelling, and inflammatory infiltration in rat kidneys [25]. In this study, we found that Pb exerted toxicity in chicken kidneys according to typical features of pathological alterations after Pb treatment, such as swollen glomeruli, inflammatory infiltration, and vascularization.
It is well known that oxidative stress is a core mechanism of Pb toxicity due to imbalance in oxidant/antioxidant homeostasis [26]. Excessive Pb was absorbed to tissues which overproduced H2O2 [27]. Similar result has revealed that H2O2 level elevated in the particulate matter component-induced oxidative stress in transformed human airway epithelial cells [28]. H2O2, a typical oxidant, is capable of diffusing throughout the mitochondria and across cell membranes and producing many types of cellular injury [29]. Moreover, Pb depletes cells antioxidants, particularly thiolcontaining compounds (GST) and antioxidant enzymes (CAT and T-AOC) during oxidative stress [27]. GST is one of the predominant antioxidant enzymes against oxidative stress in living organisms [30]. CAT is a primary defense against oxidative stress, and can catalyze the conversion of H2O2 into oxygen and water [31]. At the same time, it is a potential target of Pb [32]. T-AOC is used to measure the amount of free radical purge and evaluate antioxidant status [33]. It has been reported that changes in GST, T-AOC, CAT, and H2O2 were associated with Pb poisoning in rat kidneys [34, 35]. Pb can decrease T-AOC, GST, and CAT activities; and increase H2O2 content; and cause oxidative stress in the bursa of Fabricius of chickens [36]. In our findings, the damage was clearly demonstrated by the production of H2O2, which was accompanied by depletion in the antioxidant enzymes’ (T-AOC, GST, and CAT) activities in the chicken kidneys upon exposure to Pb compared to non-treated group, suggesting that Pb caused renal injury and oxidative stress in the chicken kidneys. These results supported the fact that Pb toxicity induced renal injury by increasing oxidative stress, and similar phenomena had been reported previously [37–39]. The consequence of the decrease in the level of T-AOC, CAT and GST was due to direct binding of Pb with their sulfhydryl groups [40], altering their function or suppressing their activities by Pb [41]. In addition, the decrease in CAT activity was also attributed to scavenging of H2O2 in Pb-intoxicated chickens. Therefore, the alterations of oxidative status, either by the overproduction of oxidants or deficit in antioxidant activity, was one of direct consequences of Pb toxicity in the chicken kidneys. In addition, we also found that T-AOC, CAT, GST, and H2O2 changed in a time-dependent effect in the Pb-induced chicken kidneys. It suggested that oxidative stress was gradually strengthened with Pb treatment duration. Our previous experiment also reported that Pb had a time-dependent effect on T-AOC, GST, and CAT activities and H2O2 content in chicken bursa of Fabricius [36].
Inflammatory response is the first line of defense in response to all forms of cellular injuries and clears cellular damage and initiates cellular repair [42]. But when inflammatory response is inappropriate it can lead to damage of surrounding normal cells. One of the events that occurred following oxidative stress is inflammatory response. It has been reported that increased oxidative stress might stimulate the expression of cytokines leading to increased inflammation [43]. IL-4 and IL-12β were proinflammatory mediators and IL-2 was anti-inflammatory one. Thus, in present study, IL-2, IL-4, and IL-12β were selected for mRNA expression analysis. We found that Pb treatment increased IL-4 and IL-12β and decreased IL-2 in the chicken kidneys, suggesting that Pb enhanced inflammatory process after oxidative stress in the chicken kidneys. As reported by Khatlab et al. (2019), reducing IL-2 expression level as the consequent of inflammatory response induced by Eimeria spp. challenge in broiler chickens [39]. The process of abnormal Pb invasion-caused oxidative stress triggered inflammatory response, through the cytokine production, such as IL-4 and IL-12β, which led to a reduction in the anti-inflammatory cytokine production, such as IL-2, and consequently, cells were damaged. In fact, inflammatory damage has been known to occur during the process of inflammation after Pb treatment, which was clearly seen from our histological results. Moreover, the increase of H2O2 level could cause structural damage to membranes. Our findings suggested a crosstalk between Pb-induced oxidative stress and inflammation. Other researchers also concluded that there was a relationship between oxidative stress and inflammation. [44] found that lipopolysaccharide decreased CAT activity and increased H2O2 content with the increase of IL-4 in chicken myocardial [44]. Abd El-Ghffar et al. (2018) reported that H2O2 content increased, GST and CAT activities decreased, and oxidative stress occurred which prompt expression of IL-4 in aspirin-treated mouse stomaches [45]. In addition, we also found that IL-2, IL-4, and IL- 12β mRNA expressed in a time-dependent effect in the Pb-induced chicken kidneys, which suggested that inflammatory response was gradually strengthened with Pb treatment duration.
Oxidative stress is also responsible for activation of heat shock response [46]. HSPs also play a role in sensing oxidative stress, are involved in restoring physiological protein conformation during and after oxidative stress, and which are characteristic features of a number of pathological conditions. In response to oxidative stress, the expression of HSPs elevates dramatically which is notable as a pervasive adaptation mechanism in organisms that enables them to survive and adapt to different environmental stressors [46]. Increased levels of HSPs were an indicative of initiation of a stress response for mediating cellular protection and indicated tissue damage [47]. Heat shock response can protect against toxicity caused by excess heavy metals [48]. Previous studies reported that Pb increased HSPs (HSP27, HSP40, HSP60, HSP70, and HSP90 mRNA expression in peripheral blood neutrophils [49] and hearts [50] of chickens. In the present study, we observed high expression of HSPs (HSP27, HSP40, HSP60, HSP70, and HSP90) mRAN and protein caused by Pb exposure in the chicken kidneys, reflected the activation of this intracellular buffer system, which responds to oxidative stress when the antioxidant enzyme (T-AOC, GST, and CAT) exhaustion occurs [46]. The findings of this study indicated that Pb exposure resulted in the activation of HSPs under burden of oxidative stress. Interestingly, increasing evidence suggests that there is a complementary regulation between HSPs and inflammation [46]. Besides, inflammation is itself a stimulus for upregulation of HSPs production [51]. Therefore, in our study, elevated HSPs, on the one hand, antagonized the mentioned Pb-induced oxidative stress, on the other hand, inhibited inflammation. In addition, we also found that HSP27, HSP40, HSP60, HSP70, and HSP90 mRNA expression increased in a time-dependent effect in the Pb-induced chicken kidneys. It suggested that HSP response was gradually strengthened with Pb treatment duration.
Autophagy is an intracellular lysosomal degradation process, which plays an important role in regulating normal cell homeostasis, and is considered as one of cellular defense against increased oxidative stress [52]. Song et al. (2017) reported that autophagy contributed to Pb-induced nephrotoxicity in primary rat proximal tubular cells [53]. Pb promoted protein levels of Beclin1, LC3-I, and LC3-II; and induced autophagy in rat hippocampi [54]. Han et al. (2017) reported that Pb increased mRNA and protein levels of ATG5, Beclin-1, Dynein, LC3-I, and LC3-II; decreased mRNA and protein levels of mTOR; and induced autophagy in chicken spleens [12]. Our present research is consistent with above studies. We found that Pb treatment promoted mRNA and protein expression of Beclin 1, Dynein, ATG 5, LC3-I, and LC3-II; and inhibited mRNA and protein expression of mTOR. As reported by Wang et al. (2019), the increased mTOR expression were detected in weaned pigs compare with dietary tributyrin supplemented weaned pigs [55]. Furthermore, we found typical features of autophagy, formation of autophagosome, through the ultrastructure of chicken kidneys. Molecular and histology evidence of our study demonstrated that Pb induced autophagy in the chicken kidneys. Therefore, we concluded that elevated HSPs (HSP27, HSP40, HSP60, HSP70, and HSP90) were also a trigger for autophagy in Pb treatment group.
Previous studies have confirmed potent antioxidative and anti-inflammatory activities of Se. Some researches demonstrated that Se mitigated Pb-induced oxidative stress by means of increasing T-AOC, GST, and CAT activities; and decreasing H2O2 content in Cyprinus carpio livers [56], chicken bursa of Fabricius [36], and chicken splenic lymphocytes [57]. Jiao et al. (2017) found that Se can mitigate increase of IL-4 and IL-12β mRNA expression, and the decrease of IL-2 mRNA expression in Pb-treated chicken bursa of Fabricius [36]. Xing et al. (2018)reported that Se alleviated the increase of IL-4 and IL-12β mRNA expression and the decrease of IL-2 mRNA expression caused by Pb in chicken neutrophils [49]. In addition, Se can mitigate Pb-caused increase of HSPs and autophagy. Wang et al. (2017) found that Pb poisoning induced mRNA expression of HSP27, HSP40, HSP60, HSP70, and HSP90; and Se administration alleviated the above HSPs changes in chicken testes [19]. Se exhibited significant antagonistic roles against Pb-induced increases of HSP (27, 40, 60, 70, and 90) mRNA expression in peripheral blood neutrophils [49] and hearts [50] of chickens. Se was reported by one of the articles to alleviate spleen toxicity in a chicken model induced by Pb via the modulation of oxidative stress, inflammation, and autophagy [12]. Se alleviated protein increase of ATG5, Beclin1, Dynein, LC3-I, and LC3-II and protein decrease of mTOR in Cd-induced chicken pancreas [58]. In our study, all alterations caused by Pb were ameliorated by treatment with Se. Such effect was attributed to kidney tissue antioxidant capacity because of better antioxidant supply, thus reducing the oxidative damage represented by the reduction of T-AOC, GST, and CAT and the rise of H2O2. The ability of Se to neutralize oxidative stress could be due to facilitating chelation with Pb in the chicken kidney tissues, resulting in reduced Pb accumulation in the body through its potential antioxidant efficacy [59]. So Se alleviated oxidative stress, which naturally alleviated these downstream events. Therefore, Se alleviates heat shock response and autophagy.