Dietary Cistanche Alleviated Acute Ileum Damage Of Ducks Ⅱ

3.1.Intestinal Morphology

In this study, the top pathological examination (H&E)and the instruction of the microstructure assessed by an ultrastructural of ileum had been demonstrated in Figure 1. Compared to the T0 group, the ileum in the T0+AFB1 group shows epithelial thick-ness reduction, villi structure damage and inflammatory cell aggregation, and the microstructure destruction, such as a large number of microvilli severely broken and mi-mitochondria well ed and shrinkage. The damage in the ileum of ducks containing structural destruction of villi and microvilli after AFB1 administration induced the appearance of inflammationan oxidation stress. As expected, dietary cistanche had the ability to protect the ileum against acute damage induced by AFB1 administration, including a few ileum villi-broken,inflammatory cell gathered and a little damage of ileum structural-rated by H&E, and decreasing the number of broken microvilli, reducing mitochondrial swelling and eliminating mitochondria shrink age assessed by annular structural shown in structure difference between the T500+AFB1group and the T0+AFB1group.

group; T500 + AFB1: cistanche + AFB1 group

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3.2. Levels of AFB1-DNA Adducts in the Plasma 

AFB1-DNA adducts in plasma of ducks was measured by indirect competitive ELISA and shown in Figure 2. Compared to the T0 group, AFB1 administration signifificantly increased the content of AFB1-DNA adducts (p < 0.001) in the plasma. As expected, dietary cistanche reduced AFB1-DNA adducts content (p = 0.001) in the plasma of ducks in the T500 + AFB1 group relative to that in the T0 + AFB1 group.

+ AFB1: cistanche + AFB1 group. Values are expressed as Mean ± SEM (n= 15), ** means < 0.01.

3.3. Antioxidant Capacity in the Plasma and Ileum 

The antioxidant capacity of the plasma was shown in Figure 3. Exposure of AFB1 led to oxidation stress, manifesting that the T-SOD (p = 0.073), GSH-PX (p = 0.034) and GSH-ST (p = 0.003) activities in the plasma were decreased in the T0 + AFB1 group than those in the T0 group. However, the T-SOD (p = 0.039), GSH-PX (p = 0.009) and GSH-ST (p = 0.003) activities were signifificantly enhanced in the T500 + AFB1 group than those in the T0 + AFB1 group. The concentration of MDA (p = 0.028) in the plasma was increased in the T0 + AFB1 group than that in the T0 group, and the concentration of MDA (p < 0.001) in the plasma was decreased in the T500 + AFB1 group than those in the T0 + AFB1. 

3.4. Expression of Genes Related to Nrf2-ARE Signaling Pathway in the Ileum AFB1 administration induced the cell oxidation stress and further resulted in the expression changes of genes including Keap1, Nrf2, CAT, SOD1, GPX, GST, NQO-1, HO-1, GCLC and GCLM in the ileum of ducks. As shown in Figure 4, AFB1 administration signifificantly increased the mRNA (p = 0.001) level of Keap1 gene, and inhibited mRNA levels of genes including Nrf2 (p = 0.171), CAT (p = 0.166), SOD1 (p = 0.121), GPX (p = 0.065), GST (p = 0.008), NQO-1 (p = 0.061), HO-1 (p = 0.068), GCLC (p = 0.800) and GCLM (p = 0.090) and Nrf2 protein content (p = 0.001) in the ileum of ducks in the T0 + AFB1 group relative to those in the T0 group. As expected, adding cistanche into diet fed ducks for 70 days signifificantly decreased the mRNA (p = 0.012) expression of Keap1 gene in ileum, signifificantly increased mRNA level of genes including Nrf2 (p = 0.042), SOD1 (p = 0.038), HO-1 (p = 0.041), NQO-1 (p = 0.047) and GCLC (p = 0.043), and improved mRNA level of genes including CAT (p = 0.229), GPX (p = 0.568), GST (p = 0.454) and GCLM (p = 0.860), and Nrf2 protein (p = 0.005) in ileum of ducks in the T500 + AFB1 group relative to those in the T0 + AFB1 group. 

* means p < 0.05, ** meansp < 0.01

3.5. Expression of Genes Related to NF-κB Signaling Pathway in the Ileum

The expression of inflflammatory genes was shown in Figure 5. Compared to the T0 group, AFB1 administration increased mRNA level of some genes such as TLR4 (p = 0.037), NF-κB (p < 0.001), TNF-α (p = 0.025), IL-6 (p = 0.072), TXNIP (p = 0.007), NLRP3 (p < 0.001) and IL-18 (p = 0.478) in the ileum of ducks in the T0 + AFB1 group. As expected, dietary cistanche signifificantly suppressed the over-expression of genes including TLR4 (p < 0.001), NF-κB (p = 0.001), TNF-α (p = 0.012), IL-6 (p = 0.007), TXNIP (p = 0.001), NLRP3 (p = 0.001) and IL-18 (p < 0.001) in the ileum of ducks in the T500 + AFB1 group relative to those in the T0 + AFB1 group. In addition, the contents of P-P 65 (p = 0.004) in the ileum increased in the T0 + AFB1 group relative to that in the T0 group. As expected, dietary cistanche signifificantly suppressed P-P 65 (p = 0.015) protein content. 

intensitiy of the target protein to the internal reference (GAPDH). Values were expressed as Mean± SEM (n = 15), and* means p < 0.05, ** means p < 0.01

4. Discussion

Intestinal morphology is one of the behavioral markers to evaluate inflammation and oxidation stress of the intestine induced by AFB1 administration. Literature on the effects of AFB1 administration on ileum morphology of ducks are scant. Yan et al. (2020) reported that AFB1 administration led to cardiac pathologic damage of Sprague-Dawley rats, inflflammatory cell infiltration and greater cardiomyocyte degeneration [29]. Catarrhal enteritis with inflflammatory cell infiltrations in the intestine of broiler chickens induced by AFB1 destroyed the structure of the intestine [12]. Luzi et al. (2002) reported acute AFB1 administration-induced ileum contractions [30]. The results in this study demonstrated that dietary cistanche is a potent protective agent of ileum against inhibiting inflammation, which may be that cistanche had the ability to inhibit anti-inflammatory in multiple inflflammatory disorders in mice [31–33]. DNA damage caused by oxidative stress will destruct the stability of DNA, which can promote the formation of various DNA adducts [34]. AFB1-DNA adduct is a biomarker to evaluate the injury degree of body which was induced by AFB1. Synthesizing and enriching of AFB1-DNA adducts destroyed the structure of tissues, then resulting in carcinogenic development [35]. AFB1 would be metabolized by cytochrome P450s isoenzymes to AFB1-8,9-epoxide (AFBO) and produce related adducts [36] and increase tissue damage, oxidative stress and DNA damage by ROS [37]. AFB1-DNA adducts can bind with the nucleoproteins and nucleic acids, thus inducing DNA and cell damage and decreasing the level of antioxidant enzymes and protein synthesis [38]. Zhang et al. (2016) reported that cistanche-supplemented inhibited liver damage induced by AFB1 by increasing the antioxidation activity of antioxidant enzymes (GPx, SOD, CAT and GST) and inhibiting AFB1-DNA production [39]. In this study, AFB1 administration increased AFB1-DNA adducts content in the plasma. Dietary cistanche signifificantly diminished this phenomenon, results demonstrated that dietary cistanche was the potential to protect the ileum in this acute AFB1 administration model which may be explained by the antioxidant effect of cistanche that improved the antioxidation capacity of the body [40,41]. Oxidation stress occurred when the imbalance of oxidation and antioxidation in bodies is induced by the decreases of antioxidant enzyme activities and the increases in lipid peroxidation levels. Antioxidant enzyme system including CAT, GSH-Px, and SOD is the first line of cell defenses against free radicals and reactive oxygen species (ROS) and is indispensable in the entire defense strategy of antioxidants in the body [42]. 

GST is a crucial enzyme to downregulate reactive oxygen species (ROS) and oxidative stress in order to achieve detoxification for bodies [43]. As shown in Figure 3, oxidation stress in the plasma and ileum of ducks occurred during acute ileum damage induced by AFB1 administration. As expected, dietary cistanche ameliorated the oxidation stress of bodies by increasing T-SOD, GSH-PX, and GSH-ST activities in the plasma and ileum after AFB1 administration. The results in this study demonstrated that cistanche is a potent anti-oxidation agent for ducks fed AFB1 administration that may be explained by the anti-oxidative capacity via suppressing lipid oxidation and increasing antioxidation enzyme activity by cistanchesupplemented [8,44,45]. These results are in line with a previous report that demonstrated that cistanche ameliorated AFB1-induced alteration in glutathione, SOD, CAT and MDA activities [8]. This may be due to the ability of cistanche to scavenge free radicals by restoring antioxidant enzyme activities and alleviating oxidative stress [15,46]. The balance between oxidation and anti-oxidation in vivo were regulated by T-SOD and GSH-Px [47]. Nrf2 was translocated into the cell nucleus and combined with t antioxidant response element (ARE) and upregulated the transcription of the antioxidant enzyme genes including SOD, CAT, GSH-Px, HO-1, NQO-1, GCLC and GCLM [47,48]. In addition, GST is upregulated by activating Nrf2 signaling way and as a kind of phase-II detoxifying enzyme involved in various detoxification in vivo to relieve oxidative stress [49,50]. Oxidative stress and lipid peroxidation were biomarkers for rats induced by AFB1 administration [51,52]. Oxidative stress was diminished by activation genes expression under Nrf2-ARE signaling pathways, such as NQO-1, HO-1 and GCLC [40]. Numerous research reported that cistanche-supplemented induced genes expression including HO-1, NQO-1, γ-GCLC, γ-GCLM, CAT and GPX via Nrf2 activation in broiler and rats [40,41] and eliminated liver damage induced by AFB1 administration [39,53]. 

The results in this study indicated that AFB1 administration may induce oxidative stress damage of the ileum via inhibiting Nrf2 signaling pathway. Dietary cistanche promoted Nrf2 downstream genes expressions such as antioxidant genes (CAT, SOD1, GPX1, GST) and phase II detoxifying enzyme genes (NQO1, HO-1, GCLC, GCLM), which demonstrated that adding cistanche into the diet for ducks inhibited the acute oxidation damage of ileum induced by AFB1 administration through activating Nrf2-ARE signaling pathway Results in this study provided evidence that dietary cistanche could be a potent ameliorating agent to protect ileum against oxidation stress induced by AFB1 administration. Oxidation stress-activated NF-κB signaling pathway then further evaluated the production of inflflammatory cytokines [54]. In this study, ileum injury induced by AFB1 administration may be due to inflflammation. AFB1 administration may induce ileum damage directly via increasing expression of inflflammatory factors, and directly activating the inflflammatory signaling pathway. There is a positive relationship between oxidation stress and inflflammation in tissues [55]. Ko et al. (2020) reported that oxidation stress-activated NF-κB signaling pathway, upregulated pro-inflammatory cytokines and caused inflflammation in rat lung [56]. The NF-κB signaling could be activated by AFB1 in the cell line 3D4/21 [57], resulting a series of inflflammatory reactions [58]. Kumara et al. (2020) found that AFB1 administration induced the inflflammatory by elevating levels of pro-inflammatory factors cytokines, TNF-α, IL-12 and IL-6 in the serum of albino mice [59]. Dietary AFB1 exposure resulted in gene over-expression of TNF-α, IL-1β and IL-6 in the liver of pigs, and the mRNA levels of inflflammatory factors (TNF-α, IL-12, IL-6) in the liver of pigs fed the diet containing 8% grape seed meal and AFB1 returned to the control levels [60]. The levels of TNF-α, IL-6 and IL-1β in the liver of rats with intra-uterine growth retardation were increased, which in the liver of IUGR rats fed with cistanche 400 mg kg−1 diet returned to the control level of normal birth weight rats [38]. The activation of NF-κB signaling pathway upregulated genes expression (NLRP3 and Caspase-1), which promoted IL-1β and IL-18 maturation and secretion and triggered an inflflammatory response [61]. Results in this study demonstrated that AFB1 administration signifificantly evaluated gene expression of inflflammation factors in the ileum of ducks, and dietary cistanche inhibited this gene expression, in line with a study that the activation of NLRP3 signaling pathway by AFB1 in rats [40]. Previous results showed that cistanche had the ability to inhibit NLRP3 protein expression via suppressing caspase-1 and IL-1β [29]. Thus, results in this study revealed that cistanche may be one of the promising feed additives to relieve inflflammation in the ileum and ileum damage induced by AFB1 administration by inhibiting the NF-κB signaling pathway

5. Conclusions

In conclusion, AFB1 administration induced ileum injury, oxidation stress and inflflammation via inhibiting the expression of downstream genes of the Nrf2-ARE signaling pathway and activating genes expression of downstream genes of the NF-κB signaling pathway. However, dietary cistanche markedly ameliorated ileum damage, oxidation stress, and inflflammation of ducks induced by AFB1 administration, possibly due to activating Nrf2-ARE and inhibit NF-κB signaling pathway (Figure 6). Results in this study provided powerful evidence that dietary cistanche is an effective feed additive to protect the ileum against acute injury induced by AFB1 administration via activating Nrf2-ARE and inhibiting NF-κB signaling pathway.