Use of let-7k-5p in preparation of medicine for treating arsenic-induced kidney injury of broilers

Abstract

The application discloses application of Let-7k-5p in preparation of a medicine for treating arsenic-induced kidney injury of broilers and belongs to the technical field of genetic engineering and toxicology. The application obtains Let-7k-5p with a nucleotide sequence as shown in SEQ ID NO. 1 by screening miRNA for targeting and regulating a chicken ChaC1 gene. Experimental results show that arsenic exposure significantly reduces the level of Let-7k-5p; up-regulation of the level of Let-7k-5p can directly target and inhibit ChaC1, restore GPX4 expression, reduce lipid peroxidation and iron accumulation, and relieve arsenic-induced ferroptosis and kidney injury; overexpression of Let-7k-5p can reverse pathological injury, abnormal kidney function or fibrosis of kidney tissue caused by arsenic accumulation. The application provides a new medicine for prevention and treatment of arsenic-induced kidney injury of broilers and provides a new detection target for diagnosis of arsenic-induced kidney injury of broilers.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and toxicology, and in particular to the application of Let-7k-5p in the preparation of drugs for treating arsenic-induced kidney damage in broilers. Background Technology

[0002] Arsenic is a widespread environmental metalloid toxicant, with the kidneys being the primary target organ for its accumulation and toxic damage. Ferroplasm, an iron-dependent, lipid peroxidation-driven regulatory form of cell death, plays a crucial role in various kidney diseases. miRNAs, as endogenous non-coding small RNAs, can negatively regulate gene expression at the post-transcriptional level and participate in environmentally induced organ damage.

[0003] Existing research confirms that arsenic exposure can induce ferroptosis in mammalian kidneys, and that some miRNAs are involved in the regulation of ferroptosis and kidney injury. ChaC1, as a glutathione-specific degrading enzyme, can inhibit GPX4 function by degrading GSH, thus promoting ferroptosis. However, the specific molecular mechanisms of arsenic-induced kidney injury in broilers remain unclear, the regulatory pathways of ferroptosis are not elucidated, and molecular markers for early diagnosis, warning, and intervention are lacking. In particular, there is a lack of research on miRNA regulation of the ChaC1 / GPX4 axis-mediated ferroptosis, and the targeted regulatory relationship between let-7k-5p and ChaC1 in arsenic-induced kidney injury in broilers has not been discovered, hindering effective prevention and mitigation of arsenic-poisoned kidney injury in broilers. Summary of the Invention

[0004] The purpose of this invention is to provide the application of Let-7k-5p in the preparation of drugs for treating arsenic-induced kidney injury in broilers, in order to solve the problems existing in the prior art. This invention has found that arsenic exposure significantly downregulates Let-7k-5p levels, and overexpression of Let-7k-5p can reverse the pathological damage to kidney tissue, abnormal kidney function, or fibrosis caused by arsenic accumulation. This provides a new drug for the prevention and treatment of arsenic-induced kidney injury in broilers, and also provides a new detection target for the diagnosis of arsenic-induced kidney injury in broilers.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides the application of Let-7k-5p in the preparation of a drug for treating kidney injury in broilers, wherein the nucleotide sequence of Let-7k-5p is shown in SEQ ID NO.1.

[0006] Furthermore, the kidney injury in the broilers is arsenic-induced kidney injury in the broilers.

[0007] Furthermore, Let-7k-5p exerts its therapeutic effect on kidney injury in broilers by targeting and regulating the expression levels of ChaC1 and GPX4.

[0008] The present invention also provides the application of Let-7k-5p in the preparation of a drug for preventing kidney damage in broilers, wherein the nucleotide sequence of Let-7k-5p is shown in SEQ ID NO.1.

[0009] Furthermore, the kidney injury in the broilers is arsenic-induced kidney injury in the broilers.

[0010] Furthermore, Let-7k-5p exerts a preventive effect against kidney damage in broilers by targeting and regulating the expression levels of ChaC1 and GPX4.

[0011] The present invention also provides the application of a reagent for detecting Let-7k-5p expression level in the preparation of products for diagnosing kidney injury in broilers, wherein the nucleotide sequence of Let-7k-5p is shown in SEQ ID NO.1.

[0012] Furthermore, the kidney injury in the broilers is arsenic-induced kidney injury in the broilers.

[0013] Furthermore, the product is a test kit.

[0014] The present invention discloses the following technical effects: This invention screened miRNAs that target and regulate the chicken ChaC1 gene, obtaining Let-7k-5p with the nucleotide sequence shown in SEQ ID NO. 1. Experimental results showed that arsenic exposure significantly downregulated Let-7k-5p levels; upregulation of Let-7k-5p levels directly targeted and inhibited ChaC1, restored GPX4 expression, reduced lipid peroxidation and iron accumulation, and alleviated arsenic-induced ferroptosis and kidney damage; overexpression of Let-7k-5p could reverse kidney tissue pathological damage, abnormal kidney function, or fibrosis caused by arsenic accumulation. This invention provides a new drug for the prevention and treatment of arsenic-induced kidney injury in broilers, and also provides a new detection target for the diagnosis of arsenic-induced kidney injury in broilers. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 Figure showing the effect of arsenic exposure on kidney histopathology (H&E) in broiler chickens; Figure 2 A stained image of fibrosis in the kidney tissue of arsenic-exposed broiler chickens. Figure 3 Ultrastructural images of arsenic-induced iron death in broiler kidneys observed by transmission electron microscopy; Figure 4 Figure 1 shows the results of the differential expression analysis of the ChaC1 gene in the kidneys of broilers induced by arsenic exposure. In the figure, A is a volcano plot / heatmap of differential expression of the ChaC1 gene screened by transcriptome sequencing; B is the reliability of ChaC1 gene expression in transcriptome sequencing verified by qPCR; C is the relative mRNA expression levels of ChaC1 and ATF4 in kidney tissue after arsenic exposure. Figure 5 Immunofluorescence images of arsenic exposure on ferroptosis marker proteins ChaC1 and GPX4 in broiler kidneys; where A is the immunofluorescence localization and expression map of ChaC1 protein in the control group and the arsenic-treated group; and B is the immunofluorescence localization and expression map of GPX4 protein in the control group and the arsenic-treated group. Figure 6 The effect of arsenic exposure on CEK cell survival and intracellular Fe 2+ The results of the horizontal effect are shown in the figure; where A is the effect of different concentrations of arsenic on the survival rate of CEK cells as detected by CCK-8 assay; B is the intracellular Fe 2+ Quantitative statistical analysis results of average fluorescence intensity; C represents Fe 2+ Fluorescent probe staining reveals the distribution of intracellular iron ion levels; Figure 7 The figure shows the effect of arsenic exposure on the expression of ChaC1 and GPX4 proteins in CEK cells; where A is the immunofluorescence image of ChaC1 after treatment with different concentrations of arsenic; B is the quantitative statistical result of the fluorescence intensity of ChaC1 protein; C is the immunofluorescence image of GPX4 after treatment with different concentrations of arsenic; and D is the quantitative statistical result of the fluorescence intensity of GPX4 protein. Figure 8 Figure showing the bioinformatics prediction results of candidate miRNAs targeting ChaC1; Figure 9 The expression levels of miRNA genes in kidney tissue and CEK cells of arsenic-exposed broiler chickens were determined. A represents the expression level of gga-let-7d in kidney tissue and CEK cells; B represents the expression level of gga-let-7k-5p in kidney tissue and CEK cells; C represents the expression level of gga-miR-15a in kidney tissue and CEK cells; D represents the expression level of gga-miR-15b-5p in kidney tissue and CEK cells; E represents the expression level of gga-miR-15c-5p in kidney tissue and CEK cells; F represents the expression level of gga-miR-16-5p in kidney tissue and CEK cells; and G represents the expression level of gga-miR-16c-5p in kidney tissue and CEK cells. Figure 10 Figure showing the results of dual-luciferase reporter assays to validate let-7k-5p targeting ChaC1; Figure 11Figure 1 shows the effect of knocking down ChaC1 on the expression levels of genes and proteins related to arsenic-induced ferroptosis and lipid metabolism in CEK cells. In the figure, A represents ChaC1 mRNA expression; B represents GPX4 mRNA expression; C represents FATP1 mRNA expression; D represents C / EBPα mRNA expression; E represents CD36 mRNA expression; F represents HMGCR mRNA expression; G represents SREBP1C mRNA expression; H represents PPARα mRNA expression; I represents Western blot bands for GPX4 and HO-1; J represents grayscale analysis of GPX4 protein; and K represents grayscale analysis of HO-1 protein. Figure 12 The figure shows the effects of ChaC1 overexpression on the expression levels of genes and proteins related to arsenic-induced ferroptosis and lipid metabolism in CEK cells. A represents ChaC1 mRNA expression; B represents GPX4 mRNA expression; C represents FATP1 mRNA expression; D represents SREBP1C mRNA expression; E represents C / EBPα mRNA expression; F represents PPARα mRNA expression; G represents CD36 mRNA expression; H represents HMGCR mRNA expression; I represents Western blot bands for GPX4 and HO-1; J represents grayscale analysis of GPX4 protein; and K represents grayscale analysis of HO-1 protein. Figure 13 The figure shows the effect of inhibiting let-7k-5p on the expression levels of genes and proteins related to arsenic-induced ferroptosis and lipid metabolism in CEK cells. In the figure, A represents ChaC1 mRNA expression; B represents GPX4 mRNA expression; C represents FATP1 mRNA expression; D represents SREBP1C mRNA expression; E represents C / EBPα mRNA expression; F represents PPARα mRNA expression; G represents CD36 mRNA expression; H represents HMGCR mRNA expression; I represents Western blot bands for GPX4 and HO-1; J represents grayscale analysis of GPX4 protein; and K represents grayscale analysis of HO-1 protein. Figure 14 The figure shows the effects of overexpression of let-7k-5p on the expression levels of genes and proteins related to arsenic-induced ferroptosis and lipid metabolism in CEK cells. In the figure, A represents ChaC1 mRNA expression; B represents GPX4 mRNA expression; C represents FATP1 mRNA expression; D represents SREBP1C mRNA expression; E represents C / EBPα mRNA expression; F represents PPARα mRNA expression; G represents CD36 mRNA expression; H represents HMGCR mRNA expression; I represents Western blot bands for GPX4 and HO-1; J represents grayscale analysis of GPX4 protein; and K represents grayscale analysis of HO-1 protein. Figure 15The figure shows the results of double overexpression validation of the effects of let-7k-5p / ChaC1 / GPX4 axis on the expression levels of ferroptosis and lipid metabolism-related genes and proteins; where A represents ChaC1 mRNA expression; B represents GPX4 mRNA expression; C represents FATP1 mRNA expression; D represents SREBP1C mRNA expression; E represents C / EBPα mRNA expression; F represents PPARα mRNA expression; G represents CD36 mRNA expression; H represents HMGCR mRNA expression; I represents GPX4 and HO-1 Western blot bands; J represents GPX4 protein grayscale analysis; and K represents HO-1 protein grayscale analysis. Detailed Implementation

[0017] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0018] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0019] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0020] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0021] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0022] The nucleotide sequence of chicken-derived Let-7k-5p (gga-let-7k-5p) of the present invention is shown in SEQ ID NO.1; SEQ ID NO. 1: UGAGGUAGUAGAUUGAAUAGUU.

[0023] The nucleotide sequence of the chicken-derived ChaC1 gene is shown in NCBI: NM_001199656.2.

[0024] Example 1 I. Experimental Materials and Methods 1. Experimental Materials One-day-old healthy Hy-Line roosters; chicken embryo kidney cells (CEK); arsenic trioxide (As2O3); gga-let-7k-5pmimics, inhibitor and negative control; ChaC1-specific siRNA and negative control; miRNA and mRNA quantitative PCR kits; Western blot reagents; immunofluorescence kit; dual-luciferase reporter gene detection system; ChaC1 overexpression vector (pcDNA3.1-ChaC1) and empty vector control.

[0025] 2. Experimental Methods 2.1 Animal Model Construction One-day-old Hy-Line roosters were selected and, after a three-day acclimatization period, randomly divided into a control group and an arsenic exposure group, with at least three replicates per group and 15 birds per replicate. The control group was fed a basal diet, while the arsenic exposure group received 2.5 mg / kg arsenic trioxide supplemented to their basal diet. Both groups had free access to feed and water for 90 days. Light, temperature, and humidity were maintained according to standard broiler rearing practices. Kidney tissue was collected on day 90 and stored at -80℃ for later use.

[0026] 2.2 Cell Model Construction Chicken embryonic kidney cells (CEKs) were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. In vitro arsenic injury models were established by treating cells with 0, 1, 4, and 8 μM arsenic trioxide for 24 h. Cell viability was determined using the CCK-8 assay to assess the IC50. 50 The concentration was approximately 8 μM, and 1 μM was used as the treatment concentration for subsequent functional experiments.

[0027] 2.3 Gene overexpression, knockdown, and transfection methods Cell transfection was performed according to the Thermo Fisher Scientific Lipofectamine® 2000 Reagent transfection kit instructions.

[0028] (1) Overexpression of let-7k-5p: let-7k-5p mimics and mimics NC were transfected into CEK cells, with a final transfection concentration of 20 nM; (2) let-7k-5p inhibition: let-7k-5p inhibitor and inhibitor NC were transfected respectively, with a final transfection concentration of 40 nM; (3) ChaC1 knockdown: Transfect ChaC1-specific siRNA (#446, #583) and siRNA NC, with a final concentration of 30 nM; (4) ChaC1 overexpression: transfect pcDNA3.1-ChaC1 recombinant plasmid and empty vector plasmid, with a final concentration of 1 μg / mL; The medium was changed 6 hours after transfection, and arsenic treatment was performed 24 hours later. Cells were then cultured for another 24 hours before being collected.

[0029] 2.4 Dual-luciferase reporter gene assay ChaC1 3'UTR wild-type (WT) and mutant (MUT) dual-luciferase vectors were constructed and co-transfected into 293T cells with let-7k-5p or NC, respectively. 24 h after transfection, fluorescence intensity was measured according to the dual-luciferase assay kit instructions. Using Renal fluorescence as an internal control, relative fluorescence activity was calculated to verify the direct targeting relationship between let-7k-5p and ChaC1.

[0030] 2.5 Gene and Protein Detection Methods Total RNA extraction, cDNA synthesis, and qPCR were performed according to the instructions of a commercially available RNA extraction and quantitative PCR kit. U6 was used as the miRNA internal control, and β-actin was used as the mRNA internal control. -ΔΔCt The relative expression level is calculated using this method.

[0031] Western blot and immunofluorescence were performed according to standard methods (published methods may be cited) to detect the expression and localization of proteins such as ChaC1, GPX4, SLC7A11, and HO-1.

[0032] 2.6 Ferrocytosis and its Relationship with Renal Function Detecting intracellular Fe 2+ The study measured renal function indicators such as serum creatinine (CRE) and blood urea nitrogen (BUN), as well as lipid peroxidation levels, antioxidant enzyme (SOD, CAT, GPx) activity, and MDA content. It also examined the characteristic morphology of mitochondrial ferroptosis using transmission electron microscopy.

[0033] 3. Experimental grouping and control setup (1) Blank control group; (2) Arsenic treatment group alone; (3) mimics NC + arsenic group; (4) inhibitor NC + arsenic group; (5) siRNA NC + arsenic group; (6) empty vector plasmid + arsenic group; (7) let-7k-5p overexpression + arsenic group; (8) let-7k-5p inhibition + arsenic group; (9) ChaC1 knockdown + arsenic group; (10) ChaC1 overexpression + arsenic group.

[0034] All experiments were independently repeated three times. Data are expressed as mean ± standard deviation. One-way ANOVA was used, and P < 0.05 was considered statistically significant.

[0035] II. Experimental Results 1. Histopathological observation of kidney tissue HE staining results are as follows Figure 1 As shown, the kidney tissue structure of the control group (Con group) broilers was clear and intact, the glomeruli were regular in shape, and the Bowman's capsule structure was clear; the renal tubular epithelial cells were tightly arranged, the cytoplasm was evenly distributed, and the cell nuclei were clearly visible. No obvious degeneration, necrosis and inflammatory cell infiltration were observed, which is consistent with the histological characteristics of normal kidneys.

[0036] In contrast, the high-dose arsenic exposure group (As group) exhibited significant degenerative pathological changes. Observations revealed widespread vacuolar degeneration of renal tubular epithelial cells, with the cytoplasm filled with numerous vacuoles of varying sizes. Some cell nuclei showed typical condensation (yellow arrows), fragmentation, or even dissolution (black arrows), leading to localized cell shedding and exposure of the basement membrane. Simultaneously, the renal interstitial region showed significant inflammatory cell infiltration, predominantly lymphocytes (green arrows), accompanied by interstitial edema, forming clearly defined inflammatory lesions.

[0037] 2. Observation of renal tissue fibrosis Masson trichrome staining results further revealed the profound impact of arsenic exposure on the process of renal interstitial fibrosis.

[0038] like Figure 2 As shown, in the control group (Con group), only a very small amount of fine blue collagen fibers were observed in the renal interstitium, which were distributed in a fine network and formed a normal connective tissue framework. No abnormal deposition was observed.

[0039] In the high-dose arsenic exposure group (As group), the amount of blue collagen fiber deposition in the renal interstitium was significantly increased, such as... Figure 2As indicated by the black arrow, the blue collagen fibers aggregate in large sheets or cords, with significantly thickened and disordered fiber structures, especially densely deposited around the renal tubules and in the intervascular spaces. This pathological phenotype is consistent with the morphological characteristics of early-stage renal stromal fibrosis, strongly demonstrating that arsenic exposure not only induces direct damage to parenchymal cells but also initiates and promotes the fibrotic remodeling process of renal tissue by activating excessive deposition of the extracellular matrix.

[0040] 3. Ultrastructural observation of kidney tissue To further investigate the microscopic effects of subchronic arsenic exposure on kidney damage in broilers at the organelle level, this invention utilizes transmission electron microscopy to conduct in-depth observation of the ultrastructure of kidney tissue samples.

[0041] The kidney cells of the control group (Con group) exhibited highly regular ultrastructural features. The nuclei of the renal tubular epithelial cells were regularly shaped, with uniform chromatin distribution, and no signs of condensation, fragmentation, or marginalization were observed. The cytoplasm contained abundant mitochondria, mostly in typical spindle or elongated oval shapes, with a clear and intact double-membrane structure and neatly and densely arranged mitochondrial cristae. The electron density of the mitochondrial matrix was uniform, and no pathological changes such as swelling, vacuolation, or membrane damage were observed. These morphological characteristics indicate that, under normal physiological conditions, renal tubular epithelial cells maintain good subcellular structural integrity and energy metabolic homeostasis.

[0042] In stark contrast, kidney cells in the arsenic exposure group (As group) exhibited significant ultrastructural damage and displayed typical morphological phenotypes of ferroptosis. In the damaged renal tubular epithelial cells, mitochondria underwent characteristic remodeling (red arrows): their shape became significantly rounded. Simultaneously, the number of mitochondrial cristae decreased sharply, their arrangement became disordered, and in some areas, the cristae structure completely disappeared. These changes are core morphological markers of ferroptosis, distinguishing it from apoptosis and necrosis. Furthermore, numerous high-electron-density lipid droplets of varying sizes and with well-defined boundaries were observed in the cytoplasm of damaged cells (yellow arrows). These round or elliptical lipid deposits clearly reveal the lipid metabolism disorder induced by arsenic exposure, providing metabolic substrates for the lipid peroxidation chain reaction during ferroptosis. Some cell nuclei also showed mild pyknosis and autophagosome aggregation, further confirming that the cells were under extreme oxidative stress and metabolic imbalance.

[0043] 4. Multidimensional verification of arsenic exposure-induced kidney ferritosis in broilers 4.1 Effects of in vivo tissue-level arsenic exposure on the renal ferroptosis pathway To further investigate the molecular mechanisms of arsenic-induced kidney damage, this invention first performed transcriptomic sequencing on kidney tissue. The results are as follows: Figure 4 As shown in Figure A, arsenic exposure induces the expression of 62 differentially expressed genes in the kidneys, among which ChaC1 is one of the mRNAs with the most significant upregulation trend.

[0044] To verify the accuracy of the RNA-Seq sequencing results, qPCR was used to verify the expression of the differentially expressed gene ChaC1 in the kidney. The results are as follows: Figure 4 As shown in Figure B, this result indicates that the expression of the selected genes is basically consistent with the analysis results of RNA-seq, demonstrating that the RNA-seq sequencing results are accurate and reliable.

[0045] The results of quantitative real-time PCR are as follows Figure 4 As shown in Figure C, the mRNA expression levels of ChaC1 and its upstream transcription factor ATF4 in the kidney tissue of the arsenic-treated group were significantly increased, indicating that arsenic exposure may activate the ATF4 / ChaC1 signaling axis at the transcriptional level.

[0046] Further immunofluorescence analysis was performed on the ChaC1 protein in the kidney tissue. Figure 5 As shown in Figure A, ChaC1 was only weakly distributed in the kidney tissue of the control group (Group C), while in the arsenic-treated group (Group H), ChaC1 protein was densely aggregated in the cytoplasm of renal tubular epithelial cells, and the fluorescence signal intensity was significantly enhanced. This is consistent with the results of transcriptomics and qPCR, confirming that arsenic significantly induced the protein expression of ChaC1.

[0047] Given that ChaC1 is involved in the regulation of ferroptosis, this invention further analyzes ferroptosis marker proteins in kidney tissue. Immunofluorescence results are as follows: Figure 5 As shown in Figure B, the GPX4 protein in the control group (Group C) showed a uniform and continuous strong positive distribution in the cytoplasm of renal tubular epithelial cells, while the GPX4 fluorescence signal in the arsenic-treated group (Group H) was significantly weakened and the distribution range was significantly reduced.

[0048] 4.2 Direct Induction of Ferroptosis by Arsenic Exposure at the In Vitro Cellular Level To further verify the direct toxic effects of arsenic on kidney cells and the related mechanisms of ferroptosis, this invention uses a primary chicken embryo kidney cell model for in vitro experiments.

[0049] First, the effect of arsenic exposure on CEK cell survival was detected using the CCK-8 assay. The results are as follows: Figure 6 As shown in Figure A, As 3+ It can significantly reduce the survival rate of CEK cells in a concentration-dependent manner, and the half-maximal inhibitory concentration (IC50) was calculated. 50 The concentration was 8 μM. Therefore, this invention selected four concentration gradients of 0, 1, 4, and 8 μM for further investigation of the mechanism.

[0050] Given the central role of iron ions in ferroptosis, this invention further investigated intracellular Fe after arsenic exposure. 2+ Changes in level. Fe 2+ Fluorescent staining results as follows Figure 6 As shown in Figure BC, with increasing arsenic treatment concentration, intracellular Fe... 2+ The fluorescence signal intensity gradually increased, showing a clear concentration-dependent upward trend. This result indicates that arsenic exposure can lead to the accumulation of iron ions in CEK cells, providing a sufficient iron source for the Fenton reaction and lipid peroxidation.

[0051] To further verify the impact of arsenic exposure on key regulatory molecules of ferroptosis, this invention performed immunofluorescence analysis on ChaC1 and GPX4 proteins in CEK cells. The results are as follows: Figure 7 As shown, GPX4 protein exhibited a uniform cytoplasmic distribution and strong fluorescence signal in the control group cells. With increasing arsenic treatment concentration, the GPX4 fluorescence signal gradually weakened, and the positive cell rate decreased. Simultaneously, ChaC1 protein showed only a weak distribution in the control group, while the arsenic-treated group showed a significantly enhanced fluorescence signal and a granular aggregated distribution. These results indicate that arsenic can induce upregulation of ChaC1 expression at the cellular level while inhibiting GPX4 expression, which is highly consistent with observations at the in vivo tissue level.

[0052] 5. Identification and validation of gga-let-7k-5p targeting negative regulation of ChaC1 expression 5.1 Bioinformatics-based prediction of candidate regulatory miRNAs for gga-ChaC1 To systematically elucidate the posttranscriptional regulatory mechanism of the gga-ChaC1 gene in broiler kidneys, this invention combines mainstream bioinformatics algorithms such as TargetScan, miRDB, and miRmap to screen and perform topological analysis on potential miRNA binding sites in the 3' untranslated region of the gga-ChaC1 transcript sequence.

[0053] Prediction results are as follows Figure 8 As shown, the 3'UTR region of gga-ChaC1 contains several evolutionarily highly conserved miRNA binding anchors. Among these, core candidate regulators point to members of the gga-let-7 family (gga-let-7k-5p, gga-let-7d) and members of the gga-miR-15 / 16 family (gga-miR-15a, gga-miR-15b-5p, gga-miR-15c-5p, gga-miR-16-5p, and gga-miR-16c-5p). Sequence alignment analysis indicates that the seed sequences of these miRNAs can achieve precise base pairing with complementary sequences on the 3'UTR of gga-ChaC1 mRNA, suggesting that these miRNAs may participate in post-transcriptional regulation of gga-ChaC1 expression levels by directly targeting the 3'UTR of gga-ChaC1.

[0054] To further screen regulatory molecules that actually respond under arsenic exposure conditions, this invention uses qPCR to detect the expression changes of the above-mentioned miRNAs in kidney tissue and CEK cells, respectively.

[0055] like Figure 9 As shown, the intersection of in vitro and in vivo experimental results revealed that only gga-let-7k-5p exhibited an opposite trend to its target gene ChaC1. Specifically, while ChaC1 expression increased after arsenic exposure, gga-let-7k-5p expression significantly decreased, consistent with the characteristics of miRNA negatively regulating target gene expression. Subsequent experiments will be conducted using arsenic-treated CEK cells to explore the underlying mechanisms.

[0056] 6. Dual-luciferase reporter assay confirmed the direct targeting effect of gga-let-7k-5p on ChaC1. To further confirm the direct molecular interaction between let-7k-5p and gga-ChaC1, this invention uses a dual-luciferase reporter gene system in HEK-293T cells for verification. HEK-293T cells are commonly used in dual-luciferase reporter assays due to their high transfection efficiency and low background interference.

[0057] Luciferase reporter vectors containing wild-type and mutant sequences of the gga-ChaC1 3'UTR binding site were constructed and co-transfected into cells with let-7k-5p mimics or mimics NC, respectively. Four groups were set up: WT+mimicsNC, WT+mimics, MT+mimics NC, and MT+mimics.

[0058] The results are as follows Figure 10 As shown, compared with the WT+mimics NC group, the relative luciferase activity of cells in the WT+mimics group was significantly reduced by approximately 40% (P<0.01); while there was no significant difference in luciferase activity between the MT+mimics group and the MT+mimics NC group (P>0.05). These results confirm that let-7k-5p can directly target and regulate its expression by binding to the ChaC1 3'UTR.

[0059] 7. Functional validation of let-7k-5p / ChaC1 / GPX4 axis-mediated arsenic-induced ferroptosis in CEK cells 7.1 Knockdown of ChaC1 antagonizes arsenic-induced ferroptosis and lipid metabolism disorders To definitively confirm the core damaging role of ChaC1 in let-7k-5p / ChaC1 / GPX4 axis-mediated arsenic-induced CEK cell cytotoxicity, this invention employs a loss-of-function strategy, utilizing specific small interfering RNA to target and knock down ChaC1 expression. The experiment was divided into a control group, an arsenic treatment group, an arsenic combined with a negative control siRNA group, and an arsenic combined with ChaC1 knockdown group. Figure 11 As shown, the RT-qPCR results indicated that there was no statistically significant difference in the mRNA levels of all detected genes between the arsenic group and the arsenic combined with negative control siRNA group (P>0.05), further confirming that the transfection reagent and non-specific nucleic acid sequence did not significantly interfere with the experimental system.

[0060] The results are as follows Figure 11 As shown in AB and IK, in the study of functional recovery of the ferroptosis defense system, 1 μM arsenic exposure significantly upregulated the expression of the pro-ferroptosis factor ChaC1, approximately 1.6 times that of the control group (P<0.001), and significantly downregulated the level of the anti-ferroptosis core enzyme GPX4, approximately 0.9 times that of the control group (P<0.05). After transfection with ChaC1-siRNA, the mRNA level of ChaC1 in the arsenic-combined ChaC1 knockdown group dropped sharply from 1.6 times that of the arsenic group to 0.4 times that of the control group (P<0.001), demonstrating extremely high knockdown efficiency. More importantly, with the effective inhibition of ChaC1, the expression level of GPX4, which was originally suppressed by arsenic, showed a highly significant compensatory recovery, reaching 1.6 times that of the control group (P<0.001). Western blot results further confirmed that at the protein level, arsenic exposure led to a decrease in GPX4 protein expression, while GPX4 protein expression was significantly restored after ChaC1 knockdown, consistent with the trend of mRNA changes. Meanwhile, arsenic exposure induced the upregulation of HO-1 protein expression, while HO-1 expression significantly decreased after ChaC1 knockdown. This phenomenon strongly demonstrates that ChaC1 is a key negative regulatory node mediating arsenic-induced downregulation of GPX4, and that targeting and silencing ChaC1 can significantly enhance the cell's antioxidant defense system, thereby antagonizing the arsenic-triggered ferroptosis cascade.

[0061] Further evaluation of the protective effect on lipid metabolism homeostasis is needed. For example... Figure 11 As shown in Figure G, in the regulation of lipid synthesis, the activation of SREBP1C expression induced by arsenic exposure (1.6-fold, P<0.01) was completely reversed after knockdown of ChaC1, reducing its expression level to 0.5-fold of the control group (P<0.001). Regarding fatty acid transport and uptake, as... Figure 11As shown in C and E, the loss of ChaC1 significantly reversed the inhibitory effect of arsenic on FATP1 and CD36, causing their expression levels to rise to 1.4 times that of the control group (P<0.001, P<0.01). Particularly noteworthy is that, as... Figure 11 As shown in Figures D, F, and H, the key genes involved in the regulation of fatty acid oxidation and cholesterol metabolism, C / EBPα, PPARα, and HMGCR, showed a strong re-expression trend in the ChaC1 knockdown group. Among them, the expression level of C / EBPα increased to 2.7 times that of the control group (P<0.001), while HMGCR showed a supercompensatory and dramatic upregulation, reaching 3.1 times that of the control group (P<0.001).

[0062] In summary, targeted knockdown of ChaC1 not only blocks arsenic-induced ferroptosis but also comprehensively repairs the damaged lipid metabolism transcriptome. The significant "super-recovery" phenotypes of HMGCR and C / EBPα, in particular, suggest that ChaC1 may be at the intersection of multiple metabolic pathways in maintaining lipid homeostasis in CEK cells. This section's results, from a functional loss-of-function perspective, provide final evidence for the let-7k-5p / ChaC1 / GPX4 axis-mediated arsenic-induced CEK cell damage. The success of this rescue experiment confirms that ChaC1 is a core functional target of arsenic-induced chicken embryo nephrotoxicity, and that intervening in ChaC1 expression is an effective biological means to alleviate arsenic poisoning damage.

[0063] 7.2 Overexpression of ChaC1 mimics and enhances arsenic-induced cell damage effects Previous studies have confirmed that ChaC1 is a direct downstream target gene of let-7k-5p using dual-luciferase reporter gene assays. To further confirm the function of ChaC1 at the cellular level and verify whether it can mediate arsenic-induced cell damage as a key effector, this invention constructed a eukaryotic overexpression vector for the ChaC1 gene and established corresponding arsenic exposure groups and arsenic-treated empty vector transfection groups as controls. Figure 12 As shown, the RT-qPCR results indicated that there were no statistically significant differences in gene expression levels between the arsenic-treated group and the arsenic-treated group (P>0.05). This result effectively ruled out the interference of plasmid transfection operation and the vector itself on the transcription profile of endogenous genes in cells, ensuring the specificity and reliability of subsequent functional experiments.

[0064] like Figure 12As shown in Figures AB and IK, in the study of regulation of ferroptosis homeostasis, arsenic exposure significantly induced the mRNA expression of ChaC1, a key pro-ferroptosis factor, in CEK cells, approximately 1.5 times that of the control group (P<0.01), accompanied by a significant inhibition of the transcriptional level of the core antioxidant enzyme GPX4, approximately 0.85 times that of the control group (P<0.01). When ChaC1 was overexpressed exogenously, the intracellular ChaC1 level in the arsenic-exposed combined ChaC1 overexpression group showed a cumulative jump on top of the arsenic exposure-induced level, with its expression significantly upregulated to 2.4 times that of the control group (P<0.001). Simultaneously, the mRNA level of GPX4 showed a dramatic negative response, further significantly downregulated to 0.6 times that of the arsenic group (P<0.001). At the protein level, arsenic exposure led to a decrease in GPX4 protein expression, with a compensatory increase in HO-1 protein expression; however, after ChaC1 overexpression, GPX4 protein expression further decreased, and HO-1 protein expression further increased. This finding indicates that the upregulation of ChaC1 has a significant pro-ferroptosis effect, which can mimic and amplify the oxidative stress damage and ferroptosis tendency caused by arsenic exposure by strongly inhibiting the antioxidant defense system of GPX4.

[0065] Further investigation into alterations in lipid metabolism pathways revealed that ChaC1 overexpression plays a specific regulatory role in arsenic-induced lipid metabolism disorders. Figure 12 As shown in Figure D, regarding lipid synthesis regulation, the expression level of SREBP1C, a core transcription factor in lipid synthesis, significantly increased from 1.4-fold to 2.3-fold in the arsenic-enhanced ChaC1 overexpression group (P<0.001), suggesting that ChaC1 can synergistically activate lipid synthesis programs through arsenic exposure. In fatty acid utilization and transport, such as... Figure 12 As shown in C and G, overexpression of ChaC1 significantly enhanced the inhibitory effect of arsenic on FATP1 and CD36, with the expression level of CD36 decreasing sharply from 0.45-fold to 0.15-fold in the arsenic group (P<0.01), indicating a more severe disruption of intracellular fatty acid uptake and homeostasis. However, as... Figure 12 As shown in Figures EF and H, this invention also observed that the downregulation of C / EBPα, PPARα, and HMGCR genes by overexpression of ChaC1 did not have a significant additive effect (P>0.05). This suggests that in the complex pathological network induced by arsenic, the aforementioned genes involved in fatty acid oxidation and cholesterol metabolism may be regulated by other downstream signaling pathways of let-7k-5p or by pathways independent of ChaC1.

[0066] In summary, high expression of the ChaC1 gene significantly mimics and enhances the damaging effects of arsenic on CEK cells, particularly in triggering the ferroptosis core cascade and disrupting specific lipid synthesis and transport pathways. The results in this section, from a functional gain perspective, confirm the important role of ChaC1 as an effector in the let-7k-5p / ChaC1 / GPX4 regulatory axis, providing direct functional evidence for a deeper understanding of the molecular mechanisms of arsenic-induced nephrotoxicity in chicken embryos.

[0067] 7.3 Inhibition of let-7k-5p exacerbates arsenic-induced ferroptosis and lipid metabolism disorders in CEK cells. To verify the regulatory role of endogenous let-7k-5p on arsenic-induced CEK cell cytotoxicity, this invention specifically downregulated let-7k-5p expression by transfection with a let-7k-5p inhibitor, and used RT-qPCR to detect changes in the mRNA expression of genes related to ferroptosis and lipid metabolism in each group of cells. Figure 13 ).

[0068] like Figure 13 As shown in Figures AB and IK, compared with the control group, the expression level of ChaC1 in the arsenic-treated group and the group co-treated with arsenic and the negative control inhibitor showed a significant upward trend, approximately 1.6-1.7 times that of the control group (P<0.001), while the mRNA level of GPX4, the core gene for ferroptosis and antioxidant activity, significantly decreased, approximately 0.8 times that of the control group (P<0.05), indicating that arsenic exposure had initially triggered differential expression of ferroptosis-related genes. After further transfection with the let-7k-5p inhibitor, the induction effect of ChaC1 was significantly amplified, with its expression level jumping dramatically to 2.4 times that of the control group (P<0.001); simultaneously, the mRNA expression level of GPX4 was further downregulated to 0.5 times that of the arsenic group (P<0.01). Western blot results further confirmed that, at the protein level, arsenic exposure led to a decrease in GPX4 protein expression and a compensatory increase in HO-1 protein expression; while inhibition of let-7k-5p further decreased GPX4 protein expression and further increased HO-1 protein expression. The results confirm that inhibiting endogenous let-7k-5p relieves its negative regulation of the target gene ChaC1, thereby exacerbating arsenic-induced ferroptosis sensitivity.

[0069] Regarding the regulation of the expression of lipid metabolism-related factors, such as Figure 13 As shown in Figure D, arsenic exposure significantly activated the expression of SREBP1C, a key transcription factor in lipid synthesis (P<0.01), and inhibited the expression of the fatty acid transporter FATP1 (e.g., ...). Figure 13 As shown in C), the uptake receptor CD36 (as shown in C) Figure 13 As shown in G), oxidation regulator PPARα (such as...) Figure 13As shown in F), cholesterol synthase HMGCR (such as Figure 13 (as shown in H) and transcription factor C / EBPα (as shown in H) Figure 13 The mRNA levels (as shown in Figure E) were significantly lower than those in the control group (P < 0.05 or P < 0.001). Notably, in the inhibitor co-treatment group, all of the above-mentioned lipid metabolism disorders showed a significant worsening trend: SREBP1C expression further increased to 2.3 times that of the control group (P < 0.01), while the expression levels of FATP1 and CD36 significantly decreased to 0.2 times (P < 0.01 and P < 0.05), respectively. The inhibitory effects of PPARα, HMGCR, and C / EBPα expression were also significantly additive on the basis of arsenic exposure. These data fully demonstrate that let-7k-5p plays an important anti-damage role in arsenic-induced CEK cytotoxicity, and its expression inhibition is a key pathological link in the arsenic-induced aggravated ferroptosis and the collapse of lipid metabolism homeostasis.

[0070] 7.4 Overexpression of let-7k-5p alleviates arsenic-induced ferroptosis and lipid metabolism disorders in CEK cells. Based on the established fact that inhibiting let-7k-5p exacerbates cell damage, this invention constructs a gain-of-function experiment by transfecting let-7k-5p mimics to verify whether exogenous compensation for let-7k-5p can protect against arsenic-induced CEK cell damage. RT-qPCR results showed no significant differences in gene expression levels between the arsenic-treated group and the co-treatment group with arsenic and negative control mimics, ruling out interference from transfection procedures.

[0071] like Figure 14 As shown in Figures AB and IK, in the expression analysis of ferroptosis-related genes, compared with the control group, arsenic exposure significantly induced the expression of the ferroptosis-promoting gene ChaC1, approximately 1.5 times that of the control group (P<0.05), and suppressed the mRNA level of the core antioxidant gene GPX4, approximately 0.6 times that of the control group (P<0.001). However, after transfection with let-7k-5p mimics, the arsenic-induced gene expression abnormalities were significantly reversed: the expression level of ChaC1 decreased significantly from 1.5 times that of the arsenic group to 0.5 times (P<0.001), while the expression level of GPX4 increased significantly from 0.6 times to 1.1 times (P<0.001). Western blot results further confirmed that at the protein level, arsenic exposure led to a decrease in GPX4 protein expression and a compensatory increase in HO-1 protein expression; however, after overexpression of let-7k-5p, GPX4 protein expression significantly rebounded, and HO-1 protein expression significantly decreased, consistent with the trend of mRNA changes. These results indicate that exogenous upregulation of let-7k-5p can antagonize arsenic-induced ferroptosis by downregulating its target gene ChaC1 and restoring the expression of GPX4.

[0072] Meanwhile, overexpression of let-7k-5p also showed a significant alleviating effect on arsenic-induced lipid metabolism disorders. Results are as follows: Figure 14 As shown in Figure D, arsenic exposure significantly activated the expression of SREBP1C, a key transcription factor in lipid synthesis (P<0.05), and inhibited a series of key genes involved in fatty acid transport, oxidation, cholesterol synthesis, and transcriptional regulation (P<0.01 or P<0.001). In the let-7k-5p mimics transfection group, overexpression of SREBP1C was significantly inhibited, decreasing to 0.4-fold of the control group (P<0.001); simultaneously, FATP1 ( Figure 14 C), C / EBPα ( Figure 14 E), PPARα ( Figure 14 (f), CD36 Figure 14 (G) and HMGCR ( Figure 14 The mRNA levels of H+ showed a statistically significant rebound (P<0.05).

[0073] The data above indicate that let-7k-5p is a key factor regulating the toxic effects of arsenic. Overexpression of let-7k-5p in CEK cells can effectively reverse ferroptosis and impaired lipid metabolism pathways caused by arsenic exposure. This further confirms, from a functional gain perspective, the core regulatory role of the let-7k-5p / ChaC1 / GPX4 axis in the development and progression of arsenic-induced CEK cell toxicity.

[0074] 7.5 Dual overexpression validation of let-7k-5p / ChaC1 / GPX4 axis regulation of ferroptosis and lipid metabolism To confirm the core regulatory role of the let-7k-5p / ChaC1 / GPX4 axis in arsenic-induced ferroptosis, this invention conducted a double overexpression functional verification experiment in CEK cells.

[0075] The results are as follows Figure 15 As shown in AB and IK, compared with the control group, the arsenic-treated group showed significantly increased ChaC1 expression (P<0.05), significantly decreased let-7k-5p expression (P<0.05), and significantly downregulated GPX4 expression (P<0.05).

[0076] Simultaneously, lipid metabolism reprogramming was observed: SREBP1C expression was significantly upregulated ( Figure 15 D, P<0.05), FATP1 ( Figure 15 C), CD36 Figure 15 G), PPARα ( Figure 15 (F), C / EBPα ( Figure 15Arsenic exposure significantly downregulated GPX4 protein expression (P<0.05). Western blot results further showed that arsenic exposure led to a decrease in GPX4 protein expression, with a compensatory increase in HO-1 protein expression. Overexpression of ChaC1 alone mimicked the effects of arsenic exposure: ChaC1 expression was significantly increased (P<0.01), let-7k-5p showed no significant change, GPX4 expression further decreased to even lower levels (P<0.01), and lipid metabolism disorder phenotypes were exacerbated (SREBP1C upregulated, FATP1 downregulated, CD36 downregulated). At the protein level, overexpression of ChaC1 further decreased GPX4 protein expression and further increased HO-1 protein expression. In the dual overexpression group (simultaneous overexpression of ChaC1 and let-7k-5p), although ChaC1 expression was still significantly higher than the control group (P<0.05), it was significantly lower than the ChaC1-only overexpression group (P<0.05); GPX4 expression significantly rebounded, approaching the control group level; and the expression of lipid metabolism-related genes tended to normalize. At the protein level, GPX4 protein expression in the double overexpression group rebounded to near the control group level, and HO-1 protein expression also tended to normalize. These results indicate that let-7k-5p can partially counteract the inhibitory effect of ChaC1 overexpression on GPX4 and alleviate lipid metabolism disorders by targeting and inhibiting ChaC1 expression, confirming that the let-7k-5p / ChaC1 / GPX4 axis is the core regulatory pathway for arsenic-induced ferroptosis and lipid metabolism reprogramming.

[0077] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of Let-7k-5p in the preparation of a drug for treating kidney injury in broilers, characterized in that, The nucleotide sequence of Let-7k-5p is shown in SEQ ID NO.

1.

2. The application as described in claim 1, characterized in that, The kidney damage in the broilers was caused by arsenic.

3. The application as described in claim 1, characterized in that, The Let-7k-5p exerts its therapeutic effect on kidney injury in broilers by targeting and regulating the expression levels of ChaC1 and GPX4.

4. The application of Let-7k-5p in the preparation of drugs for preventing kidney damage in broilers, characterized in that, The nucleotide sequence of Let-7k-5p is shown in SEQ ID NO.

1.

5. The application as described in claim 4, characterized in that, The kidney damage in the broilers was caused by arsenic.

6. The application as described in claim 4, characterized in that, The Let-7k-5p exerts its effect in preventing kidney damage in broilers by targeting and regulating the expression levels of ChaC1 and GPX4.

7. The application of a reagent for detecting Let-7k-5p expression levels in the preparation of products for diagnosing kidney injury in broilers, characterized in that, The nucleotide sequence of Let-7k-5p is shown in SEQ ID NO.

1.

8. The application as described in claim 7, characterized in that, The kidney damage in the broilers was caused by arsenic.

9. The application as described in claim 7, characterized in that, The product in question is a test kit.

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