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. 2026 Jul 30;40(8):e71056. doi: 10.1002/jbt.71056

Contrasting Effects of HT‐2 Toxin Exposure and ZIP6 Knockdown on Chondrocyte Extracellular Matrix Metabolism

Yang Liu 1, Yu Zhang 1, Ruitian Huang 1, Lulu Bai 1, Lian Liu 1, Chaowei Wang 2, Yirong Qin 2, Hui Wang 2, Xiong Guo 2,3, Xi Wang 1,2,✉, Yujie Ning 2,✉
PMCID: PMC13424907  PMID: 42533448

ABSTRACT

Kashin‐Beck disease (KBD) is a chronic, endemic osteoarticular disorder associated with T‐2 toxin exposure, which is rapidly metabolized to HT‐2 toxin in vivo. However, the role of zinc transporter ZIP6 in HT‐2 toxin‐induced extracellular matrix metabolic disturbance in chondrocytes remains unclear. This study established HT‐2 toxin intervention and ZIP6 knockdown chondrocyte models, combined with quantitative reverse transcription polymerase chain reaction (qRT‐PCR) and transcriptome sequencing, to investigate the regulatory mechanisms of ZIP6 in chondrocyte injury. Following HT‐2 toxin exposure, altered chondrocyte morphology and reduced cell viability were observed; ZIP6, COL2A1, MTF1, and MTF2 expression were significantly downregulated, whereas MMP1, MMP13, and COL10A1 were upregulated. ZIP6 knockdown significantly upregulated COL2A1, MTF1, and MTF2, downregulated MMP1 and COL10A1, but did not alter MMP13 expression. Differentially expressed genes following ZIP6 knockdown were predominantly enriched in FoxO, cAMP, and TNF signaling pathways. qRT‐PCR validation confirmed consistent expression changes in FOXO4, TNFSF10, COLEC10, UCA1, and LRRC17 with transcriptome sequencing results. Collectively, HT‐2 toxin suppresses ZIP6, MTF1, and MTF2 expression, disrupting chondrocyte extracellular matrix metabolism; conversely, ZIP6 knockdown ameliorates matrix metabolic abnormalities through modulation of relevant signaling pathways. These findings reveal differential regulatory effects of HT‐2 toxin and ZIP6 on cartilage matrix metabolism, providing experimental evidence for elucidating molecular mechanisms underlying cartilage injury‐related diseases.

Keywords: Chondrocyte, extracellular matrix, HT‐2 toxin, transcriptome sequencing, ZIP6

HT‐2 toxin triggers chondrocyte damage and matrix metabolism disorders in KBD via suppressing ZIP6. ZIP6 knockdown alleviates these dysfunctions through FoxO, cAMP and TNF pathways, clarifying the molecular mechanism of cartilage injury.

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1. Introduction

Kashin‐Beck disease (KBD) is a chronic, endemic osteoarticular disorder characterized pathologically by degenerative necrosis of epiphyseal cartilage, epiphyseal plate cartilage, and articular cartilage, ultimately resulting in joint deformity and disability [1]. Previous studies have demonstrated that T‐2 toxin contamination represents a significant environmental risk factor for this disease [2]. Following entry into the body, T‐2 toxin is rapidly metabolized to HT‐2 toxin [3]; both compounds have been shown to induce apoptosis and autophagy in human chondrocytes [4], and HT‐2 toxin additionally disrupts chondrocyte ultrastructure and cell cycle progression [5]. Nevertheless, the specific molecular mechanisms underlying HT‐2 toxin‐induced dysregulation of extracellular matrix (ECM) metabolism in chondrocytes remain to be fully elucidated.

Zinc, an essential trace element for cellular physiological function [6], plays a pivotal role in cartilage development and matrix metabolism [7]. Dysregulated expression of zinc transporters disrupts zinc homeostasis, thereby altering downstream metal‐responsive transcription factor 1 (MTF1) activity and leading to aberrant matrix metalloenzyme expression. For instance, overexpression of the zinc transporter ZIP8 in murine cartilage and synovium has been shown to upregulate MMP3 and MMP13, resulting in cartilage destruction; conversely, ZIP8 knockdown significantly attenuates cartilage damage [8]. MTF1 serves as a critical downstream effector of ZIP8; its knockdown in surgically induced osteoarthritis mice suppresses ZIP8‐driven expression of matrix‐degrading enzymes and markedly attenuates cartilage destruction [9]. Solute carrier family 39 member 6 (ZIP6) is a plasma membrane‐localized zinc ion transporter that facilitates zinc mobilization from the extracellular space or intracellular organelles into the cytoplasm [10]. Notably, ZIP6 has been identified as differentially expressed in cartilage tissue obtained from KBD patients, and its expression is significantly downregulated in a T‐2 toxin‐induced rat model of KBD [11], suggesting a potential involvement of ZIP6 in KBD pathogenesis. However, whether ZIP6 contributes to the regulation of ECM metabolism in KBD chondrocytes and the underlying mechanistic pathway through which it may operate remain to be determined.

In this study, the human chondrocyte cell line C28/I2 was utilized as an in vitro model to systematically investigate the effects of HT‐2 toxin on chondrocyte viability, morphology, and the expression of genes associated with ECM metabolism. ZIP6 expression was subsequently knocked down to elucidate its regulatory role in chondrocyte ECM metabolism and MTF gene expression. Finally, transcriptome sequencing was performed following ZIP6 knockdown to identify differentially expressed genes and delineate the signaling pathways involved. These findings provide novel experimental evidence for the molecular mechanisms underlying HT‐2 toxin‐induced chondrocyte injury and the regulatory function of ZIP6, thereby contributing to a deeper understanding of KBD pathogenesis.

2. Material and Methods

2.1. HT‐2 Toxin Preparation

A stock solution of HT‐2 toxin at a concentration of 100 μg/mL (CAS No. 26934‐87‐2, procured from China Yuanye Biotechnology Co. Ltd.) was diluted to 100 ng/mL using phosphate‐buffered saline (PBS) (HyClone, USA) and subsequently stored at 4°C.

2.2. Culture and Processing of C28/I2 Chondrocytes

The C28/I2 cell line, derived from non‐clonal T/C‐28a4 cells, was immortalized through the introduction of simian virus 40 large T antigen (Tag) using retroviral vectors. Human C28/I2 chondrocytes were cultured in DMEM/F12 medium (HyClone, USA) supplemented with fetal bovine serum (Four Seasons, China) and penicillin‐streptomycin solution (HyClone, USA) in an incubator maintained at 37°C with 5% CO2. Upon reaching 80% to 90% confluence, the cells were subjected to various treatments. Intervention group cells were cultured in media containing 1, 5, 10, 20, or 50 ng/mL HT‐2 toxin, and cell viability was assessed at 24 and 48 h. The inhibitory effect on cell viability was more pronounced at 48 h; therefore, 48 h was selected as the intervention duration for subsequent experiments (Figure 1a). The toxin concentration range was narrowed, and cells were subjected to standardized 48 h intervention with gradient concentrations of 5, 10, 15, 20, 25, and 30 ng/mL. C28/I2 chondrocytes were treated with 5, 10, or 15 ng/mL HT‐2 toxin for 48 h, followed by morphological observation and qRT‐PCR analysis. Control cells were treated with an equal volume of PBS as the vehicle control and cultured under the same conditions as the HT‐2 toxin‐treated groups. Blank wells contained culture medium without cells.

Figure 1.

Figure 1

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(a) Viability of C28/I2 chondrocytes after 24 and 48 h of exposure to HT‐2 toxin at concentrations of 0, 1, 5, 10, 20, and 50 ng/mL (n = 3); (b) Viability of C28/I2 chondrocytes after 48 h of exposure to HT‐2 toxin at concentrations of 5, 10, 15, 20, 25, and 30 ng/mL (n = 3); (c) Morphological status of C28/I2 chondrocytes after treatment with HT‐2 toxin at concentrations of 5, 10, and 15 ng/mL (100×).

2.3. Cell Viability Assay

Cell viability was assessed using the MTT assay (Phygene, China), where 20 μL of a 5 mg/mL MTT solution was added to the cells under light‐protected conditions and incubated for 4 h. A volume of 150 µL of dimethyl sulfoxide (DMSO) was utilized to solubilize the metazan product. Optical density (OD) values were measured and analyzed at a wavelength of 490 nm using a microplate reader. Cell viability was determined using the formula: (OD of experimental group ‐ OD of blank group)/(OD of control group ‐ OD of blank group) × 100%. Blank wells containing culture medium without cells were included for background correction, and the corresponding absorbance values were subtracted during the calculation of cell viability.

2.4. Construction of Lentiviral Vectors

Three coding sequences targeting distinct sites of ZIP6 (Y19145, Y19146, and Y19147) were designed based on the Human SLC39A6 transcript, alongside one non‐specific scrambled sequence (GL427NC2) (Table S1). The shRNA fragments were cloned into a lentiviral vector capable of mediating ZIP6 knockdown. The vector used in this study, pSLenti‐U6‐shRNA‐CMV‐EGFP‐F2A‐Puro‐WPRE, is illustrated in Figure S1.

2.5. Lentiviral Titre Assay

C28/I2 chondrocytes, in the logarithmic growth phase, were digested into a cell suspension using trypsin (HyClone, USA) and seeded into 96‐well plates at a density of 1 × 104 cells per well. Once the cell confluence reached 40% to 50%, the cells were prepared for infection. The multiplicity of infection (MOI) values were set at 0, 40, 60, and 80. An experimental group containing 5 µg/ml polybrene (He Yuan Biologicals, China) was included, with an additional control group of untreated cells. The old culture medium was removed from each well, and the diluted virus‐containing mixture was added, followed by gentle shaking to ensure thorough mixing. After 12–16 h, the culture medium was replaced with fresh medium, and incubation was continued. Representative fluorescence images were used to qualitatively assess lentiviral infection under different MOIs and infection durations. Based on these observations, an MOI of 60 and an infection duration of 72 h were selected for subsequent experiments (Figure 3a).

Figure 3.

Figure 3

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(a) Representative fluorescence images showing lentiviral infection at different MOIs (40, 60, and 80) and infection durations (24, 48, and 72 h). These images were used for qualitative assessment of the infection conditions; (b) Relative ZIP6 mRNA expression determined by qRT‐PCR for quantitative evaluation of ZIP6 knockdown efficiency (shctrl, negative control; Y19145, Y19146, and Y19147, three shRNA sequences targeting different sites of ZIP6); (c–h) Comparison of the expression of MMP1, MMP13, COL2A1, COL10A1, MTF1, and MTF2 between the shctrl group andshZIP6 group (n = 3. shctrl, negative control for lentivirus infection. shZIP6, ZIP6 knockdown group. * p  < 0.05, ** p  < 0.01, **** p  < 0.0001, ns, p  > 0.05).

2.6. Establishment of the ZIP6 Knockdown Model

Four groups were established: Y19145, Y19146, Y19147, and GL427NC2. C28/I2 chondrocytes were infected at an MOI of 60 following the procedure described in step 2.5 and cultured for 72 h. The knockdown efficiency of ZIP6 was subsequently quantified by qRT‐PCR. The shRNA sequence exhibiting > 80% knockdown efficiency was selected for stable cell line generation; C28/I2 chondrocytes were infected and subsequently selected with 5 µg/mL puromycin (Beyotime, China) for approximately 1 week. The resulting stable cell line was designated as the ZIP6 knockdown group (shZIP6 group). Cells without lentiviral infection served as the control (shctrl group).

2.7. Total RNA Extraction and qRT‐PCR for mRNA Determination

Total RNA was extracted from the HT‐2 toxin intervention group, as well as from the shctrl and shZIP6 groups, using the TRIzol method. The mRNA expression levels of relevant genes were measured via qRT‐PCR. In the HT‐2 toxin intervention group, the genes ZIP6, MTF1, MTF2, MMP1, MMP13, COL2A1, and COL10A1 were assessed. In contrast, the shctrl and shZIP6 groups were analyzed for MTF1, MTF2, MMP1, MMP13, COL2A1, COL10A1, FOXO4, TNFSF10, COLEC10, UCA1, and LRRC17. qRT‐PCR was performed using the SYBR® Green Pro Taq HS Premixed qPCR reagent kit (AG11701, Hunan Aikairui Bioengineering Co. Ltd.), and amplification was carried out on a Bio‐Rad real‐time PCR instrument. The primer sequences are listed in Table S2. Relative gene expression was calculated using the 2^‐ΔΔCt method based on the cycle threshold (Ct) values. Each experiment was independently performed using three biological replicates. For each biological replicate, qRT‐PCR reactions were performed in technical triplicate to minimize experimental variability, and the average Ct value was used for subsequent analysis. GAPDH served as the internal reference gene. The raw Ct values for all qRT‐PCR assays are provided in Table S3.

2.8. Transcriptome Sequencing

Differentially expressed genes (DEGs) between the shctrl and shZIP6 groups were detected using transcriptome sequencing, with each group comprising three biological replicates. Total RNA was isolated using TRIzol reagent, and mRNA was isolated from the total RNA using Dynabeads Oligo (dT) (Thermo Fisher, CA, USA). The mRNA was denatured at 94°C and sequentially reverse transcribed into first and second‐strand cDNA. Following this, adenine bases were added to the blunt ends of each strand to facilitate their ligation to index adapters. The aptamers were purified using 100 μl of AMPure XP Beads, and the ligation products were amplified via PCR, with an initial denaturation at 95°C for 3 min, followed by 12 cycles of denaturation at 9°C for 10 s, annealing and extension at 65°C for 75 s, and a final extension at 65°C for 5 min. Subsequently, 2 × 150 bp paired‐end sequencing (PE150) was conducted on the Illumina NovaSeq™ 6000 platform (LC‐Bio Technology Co. Ltd, Hangzhou, China) in accordance with the supplier's protocol.

Differences between biological replicates were analyzed using the limma package, with a selection criterion of |log2Fc | ≥ 1 and p ≤ 0.05. Gene expression for each sample was annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG), ENSEMBL, NCBI, and Gene Ontology (GO) databases. GO functional enrichment analysis and KEGG signaling pathway enrichment analysis of DEGs were conducted utilizing the Lianchuanbio OmicStudio cloud platform. The Benjamini‐Hochberg method was adopted to correct multiple testing bias, and GO terms and KEGG pathways with adjusted p‐value (FDR) < 0.05 were regarded as significantly enriched.

2.9. Statistical Analysis

Statistical analyses were performed using SPSS version 18.0, and the results are presented as mean ± standard deviation (Mean ± SD). Prior to statistical analysis, data normality and homogeneity of variance were assessed using the Shapiro–Wilk test and Levene's test, respectively. Comparisons between two groups were performed using Student's t‐test, whereas comparisons among three or more groups were analyzed using one‐way analysis of variance (ANOVA). When the assumptions of normality and homogeneity of variance were satisfied, and the overall ANOVA was significant, Fisher's least significant difference (LSD) test was used for post hoc pairwise comparisons. If these assumptions were not satisfied, the corresponding nonparametric tests would be applied. A p‐value < 0.05 was considered statistically significant.

3. Results

3.1. HT‐2 Toxin Alters Chondrocyte Survival Rate and Chondrocyte Morphology

As shown in Figure 1b, cytotoxicity was dose‐dependently enhanced with increasing HT‐2 toxin concentration, resulting in decreased cell viability. Notably, pairwise comparisons of cell viability at 5, 10, and 15 ng/mL revealed statistically significant differences (p < 0.05), with corresponding survival rates of 79.50% (5 ng/mL), 46.85% (10 ng/mL), and 24.23% (15 ng/mL). In contrast, no significant differences were observed between 20, 25, and 30 ng/mL.

Cellular morphology following intervention with 5, 10, and 15 ng/mL HT‐2 toxin is presented in Figure 1c. Cytotoxic damage was progressively exacerbated with increasing toxin concentration, accompanied by reduced C28/I2 chondrocyte density. Whereas control cells maintained normal oval or round morphology, intervention group cells exhibited marked morphological changes characterized by fibroblast‐like spindle shapes.

3.2. Effects of HT‐2 Toxin on Cartilage ECM Metabolism and the Genes of ZIP6 and MTFs

To investigate the mechanisms underlying HT‐2 toxin‐induced chondrocyte injury and its association with ZIP6 and MTFs, cells were treated with 5, 10, and 15 ng/mL HT‐2 toxin. Expression levels of ECM metabolic marker genes, ZIP6, MTF1, and MTF2 in treated chondrocytes are presented in Figure 2a–g. The findings indicated a significant reduction in the expression level of COL2A1 (p = 0.0244, p = 0.0018) compared to the control group, while the mRNA levels of MMP1 (p = 0.0005), MMP13 (p = 0.0038), and COL10A1 (p < 0.0001) were significantly elevated, with these differences being statistically significant. Furthermore, the mRNA expression levels of ZIP6 (p = 0.0225, p = 0.0255), MTF1 (p = 0.0138, p = 0.0189), and MTF2 (p = 0.0129) exhibited statistically significant decreases. These results suggest that HT‐2 toxin induces chondrocyte damage by promoting the abnormal expression of ECM‐degrading enzymes.

Figure 2.

Figure 2

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Expression of (a) MMP1, (b) MMP13, (c) COL2A1, (d) COL10A1, (e) ZIP6, (f) MTF1, and (g) MTF2 in C28/I2 chondrocytes of the normal control group and those treated with HT‐2 toxin at 5, 10, and 15 ng/mL. (n = 3, * p  < 0.05, ** p  < 0.01, *** p  < 0.001, **** p  < 0.0001).

3.3. Effects of Low ZIP6 Expression on ECM Metabolism in Cartilage

As shown in Figure 3b, all three interference sequences effectively suppressed ZIP6 mRNA levels compared with the shctrl control group (p < 0.0001). The knockdown efficiencies were 77.24% for Y19145, 79.58% for Y19146, and 83.48% for Y19147. Y19147 was selected for the generation of the ZIP6 knockdown group. The expression levels of MMP1, MMP13, COL2A1, and COL10A1, together with MTF1 and MTF2, were subsequently evaluated to assess the effect of ZIP6 knockdown on ECM metabolism. The results indicated that, in comparison to the shctrl group, the mRNA expression levels of MMP1 (p < 0.0001) and COL10A1 (p < 0.0001) were significantly decreased, whereas the expression of COL2A1 (p = 0.0086) was significantly increased. However, the increase in MMP13 expression was not statistically significant (p > 0.05). Furthermore, MTF1 (p = 0.0243) and MTF2 (p = 0.0045) exhibited significantly elevated expression levels, as depicted in Figure 3c–h. In contrast to the downregulation of MTF1 and MTF2 induced by HT‐2 toxin, ZIP6 knockdown resulted in increased expression of both genes, suggesting that additional regulatory mechanisms may be involved.

3.4. Transcriptome Sequencing Analysis to Screen for Differentially Expressed Genes

Transcriptome sequencing was performed to compare differentially expressed genes before and after ZIP6 knockdown, thereby elucidating the role of ZIP6 in cellular function and signaling pathways. A total of 185 differentially expressed genes were identified, comprising 77 up‐regulated and 108 down‐regulated genes. GO enrichment analysis revealed that these differentially transcribed genes were predominantly associated with cellular components and molecular processes. Within biological processes, the focus was primarily on signal transduction, multicellular organism development, and the negative regulation of transcription by RNA polymerase II. Regarding cellular components, emphasis was placed on the cellular membrane, membrane components, the cytoplasm, the nucleus, and the extracellular interstitial space. In terms of molecular functions, the primary focus was on protein, metal ion, and nucleotide binding. Among the identified genes, those involved in cell membrane function and protein binding exhibited the highest levels of differential transcription, as illustrated in Figure 4a. According to the KEGG pathway analysis, the differentially transcribed genes are predominantly associated with the FoxO signaling pathway, tumor‐related pathways, the TNF signaling pathway, aldosterone synthesis and secretion, the cAMP signaling pathway, vitamin B6 metabolism, cytokine‐cytokine receptor interactions, C‐type lectin receptor signaling, and the pentose phosphate pathway, as shown in Figure 4b. Table 1 delineates the top 20 genes exhibiting the most significant differences between the shctrl and shZIP6 groups. It should be noted that several of the top‐ranked differentially expressed genes are pseudogenes or read‐through/fusion transcripts, or have limited functional annotation in chondrocytes. Therefore, subsequent analyses focused primarily on candidate genes with established biological relevance. To assess the reliability of the sequencing data, five representative differentially expressed genes were selected for validation by qRT‐PCR. Compared to the negative control group, low expression of ZIP6 significantly down‐regulated the mRNA expression levels of FOXO4 (p = 0.0003) and TNFSF10 (p = 0.0114) in chondrocytes, while significantly up‐regulating the mRNA expression levels of COLEC10 (p = 0.0036), UCA1 (p = 0.0006), and LRRC17 (p = 0.0013). These findings were consistent with the trends observed in the transcriptome sequencing data, thereby demonstrating high precision and confidence in the sequencing results, as shown in Figure 4c.

Figure 4.

Figure 4

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(a) GO term annotation plot of shctrl group versus shZIP6 group; (b) KEGG enrichment scatter plot of shctrl group versus shZIP6 group; (c) Comparison of transcriptome sequencing results with mRNA expression levels of UCA1, COLEC10, LRRC17, TNFSF10, and FOXO4 detected by qRT‐PCR. (n = 3.*p ＜ 0.05, qRT‐PCR group vs. shctrl group).

Table 1.

Differentially expressed gene TOP20 in shctrl group vs shZIP6 group.

Gene Name log2(Fc) p value
HSPE1‐MOB4 13.62 1.81 × 10−8
TRIM39‐RPP21 12.25 3.56 × 10−8
FMC1‐LUC7L2 11.56 8.62 × 10−8
TREML3P 11.29 7.90 × 10−5
MTCO2P16 10.99 2.60 × 10−3
TMEM240 9.48 2.03 × 10−3
ATP6V0A4 8.72 9.42 × 10−4
NLRP3 8.30 1.72 × 10−3
FAM9B 8.22 2.81 × 10−4
PTCHD1 6.70 3.65 × 10−3
TMEM189‐UBE2V1 −16.54 5.11 × 10−12
RPL17‐C18orf3 −15.71 3.14 × 10−21
INO80B‐WBP1 −14.34 3.66 × 10−10
ISY1‐RAB43 −12.85 5.74 × 10−10
GUSBP4 −11.07 2.93 × 10−3
NUS1P2 −10.94 1.18 × 10−3
CHORDC1P1 −10.54 2.78 × 10−3
CORO7‐PAM16 −10.17 9.21 × 10−7
ASCL2 −10.07 5.04 × 10−4
NCKAP5‐AS2 −9.75 2.08 × 10−3
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4. Discussion

As a significant risk factor for KBD, the impact of T‐2 toxin on ECM degradation in chondrocytes has been well established in previous studies. However, the specific mechanisms by which its primary metabolite, HT‐2 toxin, induces ECM metabolic dysregulation remain incompletely understood, and the role of zinc homeostasis and its regulatory factors has not been thoroughly investigated. Accordingly, the present study systematically examined cellular viability, ECM metabolism, and ZIP6 expression in response to HT‐2 toxin exposure. MTT assay results demonstrated that C28/I2 chondrocyte viability was decreased in a dose‐dependent manner with increasing HT‐2 toxin concentrations, accompanied by corresponding alterations in cell density and growth status, consistent with previous reports indicating that HT‐2 toxin suppresses chondrocyte proliferation [12]. Notably, no statistically significant difference in cell viability was observed at concentrations of 20 ng/mL and above. Similarly, it has been reported that HT‐2 concentrations exceeding 100 nM failed to induce further reductions in cell viability at any time point examined, suggesting that the cytotoxic effect of HT‐2 toxin on chondrocytes may be subject to a threshold effect [13]. This observation may result from saturation of cytotoxic signaling or activation of adaptive cellular responses at higher toxin concentrations; however, the underlying mechanism remains to be elucidated.

Arthritis development is commonly associated with matrix degradation mediated by multiple matrix metalloproteinases (MMPs) [14]. Among these, MMP1 and MMP13 are the collagenases most closely implicated in cartilage collagenolysis [15, 16]. Additionally, COL2A1 encodes the α1 chain of type II collagen, which is essential for maintaining structural integrity and regulating chondrocyte proliferation, metabolism, and differentiation [17, 18], whereas COL10A1 encodes the α1 chain of type X collagen and is expressed during chondrocyte hypertrophic differentiation [19]. In the present study, treatment with 15 ng/mL HT‐2 toxin for 48 h resulted in significant upregulation of MMP1, MMP13, and COL10A1, while COL2A1 was markedly downregulated at both 10 ng/mL and 15 ng/mL concentrations. These findings suggest that HT‐2 toxin promotes cartilage matrix degradation, mineralization, and aberrant differentiation through the induction of dysregulated ECM‐degrading enzyme metabolism. Previous studies have similarly demonstrated elevated mRNA levels of MMP1, MMP3, and MMP13, alongside reduced COL2A1 expression, following HT‐2 toxin exposure [13]. Notably, the expression levels of ZIP6, MTF1, and MTF2 were also significantly decreased after HT‐2 toxin treatment. ZIP6 is known to modulate intracellular zinc homeostasis, thereby influencing metalloproteinase activity and transcription factor function [20]. MTF1, a zinc‐sensing transcription factor, regulates the expression of genes involved in zinc homeostasis and metal metabolism [21]. In contrast, the role of MTF2 in zinc homeostasis has not been clearly established. The coordinated downregulation of these genes suggests that HT‐2 toxin may induce chondrocyte injury through the disruption of zinc homeostasis.

The impact of ZIP6 downregulation on chondrocyte ECM was further examined in this study. ZIP6 knockdown resulted in significantly decreased mRNA levels of MMP1 and COL10A1, whereas COL2A1 expression was markedly elevated. These findings indicate that HT‐2 toxin‐induced ECM degradation is not solely mediated by ZIP6 downregulation. Additionally, MTF1 and MTF2 expression was significantly upregulated following ZIP6 knockdown, suggesting a negative regulatory role of ZIP6 on MTF1 and MTF2—a pattern contrasting with their downregulation observed under HT‐2 toxin exposure. The opposite expression patterns of MTF1 and MTF2 following HT‐2 toxin treatment and ZIP6 knockdown suggest that HT‐2 toxin may regulate MTF1 and MTF2 through ZIP6‐independent mechanisms, whereas ZIP6 knockdown may trigger compensatory feedback responses. However, this hypothesis remains to be experimentally validated. Collectively, these results demonstrate that reduced ZIP6 expression exerts a partial protective effect on select ECM genes.

To comprehensively identify ZIP6‐regulated pathways in chondrocytes, transcriptome sequencing was performed following ZIP6 knockdown. First, the NLRP3 inflammasome promotes the production of inflammatory cytokines (including IL‐1β and TNF‐α) and matrix‐degrading enzymes such as MMP3, thereby driving cartilage degeneration and synovial inflammation. Significant upregulation of NLRP3 was observed in this study, suggesting its close involvement in chondrocyte injury [22]. Second, FoxO transcription factors respond to oxidative stress signals to regulate ROS levels, and excessive ROS can activate MMPs to degrade the ECM [23, 24]. Downregulation of FOXO4 was observed following ZIP6 knockdown, indicating that ZIP6 downregulation may attenuate cartilage matrix degradation through the suppression of ROS production and apoptosis. ZIP6 knockdown also significantly affected the cAMP signaling pathway. Elevated cAMP levels in articular cartilage not only promote proteoglycan 4 production but also reduce MMP expression and activity, thereby inhibiting cartilage matrix degradation [25]. The observed decrease in MMP1 expression following ZIP6 knockdown was consistent with this protective effect. However, MMP13 expression was not reduced, suggesting differential regulatory mechanisms between these two enzymes. Previous studies have demonstrated that JUN knockdown decreases ATF3 levels, thereby suppressing MMP13 expression without affecting MMP1 [26]. Transcriptome sequencing revealed increased JUN expression following ZIP6 knockdown, which may explain the differential regulation of MMP1 and MMP13. This hypothesis remains to be experimentally validated, and future studies should investigate the roles of JUN and ATF3 in this regulatory process. Additionally, ZIP6 downregulation impacted the TNF signaling pathway. The diverse biological effects of TNF are mediated through NF‐κB [27], which plays critical roles in both cartilage homeostasis maintenance and pathological degradation [28]. Transcriptome analyses of KBD patients have similarly confirmed the involvement of the TNF signaling pathway in disease pathogenesis [29]. In summary, reduced ZIP6 expression may participate in chondrocyte injury through the modulation of NLRP3 inflammasome, FoxO, cAMP, and TNF signaling pathways.

This study has several limitations. First, all experiments were conducted using the immortalized human chondrocyte cell line C28/I2, which cannot fully recapitulate the physiological cartilage microenvironment in vivo. Future studies incorporating primary chondrocytes and animal models are needed to further validate the biological role of ZIP6 in cartilage degeneration. Second, although transcriptomic analysis suggested that ZIP6 may regulate the FoxO, cAMP, and TNF signaling pathways, no pathway perturbation, rescue, protein‐level, or pathway activity analyses were performed, leaving the underlying mechanisms to be further elucidated. In addition, although RNA sequencing identified numerous differentially expressed genes, only five were validated by qRT‐PCR, and their functional roles in ECM metabolism were not further validated by mechanistic cell‐based experiments. Future studies should investigate these candidate genes using gain‐ and loss‐of‐function approaches, followed by validation in animal models. Furthermore, ZIP6 knockdown was achieved using a single shRNA construct, and potential off‐target effects were not evaluated. Future studies using multiple independent shRNAs together with rescue experiments will further strengthen the specificity and reliability of these findings.

5. Conclusion

In this study, the effects of HT‐2 toxin on chondrocyte injury and the underlying molecular mechanisms were systematically investigated through in vitro exposure of C28/I2 chondrocytes to HT‐2 toxin, combined with ZIP6 knockdown and transcriptome sequencing. HT‐2 toxin was found to inhibit chondrocyte viability in a dose‐dependent manner, induce ECM metabolic dysregulation (characterized by upregulation of MMP1, MMP13, and COL10A1 and downregulation of COL2A1), and significantly suppress the expression of ZIP6, MTF1, and MTF2, suggesting the involvement of zinc homeostasis disruption in cartilage injury. ZIP6 knockdown exerted a protective effect on select ECM genes, and transcriptome sequencing revealed that ZIP6 downregulation affected multiple signaling pathways, including the NLRP3 inflammasome, FoxO, cAMP, and TNF pathways. These findings provide novel experimental evidence for elucidating the pathogenic mechanisms of Kashin‐Beck disease.

Author Contributions

Yang Liu: writing – original draft, writing – review and editing, validation, investigation, software, data curation. Yu Zhang: visualization, writing – original draft. Ruitian Huang: writing – review and editing. Lulu Bai: visualization, writing – review and editing. Lian Liu: validation. Chaowei Wang: formal analysis. Yirong Qin: formal analysis. Hui Wang: formal analysis. Xiong Guo: formal analysis. Xi Wang: writing – original draft, writing – review and editing, conceptualization, validation, funding acquisition. Yujie Ning: writing – original draft, writing – review and editing, investigation, funding acquisition.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File

JBT-40-e71056-s001.docx (179.5KB, docx)

Acknowledgments

This research was funded by the National Natural Science Foundation of China (81803179, 82273752 and 82373700).

Contributor Information

Xi Wang, Email: wn18andlife@xjtu.edu.cn.

Yujie Ning, Email: ning461871077@xjtu.edu.cn.

Data Availability Statement

All data generated or used during the study are available from the corresponding author and first author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting File

JBT-40-e71056-s001.docx (179.5KB, docx)

Data Availability Statement

All data generated or used during the study are available from the corresponding author and first author upon reasonable request.

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