Knockdown of LSD1 alleviates the IL-1β-induced chondrocyte apoptosis, inammation and ECM degradation via TRIM32-mediated autophagy

Osteoarthritis (OA) is a common joint disease with characteristics of chronic inammation and articular cartilage degeneration. It has been proved that LSD1 was up-regulated in OA cartilage tissues, but its role and regulatory mechanism in OA are unclear. Herein, interleukin 1 beta (IL-1β)-treated human chondrocytes was performed as a cell model of OA. Then, LSD1 expression was found that up-regulated in OA cartilage tissues and IL-1β-induced chondrocytes. Knockdown of LSD1 increased cell viability, while decreased apoptosis rate and inammatory cytokines secretion levels in IL-1β-induced chondrocytes. In addition, knockdown of LSD1 reduced the expression of catabolic proteins (MMP-13 and ADAMTS-5) and enhanced the expression of anabolic proteins (Collagen II and Aggrecan) in chondrocytes after IL-1β stimulation. Moreover, overexpression of TRIM32 repressed chondrocyte viability, while promoted IL-1β-induced chondrocyte apoptosis, inammation and ECM degradation. The expression of LSD1 and TRIM32 in OA cartilage was positively correlated, and knockdown of LSD1 down-regulated TRIM32 expression of chondrocytes. Our data further indicated that LSD1 regulated autophagy of chondrocytes through modulating TRIM32. Overexpression of TRIM32 reduced the effect of LSD1 knockdown on IL-1β-induced chondrocytes, while activating autophagy by Rapamycin further reversed this reduction. Therefore, our study shows that knockdown of LSD1 inhibited IL-1β-induced chondrocyte apoptosis, inammation and ECM degradation via TRIM32-mediated autophagy.


Introduction
Osteoarthritis (OA) is a common degenerative joint disease with a signi cant increase in incidence with age, and is mainly manifested by joint swelling and long-term chronic pain, which seriously affects the quality of life of patients [1,2]. OA is always characterized by the reduction of chondrocytes and the degradation of cartilage extracellular matrix (ECM) with tissular in ammation [3,4]. In the onset of OA, abnormal pathological changes in articular cartilage tissue break the balance of anabolism and catabolism in chondrocytes, and ultimately lead to the degradation of ECM [5][6][7]. Therefore, maintaining chondrocyte viability is one of the key factors to prevent OA.
However, the role and underlying mechanisms of LSD1 in OA have not been clari ed. TRIM32 (Tripartite motif 32) is a transcription factor with E3 ubiquitin linkase activity [15,16]. Its protein structure contains the characteristic zinc nger domain, B-boxes domain and coiled domain of the TRIM family, as well as characteristic 6 A repetitive NHL domain [17][18][19]. Studies have found that it participates in the occurrence and development of a variety of tumors and neurological diseases by regulating cell proliferation, differentiation, development, and apoptosis [19][20][21]. A recent paper reveals that TRIM32 comtributes to the in ammation of rheumatoid arthritis [22]. Whereas, whether TRIM32 plays a role in the pathogenesis of OA remains unknown.
In this study, we examined the expression of LSD1 and TRIM32 in normal and OA cartilage cartilage, and investigated the functional role and underlying mechanisms of LSD1 and TRIM32 in IL-1β-induced OA model, which might provide potential therapeutic targets for OA treatment.

tissue samples
The OA cartilage tissues were obtained from 14 patients who underwent arthroplasty for OA between June 2019 and July 2020 at Yantaishan Hospital. The normal cartilage tissues were obtained from 10 patients who suffered from a femoral neck fracture without OA or rheumatic arthritis at the hospital. The study was approved by the institutional ethics committee of Yantaishan Hospital, and written informed consents were obtained from all patients.
Brie y, Chondrocytes (5x10 3 cells/well) were cultured in a 96-well plate and treated with a medium supplemented with 10% CCK-8 solution at 37°C for 4 hours. Then, the absorbance of each well was assessed by a microplate reader (BIOTEK, Winooski, Vermont, USA) at 450 nm.

Apoptosis assay
The total number of apoptotic chondrocytes was quanti ed by ow cytometry with uorescein isothiocyanate (FITC)-labeled Annexin V and propidium iodide (PI) (Enzo Life Sciences Inc., Farmingdale, NY) according to manufacturer's protocol.
Enzyme-linked-immunosorbent-assay (ELISA) The cell culture supernatant was collected and the secretion levels of in ammatory cytokines, including IL-8, IL-6 and TNF-α in chondrocytes were detected using ELISA kits according to the manufacturer's instructions.

Statistical analysis
All experiments were repeated three times, and the data was analyzed by SPSS 20.0 statistical software.
Data are means ± standard deviation (SD). The correlation was analyzed by Pearson' correlation test. Statistical signi cance was assessed using student's t-test and one-way ANOVA, and P < 0.05 was considered to be statistically signi cantly relevant.

Results
1 LSD1 was overexpressed in OA cartilage tissues and IL-1β-induced chondrocytes Quantitative real-time (qRT)-PCR and Western blot assay were performed to examine the expression of LSD1 in OA (n = 14) and normal cartilage tissues (n = 6). As shown in Fig. 1A and 1B, the expression of LSD1 was up-regulated in OA cartilage tissues. Besides, a cell model of OA was established by IL-1βinduced human chondrocytes. After IL-1β stimulation, the LSD1 expression of chondrocytes increased in a dose-dependent manner ( Fig. 1C and 1D).
3 Overexpression of TRIM32 enhanced IL-1β-induced apoptosis, in ammation and ECM degradation As shown in Fig. 3A, TRIM32 was highly expressed in OA cartilage tissues, compared with normal control group. Then, the expression of TRIM32 was elevated by its overexpression vectors (pcDNA-TRIM32) in IL-1β-induced chondrocytes ( Fig. 3B and 3C). The data of functional experiments further revealed that pcDNA-TRIM32 repressed cell viability while facilitated apoptosis, in ammatory response, and ECM degradation in IL-1β-induced chondrocytes.

Discussion
OA is a clinically common chronic degenerative disease, which mainly manifests as joint pain, swelling and/or restricted movement, and is the main cause of decreased mobility and dysfunction in the elderly [26,27]. Damage and loss of articular cartilage is the key to the pathological process in OA [28,29].
In ammatory cytokines such as IL-1β can cause a series of in ammatory cascade reaction in chondrocytes, and ultimately leads to the destruction of chondrocytes and degradation of EMC [30,31]. Therefore, IL-1β-stimulated chondrocytes was used to establish a cell model of OA in this study. It has been reported that LSD1 expression is increased in OA cartilage compared with normal cartilage [11], and our data con rmed this. Besides, our data also found that LSD1 expression was up-regulated in IL-1βinduced OA model in vitro.
Autophagy plays an important role in the homeostasis maintenance of chondrocytes for clearing dysfunctional organelles and macromolecules [32,33]. With the degeneration of cartilage, autophagy is inhibited and induces the homeostasis imbalance of chondrocytes, eventually leading to cell death [34,35]. Consistent with previous studies[36, 37], we found that autophagy was down-regulated in IL-1βinduced chondrocytes. mTOR, a serine/threonine protein kinase, is a key repressor of autophagy, which is modulated by multiple upstream signaling pathways to regulate autophagy level [24,38]. It has been found that parathyroid hormone (PTH)-(1-34) and Astragaloside IV ameliorate chondrocyte apoptosis via increasing autophagy by repressing mTOR [7,39]; Xue et al. and Kong et al. have reported that inhibition of mTOR attenuates in ammation of chondrocytes in rats with OA [40,41]. What is more, numerous experimental data indicate that activating mTOR-mediated autophagy can inhibit the ECM degradation in IL-1β-induced chondrocytes [39,40,42]. These evidences suggest that mTOR-mediated autophagy plays an important role in the progression of OA.
LSD1 is the rst con rmed histone demethylase that can speci cally remove the methyl modi cation of H3K9me1/2 and restore it to the unmethylated state, thereby affecting the transcriptional activation of target genes [8,12]. Previous studies have shown that LSD1 regulates the level of autophagy to programme oocyte death and reduce cancer cell viability via modulating p62 expression [43,44]. Furthermore, Wei et al. found that LSD1 mediates autophagy to regulate ovarian cancer progression through the mTOR signaling pathway [45]. Shi et al. reported that LSD1 dowm-regulates autophagy of myoblast cells via the activity the mTOR signaling pathway [46]. A recent study has revealed that knockdown of LSD1 attenuates ox-LDL-induced in ammation of RAW264.7 cells by promoting mTORmediated autophagy [47]. In this study, we found that knockdown of LSD1 suppressed IL-1β-induced chondrocyte apoptosis, in ammation and ECM degradation by activating mTOR-mediated autophagy.
TRIM32 has been reported that be overexpressed in broblast-like synoviocytes (FLS) of OA patients [22], and the present study indicated that TRIM32 expression was up-regulated in OA cartilage tissues and IL-1β-induced chondrocytes. It is noted that LSD1 modulates TRIM37 expression in luminal breast cancer cells [12], our study showed that knockdown of LSD1 down-regulated TRIM32 expression in IL-1β-induced chondrocytes. Recently, papers have found that TRIM32 contributes to autophagy in muscle cells through the regulation of p62 activity [48]. Moreover, a in vivo experimental data shows that TRIM32 de ciency mice increases autophagy level by reducing mTOR activity [23]. In this study, we found that LSD1 knockdown activated mTOR-mediated autophagy by modulating TRIM32, and that may be the molecular mechanism of LSD1 knockdown on chondrocyte apoptosis, in ammation and ECM degradation.
In conclusion, our study found that LSD1 and TRIM32 were overexpressed in OA cartilage tissues and IL-1β-induced chondrocytes, and their expression showed a strong positive correlation. Knockdown of LSD1 enhanced chondrocyte viability, and repressed apoptosis, in ammation and ECM degradation via activating mTOR-mediated autophagy by regulating TRIM32. Therefore, our study may provide potential therapeutic targets for OA therapy.

Ethics approval and consent to participate
The study was approved by the institutional ethics committee of Yantaishan Hospital, and written informed consents were obtained from all patients.

Consent for publication
Not applicable

Con icts of Interest
The authors declare no con ict of interest.