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Andrographolide suppresses fibrogenic phenotype of chondrocytes and ameliorates osteoarthritis by regulating miR-137/BMP7 axis
⁎Corresponding author: En Song. huxidan@ydyy.cn
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Received: ,
Accepted: ,
This article was originally published by Reed Elsevier India Pvt. Ltd. and was migrated to Scientific Scholar after the change of Publisher.
Abstract
Abstract
Pathogenic degeneration of cartilage and the generation of fibrotic cartilage are crucial characteristics linked to the progression of osteoarthritis (OA). The current research aims to explore the potential function of the miR-137/BMP7 pathway in regulating the fibrogenic transition of chondrocytes associated with OA, as well as assess the therapeutic potential of andrographolide.
Samples of cartilage from the knees of patients with OA and individuals without OA were gathered to investigate the expression patterns of miR-137, BMP7, and markers associated with fibrosis. A cell model using primary chondrocytes stimulated with interleukin (IL)-1β was developed to study the involvement of the miR-137/BMP7 axis during the fibrogenic transition of these cells. Additionally, we utilized an animal model of OA in order to assess the beneficial effects of the anti-inflammatory natural compound andrographolide on the fibrogenesis induced by OA in vivo.
Elevated levels of fibrogenic and inflammatory factors were linked to decreased miR-137 expression in OA samples. In IL-1β-treated chondrocytes, there was an upregulation of fibrogenic markers alongside a reduction in miR-137 levels. The overexpression of miR-137 inhibited fibrogenesis through the negative regulation of BMP7. Additionally, treatment with andrographolide was effective in attenuating the fibrogenic phenotype in chondrocytes and mitigating OA pathogenesis via modulating the miR-137/BMP7 pathway.
miR-137 downregulation and BMP7 overexpression might contribute to the fibrogenic features in OA-related chondrocytes. Andrographolide attenuates fibrogenic phenotype in chondrocytes and alleviates the severity of OA by modulating the miR-137/BMP7 axis.
Keywords
Osteoarthritis
Chondrocytes
Fibrosis
miR-137
BMP7
1 Introduction
Osteoarthritis (OA) is a pervasive inflammatory condition affecting the joints, featured by cartilage deterioration, synovial fibrosis, and joint stiffness.1–3 The compromised biomechanical functionality of both the joint and cartilage results in diminished mobility and persistent pain 4. This condition is marked by the pathological buildup of proteins that destroy the extracellular matrix, including aggrecanases or matrix metalloproteinases (MMPs), contributing to cartilage deterioration.5,6 Additionally, abnormal chondrocyte activation can result in a fibrogenic phenotype.7–9 This transformation is linked to the heightened synthesis of collagen types I and III and an overexpression of alpha smooth muscle actin (α-SMA) or MMP inhibitory factors.10–14 Numerous risk factors, such as genetic predisposition, excessive joint loading, trauma-related articular cartilage injury, aging, and diabetes, can facilitate the progression of OA.15,16 Nonetheless, the precise molecular mechanisms that drive the formation of fibrogenic chondrocytes during the advancement of OA remain poorly understood.
The deregulation of microRNAs (miRNAs) is linked with a broad spectrum of pathophysiological disorders, such as osteoarthritis (OA).17 These RNAs negatively influence mRNA activity by hindering the translation process or facilitating the degradation of specific mRNAs.18 For instance, the overexpression of miR-378 worsens osteoarthritis by inhibiting autophagy and promoting chondrocyte hypertrophy in a mouse model of OA.19 Conversely, miR-335-5p mitigates inflammation related to OA by enhancing autophagy within chondrocytes.20 It has also been reported that miR-137 inhibits TCF4, leading to reduced inflammation and apoptosis in chondrocytes.21 Additionally, miR-137 has been shown to diminish the growth and mobility of synoviocytes in inflammatory bone condition.22 Despite the reported roles in chondrocyte inflammation, the plausible function of miR-137 in contributing to the fibrogenic characteristics of chondrocytes remains unexplored.
Numerous growth mediators have been identified as candidate therapeutic agents to mitigate OA pathogenesis. Bone morphogenetic protein 7 (BMP7) serves as a crucial factor influencing the differentiation status of chondrocytes and cartilage.23 Prior research has shown that administering BMP7 can reduce MMP13 levels in chondrocytes stimulated by interleukin (IL)-1β,24 enhance proteoglycan production in these cells,25 and reduce inflammation.26 The bone morphogenetic protein receptor type 2 (BMPR2) was initially recognized for its function in mediating the development and maturation of cartilage and bone.27 Furthermore, the absence of BMPR2 results in increased bone mass due to heightened osteogenesis.28 Nevertheless, the functions of BMP7 and BMPR2 in chondrocytes during inflammatory conditions associated with OA remain largely unexplored.
This research involved the collection of cartilage samples from the knees of 10 patients with osteoarthritis (OA) and non-OA individuals to examine the patterns of miR-137, BMP7, and BMPR2 expression. We investigated the role of the miR-137/BMP7/BMPR2 axis in the fibrogenic characteristics of chondrocytes, using IL-1β-challenged primary chondrocytes and an animal model of OA. Additionally, we assessed the potential impact of andrographolide, a natural anti-inflammatory product, on inflammation-induced fibrogenesis in chondrocytes and the OA animal model.
2 Methods
2.1 Clinical specimens
All participants signed an informed consent in a written form, and clinical specimen use gained the approval by the Ethics Committee of First Affiliated Hospital of Kunming Medical University (NO. 2024L184). The articular cartilage specimens were obtained from two groups: 10 OA patients undergoing knee arthroplasty surgery and 10 control subjects with knee trauma but no OA diagnosis. All collected specimens were immediately frozen in liquid nitrogen and kept at −80 °C for subsequent examination.
2.2 Cell culture and transfection
Human chondrocytes were provided by Procell (CP-H107, Wuhan, China). The cells were cultivated with specialized chondrocyte culture medium (CM-H107, Procell, Wuhan, China) containing fetal bovine serum, chondrocyte growth supplement and Penicillin/Streptomycin at 37oC and 5 % CO2. For the establishment of inflammtory cell model, cells were challenged with 10 ng/ml recombinant IL-1β (abs04626, Absin, Shanghai, China) for 48 h miR-137 mimic, antagomir and corresponding controls were produced by RiboBio (Guangzhou, China). Andrographolide was procured from MedChemExpress (HY-N1490, Shanghai, China) and applied at 20 μM. The delivery of above molecules into cells was achieved with Lipofectamine 3000 reagent (L30000-008, Invitrogen, Carlsbad, CA, USA).
2.3 Animal model of OA
Balb/C mice (6 weeks old, male, 20g) were housed in the pathogen-free animal facilities with a light/dark cycle of 12 h. The animals were assigned into five experimental conditions (n = 6 animals in each group): sham group; OA model group; OA + andrographolide group; OA + andrographolide + miRNA inhibitor negative control group; and OA + andrographolide + miR-137 inhibitor group. The OA condition was induced by transecting the anterior cruciate ligament in conjunction with medial meniscectomy.29 The sham operation was performed by exposing the muscle tissues of the knee without transection. miRNA inhibitor and the negative controls were injected into the knee at a dose of 0.5 mg/kg/week. Andrographolide was administrated by intraperitoneal route. Four weeks after the surgical procedures, all the animals were euthanized by CO2 asphyxiation and the knee samples and serum specimens were harvested for further analyses. The animal protocols gained the approval by the Ethical Review Committee for Animal Experiments of Kunming Medical University (NO. kmmu20230468).
2.4 RT-qPCR
RNA sample was purified from cellular or tissue specimens employing the TRIzol Reagent (15596026, Invitrogen, Shanghai, China) following specific protocols. For the synthesis of cDNA from a 1 μg RNA sample, the MiRNA Stem Ring Reverse Transcription Kit (IPD-X049, Hubei Apti Bioengineering Co., Ltd, Wuhan, China) was utilized. The quantification of qPCR was conducted with the SYBR Premix Ex TaqII kit (RR82WR, Takara, Tokyo, USA) on the CFX96 qPCR platform (CFX96, Bio-Rad, Hercules, CA, USA). To assess gene expression levels, the 2−△△CT approach was applied, with β-actin serving as the endogenous control.
2.5 Western blot
Isolation of protein samples was achieved using RIPA buffer supplemented with phosphatase and proteinase inhibitors (K10034, KeyGen, Beijing, China). Following separation via a 10 % polyacrylamide gel, the separated samples were subsequently transferred onto a PVDF membrane. After blocking the membrane for 1 h, it was probed for 24 h at 4 °C with the following primary antibodies (Abcam, Cambridge, UK): anti-collagen 1A1 (1:1000; Abcam, ab260043), anti-collagen 3A1 (1:1000; Abcam, ab184993), anti-α-SMA (1:1000; Abcam, ab5694), anti-BMP7 (1:1000; Abcam, ab84684), and anti-BMPR2 (1:1000; Abcam, ab96826). The membrane was then labeled with the secondary antibody (1:5000; 61–6520, Invitrogen, Shanghai, China), and the relative band intensities were assessed using ImageJ Software Version 1.53t (NIH, Bethesda, MD, USA) after signal development.
2.6 Luciferase reporter assay
The construction of miR-137/BMP-7 wild-type reporter and miR-137/BMP-7 mutated reporter was carried out by Sangon Biotech (Shanghai, China). The WT or MUT reporter was introduced into cells in the presence of mi-137 mimic or the control. 48 h post-transfection, the luciferase activity was quantified using the GeneLight™ Dual Luminescence Reporter Assay Kit (G098, GenLight Biotech, Shanghai, China).
2.7 Cell proliferation assay
Cell growth potential was examined by a CCK-8 assay kit (HY-K0301, MedChemExpress, Shanghai, China) according to the supplier's protocol. A density of 2000 cells per well was established by introducing cells into a 96-well plate, which were subsequently cultured for specified durations. Following this, 15 μL of CCK-8 reagent was introduced into each well for incubation at 37 °C for an extra 3 h before the signal measurement at 450 nm using a microplate reader.
2.8 ELISA
The detection of IL1-β, TNF-α and TGF-β1 was performed with commercial ELISA kits (Biovision, CA, USA). 1x10^6 cells and 0.5 g of tissue were lysed in 500 μl of lysis buffer on ice for 20 min. Subsequently, 50 μl of either the lysate or standard was placed into assay wells for 2-h incubation at 37 °C. Following this incubation, each well received 50 μl of biotin-conjugated antibody for an additional hour at 37 °C. Subsequently each well was further probed with 50 μl of streptavidin-HRP solution for 1 h. Finally, after the addition of signal development solution for 15 min, the light signal of each specimen was recorded using a photometer at 450 nm.
2.9 Histological staining
The joint tissue samples underwent fixation with formalin (10 %) and were decalcified using ethylenediaminetetraacetic acid (10 %; pH = 7.4, Beyotime, Beijing, China) over a period of 21 days. Staining was conducted on tissue sections that were 6 μm thick. Following deparaffinization and rehydration, the sections were subjected to staining using H&E (hematoxylin and eosin) kit (ab245880, Abcam, Cambridge, UK) and Safranin-O and Fast Green FCF staining solutions (Sigma-Aldrich, St. Louis, MO, USA), adhering to the manufacturer's guidelines. The stained tissue sections were then examined under the Zeiss Axiovert 200 bright-field microscope (Carl Zeiss, Jena, Germany).
2.10 Statistics
The determination of data significance was performed using GraphPad 8.0 software (GraphPad software, CA, USA). Experimental data were displayed as mean and standard deviation. Data from two experimental conditions were examined by unpaired Students’ t-test, and the statistics for multiple group comparisons were determined by one-way ANOVA test. P value less than 0.05 was set as the statistical threshold for significance analysis.
3 Results
3.1 Increased levels of fibrogenic and inflammatory factors and reduced miR-137 expression in OA samples
The knee articular cartilage specimens were harvested from individuals with osteoarthritis (OA) who had arthroplasty, as well as from individuals with knee trauma, which served as a control group. Analysis using RT-qPCR indicated a clear reduction in miR-137 levels within the cartilage tissues of OA patients (Fig. 1A). Furthermore, immunoblotting assay demonstrated a marked elevation in fibrogenic markers, specifically COL1A1, COL3A1, and α-SMA, in the OA patient samples (Fig. 1B). Additionally, the protein expressions of BMP7 and BMPR2, which play roles in cartilage repair, were found to be elevated (Fig. 1B). In parallel, the concentrations of inflammatory mediators (IL1-β and TNF-α), along with the fibrogenic factor TGF-β1, became significantly increased in OA samples (Fig. 1C).
3.2 IL-1β induces the downregulation of miR-137 in human chondrocytes
To enable to perform the mechanistic study of miR-137, an OA cellular model was developed by treating primary human chondrocytes with the inflammatory factor IL-1β.30 Stimulation with IL-1β led to a reduction in miR-137 expression (Fig. 2A). This decrease was concomitant with elevated expression of fibrogenic markers, including COL1A1, COL3A1, and α-SMA, as well as BMP7 and BMPR2 (Fig. 2B). Additionally, the proliferation of cells was significantly diminished following the challenge with IL-1β (Fig. 2C). Furthermore, IL-1β stimulation induced the release of TNF-α and TGF-β1 in human chondrocytes (Fig. 2D).
3.3 miR-137 overexpression attenuates IL-1β-triggered fibrogenesis and inflammation
To explore the role of miR-137, human chondrocytes were introduced to either a miR-137 analogue (mimic) or miR-NC prior to stimulation with IL-1β. The application of the miR-137 mimic significantly elevated the miR-137 level in chondrocytes subjected to IL-1β stimulation (Fig. 3A). Following overexpression of miR-137, the increase in fibrogenic markers (COL1A1, COL3A1, and α-SMA), along with BMP7 and BMPR2, induced by IL-1β, was mitigated (Fig. 3B). Furthermore, the impairment in cell proliferation induced by IL-1β treatment was rescued through the introduction of miR-137 mimic (Fig. 3C). Additionally, miR-137 mimic expression also dampened the secretion of TNF-α and TGF-β1 in response to IL-1β challenge (Fig. 3D). The above results indicate that miR-137 functions as a protective agent against inflammation and fibrogenesis in chondrocytes.
3.4 miR-137 negatively regulates BMP7 to dampen IL-1β induced fibrogenesis in chondrocytes
Utilizing bioinformatics predictions from the TargetScan database, we found that the 3’ untranslated region of BMP7 mRNA features possible binding sites for miR-137 (see Fig. 4A). To confirm this interaction, we created luciferase reporters with either wild type (WT) or mutated (MUT) interacting sequences and performed a luciferase activity assay. The overexpression of miR-137 resulted in a marked reduction in WT reporter activity, whereas there was no effect on the MUT reporter activity (refer to Fig. 4B). This indicates that miR-137 interacts with BMP7 through the predicted WT sequences. Moreover, introducing the miR-137 mimic resulted in decreased levels of BMP7 protein in chondrocytes (Fig. 4C). The above data imply that miR-137 acts as a negative regulator of BMP7.
To explore the function of BMP7 in fibrogenesis induced by IL-1β, chondrocytes underwent transfection with either control siRNA (si-NC) or BMP7-targeting siRNA (si-BMP7) prior to IL-1β stimulation. Following the treatment with IL-1β, the increase in BMP7 protein levels was significantly diminished after si-BMP7 transfection (Fig. 4D). Additionally, the knockdown of BMP7 caused a reduction in fibrogenic markers, as well as BMPR2 following the IL-1β stimulus (Fig. 4E). Moreover, silencing BMP7 promoted cell growth in chondrocytes exposed to IL-1β (Fig. 4F). Furthermore, BMP7 knockdown resulted in the reduced secretion of TNF-α and TGF-β1 in response to IL-1β treatment (Fig. 4G). Collectively, these findings suggest that miR-137 negatively modulates BMP7, thereby inhibiting fibrogenesis in chondrocytes.
3.5 Andrographolide suppresses IL-1β-mediated fibrogenesis by modulating the miR-137/BMP7 axis
Andrographolide has been extensively documented as an anti-inflammatory compound.31 We subsequently investigated whether this compound also mitigates IL-1β-triggered fibrogenesis through the modulation of the miR-137/BMP7 pathway. Human chondrocytes were subjected to treatments with IL-1β, a combination of IL-1β and andrographolide (20 μM), IL-1β along with a negative control miRNA inhibitor (Inh NC), or IL-1β in conjunction with a miR-137 inhibitor. Analysis by RT-qPCR revealed that andrographolide could enhance miR-137 expression upon IL-1β stimulation, and treatment with the miR-137 inhibitor nullified this upregulation caused by andrographolide (Fig. 5A). Furthermore, andrographolide treatment reduced fibrogenic marker levles, as well as BMP7 and BMPR2 expression following IL-1β treatment, while this effect was reversed upon miR-137 inhibitor transfection (Fig. 5B). The enhancement of cell proliferation in IL-1β-treated cells by andrographolide was also diminished after miR-137 inhibitor co-treatment (Fig. 5C). Additionally, treatment with andrographolide reduced the secretion of TNF-α and TGF-β1 in the presence of IL-1β, an effect that was negated by miR-137 inhibition (Fig. 5D). Therefore, it can be concluded that andrographolide suppresses IL-1β-mediated fibrogenesis through modulation of the miR-137/BMP7 axis.
3.6 Andrographolide attenuates OA progression in vivo by modulating miR-137/BMP7 axis
In order to further confirm the positive impact of andrographolide on OA, we established the following animal groups: a sham group; an OA model group; an OA + andrographolide group; an OA + andrographolide + miRNA inhibitor negative control group; and an OA + andrographolide + miR-137 inhibitor group. After four weeks following OA induction, the cartilage and joint tissues from the knees were harvested for analysis. The results from RT-qPCR indicated that OA induction caused a reduced miR-137 expression within the cartilage tissues, whereas treatment with andrographolide restored the levels of miR-137. The application of miR-137 inhibitor led to a lowered miR-137 level subsequent to andrographolide treatment (Fig. 6A). Histological examination using H&E staining illustrated that andrographolide facilitated the healing of joints affected by OA-related damage, while the inhibition of miR-137 negated this benefit (Fig. 6B). Additionally, Safranin-O/fast green staining revealed cartilage layer degeneration following OA induction, but treatment with andrographolide enhanced cartilage restoration in the OA model group, in contrast to the suppressive effect resulting from miR-137 inhibition (Fig. 6C). We also evaluated inflammatory and fibrogenic markers in the cartilage tissues through ELISA and Western blotting. The treatment with andrographolide resulted in reduced protein levels of TNF-α, TGF-β1, COL1A1, COL3A1, α-SMA, BMP-7, and BMPR2 in the OA model group; conversely, the administration of the miR-137 inhibitor resulted in an upregulation of these proteins (Fig. 6D and E). To sum up, these data imply that andrographolide mitigates the progression of OA by influencing the miR-137/BMP7 pathway in cartilage tissues.
4 Discussion
In the current investigation, we have found the concomitant changes of elevated levels of fibrogenic and inflammatory factors and the reduced expression of miR-137 in OA specimens. Specifically, our findings indicate that in chondrocytes stimulated with IL-1β, there is an observable increase in the expression of fibrogenic markers accompanied by a repression of miR-137 levels. These findings indicate that the inflammatory environment characteristic of OA is implicated in modulating miR-137 expression. Conversely, our experiments showed that miR-137 notably mitigated both the fibrogenic response and inflammatory reactions triggered by IL-1β in chondrocytes, which pinpoints miR-137 as a vital regulatory element whose enhancement could counteract the adverse effects triggered by inflammatory stimuli. Additionally, we elucidated that miR-137 plays a negative regulatory role in BMP7 expression, thereby mitigating the fibrogenic processes. Moreover, andrographolide effectively inhibited the fibrogenic phenotype in chondrocytes, contributing to the attenuation of OA progression through its action on the miR-137/BMP7 signaling axis. Our research not only shows compelling evidence for the pivotal function of the miR-137/BMP7 interaction in influencing the fibrogenic characteristics of chondrocytes but also emphasizes the therapeutic potential of andrographolide for OA treatment.
An expanding collection of research indicates that the variety of chondrocyte phenotypes in osteoarthritis (OA) articular cartilage is influenced by the transcriptomic landscape.10,32 Analyses using single-cell RNA sequencing have uncovered the presence of hypertrophic and fibrogenic chondrocyte phenotypes throughout the progression of OA. Fibrogenic chondrocytes synthesize fibrocartilage, which exhibits greater mechanical stiffness compared to hyaline cartilage; this deterioration in the biomechanical properties of fibrocartilage exacerbates OA symptoms.33–35 These fibrogenic chondrocytes are noted for their elevated production of collagens, in addition to the upregulation of α-SMA, TIMP1, and Hyaluronidase.10–14 Our research findings also demonstrated an increase in COL1A1, COL3A1, and α-SMA levels in both cartilage samples related to OA and IL-1β-treated chondrocytes. The transition of chondrocytes towards a fibrogenic phenotype may be associated with persistent inflammatory stimuli.
We additionally demonstrated a decrease in miR-137 levels in cartilage samples related to OA and in chondrocytes stimulated by IL-1β, aligning with earlier research.21,36,37 miR-137 has been shown to negatively influence the expression of TCF4, thereby alleviating inflammation and apoptosis in chondrocytes.21 Moreover, there is support for the idea that miR-137 defends against extracellular matrix digestion by targeting ADAMTS-5 in chondrocytes.36 Additionally, it was observed that miR-137 reduces the cell growth and mobility of fibroblast-like synoviocytes in cases of rheumatoid arthritis.22 We also demonstrated that overexpressing miR-137 mitigates fibrogenesis and IL-1β-induced inflammation in chondrocytes. Collectively, our findings, along with previous studies, suggest that miR-137 has a protective role against OA pathogenesis by modulating chondrocyte phenotype.
We further observed that an increased miR-137 level could lead to a reduction in BMP7 levels, which mitigated IL-1β-induced fibrogenesis in chondrocytes. Furthermore, it appeared that BMP7 positively influences BMPR2 expression, as silencing BMP7 resulted in a lower BMPR2 expression. Both BMP7 and BMPR2 have been linked to the differentiation processes of chondrocytes and osteogenic cells.23,27,28 Previous research indicates that BMP7 may serve as a growth factor that slows the progression of OA by decreasing MMP13 levels in chondrocytes stimulated with IL-1β,24 enhancing proteoglycan synthesis in chondrocytes,25 and reducing inflammation.26 Additionally, there is support for the role of BMP7 in mitigating fibrosis in various other pathological contexts, including renal and liver fibrosis.38,39 However, our findings suggest that the abnormal upregulation of BMP7, stemming from the downregulation of miR-137, may promote the fibrogenic conversion of chondrocytes in OA. Indeed, BMP7 levels in the plasma of OA individuals are significantly elevated in comparison to healthy controls, and levels of BMP7 in knee synovial fluid correlate with the severity of OA,40 implying that increased BMP7 may worsen OA progression. Our results also revealed heightened BMP7 levels in cartilage associated with OA and in chondrocytes stimulated by IL-1β. Consequently, while BMP7 is suggested as a candidate therapeutic agent for OA,41 it is crucial to exercise with caution regarding the possible risk of synovial fibrosis.42
Andrographolide is a naturally occurring compound known for its widely recognized anti-inflammatory properties.31 Additionally, this compound aids the body's antioxidant defense by counteracting free radicals.43 The therapeutic capabilities of andrographolide have been shown across a variety of pathophysiological conditions. For instance, andrographolide was reported to inhibit the inflammatory responses of macrophages in a murine model of acute colitis.44 Furthermore, andrographolide also demonstrated beneficial activities in an autoimmune myocarditis rat model.45 It also plays a role in preventing inflammatory bone loss by inhibiting osteoclastogenesis.46 Recent research has highlighted the compound's potential anti-fibrotic effects in cases of pulmonary fibrosis.47,48 Our findings indicated that andrographolide reduced the fibrogenic phenotype in chondrocytes and mitigated OA severity by targeting the miR-137/BMP7 pathway. However, it is still unclear whether the anti-fibrogenic effects of andrographolide arise from its anti-inflammatory or antioxidant activities.
5 Conclusion
Our data unveil that miR-137 downregulation along with BMP7 overexpression could play a role in the fibrogenic characteristics observed in chondrocytes associated with OA. We illustrated that andrographolide inhibited the fibrogenic phenotype in these cells and mitigated the advancement of OA by affecting the miR-137/BMP7 pathway. This research presents new insights into the involvement of the miR-137/BMP7 axis in influencing the fibrogenic transition in chondrocytes and highlights the potential of andrographolide as a therapeutic agent for alleviating OA pathogenesis.
CRediT authorship contribution statement
Yaoyu Xiang: completed the main experimental procedure and wrote the manuscript and. Xidan Hu: completed the main experimental procedure and wrote the manuscript. Xianguang Yang: researched data and contributed to the discussion, All authors contributed to the article and approved the submitted version. Guoliang Wang: researched data and contributed to the discussion, All authors contributed to the article and approved the submitted version. Yanlin Li: researched data and contributed to the discussion, All authors contributed to the article and approved the submitted version and. Fei Sun: researched data and contributed to the discussion, All authors contributed to the article and approved the submitted version. En Song: is responsible for the project planning, the fund support and the manuscript proofreading.
Patient consent for publication
All patients involved in this study agreed to this publication.
Availability of data and materials
The data generated in this study are available upon request to the corresponding author.
Ethics approval and consent to participate
The acquisition of all clinical materials had been approved by the Ethics Committee of First Affiliated Hospital of Kunming Medical University (NO. 2024L184). In addition, all the recruited subjects had provided informed consent. All the sample handling and data processing steps were following the Declaration of Helsinki. The experimental of animal procedures were approved by the Ethical Review Committee for Animal Experiments of Kunming Medical University (NO. kmmu20230468).
Funding
This work was supported by Yunnan Science and Technology Programme: Basic Research Special-Youth Project (202201AU070075); National Natural Science Foundation of China (grant No.8216020326).
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