Reagents and chemical substances
NaHS and lipopolysaccharide (LPS) have been purchased from Sigma-Aldrich (St. Louis, MO, USA). Trypsin, Dulbecco’s Modified Eagle Medium (DMEM), and 1% Penicillin-Streptomycin have been procured from Hyclone Laboratories (South Logan, UT, USA). Fetal bovine serum (FBS) was bought from Boster Organic Expertise co. Itd (Wuhan, China). BeyoClick™ EdU Cell Proliferation Equipment with Alexa Fluor 488, Western and IP Lysis Buffer, and Proteinase inhibitor have been bought from Beyotime (Shanghai, China). Cell Counting Equipment-8 (CCK-8), hochest and DAPI dye was obtained from Biosharp Life Sciences (Beijing, China).
Fabrication of NaHS@Cy5@MS@SP nanoparticles
Firstly, mesoporous silicon and CY5 have been dissolved in ethanol, stirred at room temperature in a single day, centrifuged to take away ethanol, washed in deionized water, and dispersed in deionized water to acquire CY5@SiO2. Then, CY5@SiO2 and sodium hydrosulfide (NaHS) have been dissolved in water, stirred at room temperature in a single day, centrifuged to take away ethanol, washed in deionized water, and dispersed in deionized water to acquire NaHS@CY5@SiO2. Lastly, SPC, DSPE-PEG-polypeptide, ldl cholesterol and DIL have been dissolved collectively in 3 mL of chloroform, evaporated into a movie underneath decreased stress in pattern bottle, supplemented with a small quantity of deionized water, sonicated with liposome extruder (polycarbonate movie with a pore dimension of 100 nm), after which sonicated in a water bathtub for five min after including NaHS@CY5@SiO2. NaHS@Cy5@mesoporous silic@LNP-targeted peptide-DIL (NaHS@Cy5@MS@SP) was obtained by utilizing a nanodialysis machine (polycarbonate membrane, pore dimension 30 nm).
Characterizations of NaHS@Cy5@MS@SP
Dynamic gentle scattering NanoZS ZEN3600 (Malvern, UK) was used to measure the scale and zeta potential of NaHS@Cy5@MS@SP. Fourier remodel infrared (FT-IR) spectra (4000–400 cm− 1) have been recorded by an FT-IR spectrophotometer (Bruker Company, FT-IR TENSORII, Berlin, Germany). Transmission electron microscopy (TEM, Tokyo, Japan) was used to watch the micro-morphology of NaHS@Cy5@MS@SP. Ultraviolet-visible spectrometers (Synergy H4, Bio Tek, America) have been used to detect the attribute teams of NaHS@Cy5@MS@SP.
Animals
Male DBA/1J mice have been bought from GemPharmatech Co., Ltd (Nanjing, China). The animals have been housed within the Laboratory Animal Heart, Faculty of Drugs, Jiangnan College, at a temperature of twenty-two ± 2 °C and a humidity of 40–70% with 12-h alternating gentle and darkish. The animal experimental ethics was accepted by the Experimental Animal Ethics Committee of Jiangnan College (JN.No20230330d0400930[98]). Forty-five DBA/1J mice have been divided into 5 teams with 9 mice in every group, which have been divided into management group, mannequin group, Cy5@MS@SP group, NaHS@Cy5@MS@SP group (75 mg/kg), and NaHS group (50 µM/kg), respectively. The doses of NaHS@Cy5@MS@SP and NaHS have been chosen in keeping with earlier experiences [21, 25, 30,31,32]. The management group didn’t endure collagen induction remedy, and the opposite teams underwent collagen induction for a second time 7 days after the primary bovine collagen II induction to assemble CIA mice. The intervention was administered on day 15, and the blood and inner organs of mice have been collected on the forty fifth day for the detection of related indicators. The medical scoring standards are: no erythema or swelling, 0 factors; Solely ankle erythema or delicate ankle swelling, 1 level; Ankle joint erythema with delicate swelling, 2 factors; Ankle to metatarsal joint erythema and reasonable swelling, 3 factors; Ankle, toe or limb redness and extreme swelling, 4 factors.
Mobile uptake
RAW264.7 cells and FLSs have been cultured in 6 confocal dishes with a density of 1 × 105 cells, and cultured at 37 °C and 5percentCO2 for six h. After cell adhesion, the cell media have been changed with 200 µL contemporary media containing NaHS@Cy5@MS@SP (0.28 mg/mL). The doses of NaHS@Cy5@MS@SP in mobile experiments have been chosen in keeping with earlier experiences [25, 32]. Cells have been incubated for 1, 4, and 24 h. Subsequently, cells have been mounted with 4% paraformaldehyde for 10 min, washed thrice with PBS, stained with DAPI for 10 min, and resuspended in 200 µL PBS. Fluorescence imaging was then carried out.
In vivo biodistribution
CIA mice (23–25 g) have been randomly divided into 6 teams and administered NaHS@Cy5@MS@SP (75 mg/kg) through tail vein injection. Entire-body fluorescence imaging and ex vivo organ imaging have been recorded at 1, 2, 4, 8, 12, and 24 h post-injection utilizing the IVIS spectrum in vivo imaging system underneath anesthesia at λ ex (650 nm)/λ em (670 nm) (3 mice per time level). All photos have been analyzed and normalized utilizing Dwelling Imaging software program.
Pharmacokinetics evaluation
Free NaHS (50 µM/kg) and NaHS@Cy5@MS@SP (75 mg/kg) have been injected into CIA mice through the tail vein, and blood samples have been collected from the eyeballs at 1, 2, 4, 8, 12, and 24 h after injection (3 mice have been taken at every time level). The H2S content material within the blood was then detected utilizing a H2S assay equipment.
Cell tradition and remedy
FLSs have been maintained in DMEM containing 10% FBS and 1% penicillin/streptomycin (Gibco, Carlsbad, CA, USA) in a 5% CO2 incubator (HeracellVIOS160i, ThermoScientific, USA) at 37℃. FLS cells have been pre-treated with NaHS@Cy5@MS@SP (0.1 mg/mL) and NaHS (100 µM) for 30 min, adopted by publicity to LPS (1 µg/mL) for twenty-four h. To discover the involvement of the Hedgehog signaling pathway, FLS cells have been pre-incubated with SAG for 30 min, after which stimulated with LPS (1 µg/mL) for twenty-four h.
Cell counting kit-8 (CCK-8) assay
FLS cell viability was assessed utilizing CCK-8 assay (Biosharp, Beijing, China). In short, 10 µL of the CCK-8 reagent was added to every properly of a 96-well plate. The plate was then incubated at 37℃ for 1 h. Subsequently, the absorbance at 450 nm was detected by a microplate reader (Winooski, VT, USA).
Micro-CT detection of femur and paw
CIA mice have been repeatedly anesthetized with isoflurane. After anesthesia, the imaging was carried out on a small animal imaging mattress. Micro-CT (quantum GXII, PE, Germany) was carried out to detect the paw deformity, and bone quantity fraction (BV/TV) of the femur by evaluation 12.0 software program. Micro-CT scanning voltage: 90 kv, scanning present: 80 µA, scanning discipline of view: 86 mm, scanning time: 4 min, scanning mode: excessive decision, filter: Cu 0.1 mm, scanning slice thickness: 36 μm.
Masson staining
Firstly, dewaxing was carried out on the paraffin sections of the knee joint, adopted by gradient hydration. Then, Weigert hematoxylin staining answer was used for staining for 8 min. After washing with water, Masson blue staining answer was used for five min, adopted by 5 min of Lichun pink staining, 1 min of phosphomolybdic acid washing, and a pair of min of aniline blue staining. Lastly, carry out dehydration transparency, movie sealing, and microscopic commentary.
Safranin quick inexperienced staining
After the paraffin part of the knee joint was dewaxed and hydrated, the nucleus was stained with hematoxylin, after which the eosinophilic bone tissue was stained with strong inexperienced dye answer for five min. After cleansing, the cartilage was stained with safranin dye answer for five min. Lastly, impartial gum was used to seal and solidify, after which photographed and noticed underneath a microscope.
HE staining
Mice knees have been mounted in 4% paraformaldehyde for 10 d, adopted by a 4-week decalcification course of. The decalcified knees have been then embedded in paraffin and lower into 5 μm thick sections. After deparaffinization, the tissue sections have been hydrated utilizing gradient ethanol and stained utilizing a hematoxylin-eosin staining equipment. The pathological modifications of the knee joint have been noticed utilizing a digital slice scanner (3DHISTECH, Hungary).
Immunohistochemical (IHC) staining
IHC staining of IL-6 and TNF-α within the knee joint of rats was carried out. The knee joint was first dewaxed, adopted by gradient alcohol hydration, citric acid antigen restore, peroxidase inactivation, and BSA closure. Subsequently, the slices have been incubated with particular major antibodies (dilution 1:200) at 4 °C in a single day. On the second day, the tissue sections have been rewarmed at 37 °C for 1 h, washed with PBS buffer answer, after which stained with horseradish peroxidase (HRP) labeled secondary antibody at room temperature for two h. Lastly, a digital slide scanner (3DHISTECH, Hungary) was used to watch the expression of inflammatory elements within the knee joint of rats.
5-ethynyl-2’-deoxyuridine (EdU) staining
The detection of cell proliferation was used BeyoClick™ EdU Cell Proliferation Equipment with Alexa Fluor 488 (Beyotime, Shanghai, China). Briefly, the handled FLS cells have been mounted with 4% paraformaldehyde (Solarbio, Beijing) for 1 h. Following washing thrice, cells have been pretreated with 0.5% TritonX-100 for 15 min, after which incubated with 50 mM EdU for two h underneath ambient setting. The nuclei have been dyed with Hoechst 33,342 and imaged with a ZEISS fluorescence microscope (Zeiss Axio imagar 2, Germany).
Cell migration assay
To evaluate the migration and invasion potential of FLS cells, serum-free DMEM was added to the higher chamber, whereas 750 µL medium containing 10% FBS was added to the decrease chamber. After stimulation for twenty-four h, FLS cells on the higher floor of the transwell filters have been wiped with a cotton swab. The migrated cells have been stained with 1% crystal violet. The photographs have been noticed and photographed underneath a microscope (Nikon, Japan).
Immunofluorescence staining
The joint sections underwent fixation, paraffin embedding, deparaffinization, hydration, and heat-induced antigen retrieval. The FLS cells have been mounted with 4% paraformaldehyde at 4℃ for 1 h. After washing with PBS thrice, the samples have been permeabilized by 0.1% Triton X-100 for 15 min, and subsequently blocked by 5% BSA at 37℃ for 1 h. After three PBS washes, the samples have been incubated in a single day with the indicated antibody at 4℃. On the following day, samples have been stained with fluorescently labeled secondary antibodies (Invitrogen) at 37℃ for 1 h underneath light-protected setting. The nuclei have been visualized with 4’,6-diamidino-2-phenylindole (DAPI). The photographs have been noticed and captured utilizing a ZEISS fluorescence microscope (Zeiss Axio imagar 2, Germany).
Enzyme-linked immunosorbent assay (ELISA)
The degrees of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and vascular endothelial progress issue (VEGF) have been decided by ELISA kits (Elabscience, Wuhan, China) underneath the rule of producer’s protocols. The absorbance values have been learn utilizing a microplate instrument (Synergy H4, BioTek, Winooski, VT, USA) at 450 nm.
Western blotting
After samples have been lysed by Ultrasonic Cell Crusher (Xinchen, Nanjing, China), complete protein concentrations have been measured by BCA Protein Quantification Equipment (Abbkine, Wuhan, China). An equal amount of protein underwent sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and was subsequently transferred onto the polyvinylidene difluoride (PVDF, Millipore Darmstadt, Germany) membrane. After that, the membranes have been blocked by 5% skim milk (Beyotime, Shanghai, China), adopted by incubated with the indicated major antibodies in a single day at 4℃. After washing with TBST, the blots have been uncovered to horseradish peroxidase (HRP)-coupled secondary antibodies at room temperature for an hour. The next antibodies have been used on this examine: anti-CSE (60234-1-Ig, Proteintech), anti-CBS (67861-1-Ig, Proteintech), anti-3-MST (ab96705, Abcam), anti-SHH (20697-1-AP, Proteintech), anti-SMO (20787-1-AP, Proteintech), anti-GLI1 (66905-1-Ig, Proteintech), anti-P-P38 (9211, Cell signaling), anti-T-P38 (9212, cell signaling know-how) and anti-β-actin (66009-1-Ig, Proteintech), HRP-linked anti-rabbit IgG (SA00001-2, Proteintech), HRP-linked anti-mouse IgG (SA00001-1, Proteintech). Finally, the blots have been visualized utilizing enhanced chemiluminescence answer (ECL, Biosharp, Shanghai, China). ImageJ software program (model 1.53t) was utilized to investigate the densities.
Reverse transcription-polymerase chain response (RT-PCR)
Whole RNA from FLS cells was extracted utilizing TRIzol Reagent (Vazyme BioTech Co., Ltd, Nanjing) in keeping with the producer’s directions. RNA focus and purity have been measured utilizing ultramicro spectrophotometer (Nanodrop one, Thermo Fisher Scientific, USA). RNA (1 µg) of every pattern was subjected to reverse-transcription with Hifair® AdvanceFast 1st Strand cDNA Synthesis Equipment (Yeasen, Shanghai, China). Actual-time PCR was carried out utilizing Hieff® qPCR SYBR® Inexperienced Grasp Combine (Yeasen Biotechnology, Shanghai, China) underneath the LightCycler 480 II PCR System (Roche, USA). The relative expression ranges of indicated genes have been normalized to β-actin and calculated utilizing 2–△△Ct technique. Gene-specific primers have been proven in Desk 1.
Transcriptome sequencing evaluation
Transcriptome sequencing and evaluation have been carried out by OE Biotech Co., Ltd. (Shanghai, China), as beforehand reported [16, 33, 34]. In short, complete RNA in every pattern was extracted utilizing TRIzol reagent (Invitrogen, CA, USA) in keeping with the producer’s protocols. RNA purity and focus have been assessed with a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA), whereas RNA integrity was evaluated utilizing the Agilent 2100 Bioanalyzer (Agilent Applied sciences, Santa Clara, CA, USA). RNA libraries have been ready utilizing the VAHTS Common V6 RNA-seq Library Prep Equipment following the producer’s directions. Transcriptome sequencing and subsequent evaluation have been carried out by OE Biotech Co., Ltd. (Shanghai, China), with sequencing carried out on the Illumina NovaSeq 6000 platform utilizing 150 bp paired-end reads. The uncooked reads per pattern have been generated. Uncooked reads in fastq format have been processed with fastp1, and low-quality reads have been eliminated to yield clear reads. After filtering, the clear reads per pattern have been retained for additional evaluation. Clear reads have been aligned to the reference genome utilizing HISAT22. Gene expression was quantified by calculating FPKM values for every gene, and gene learn counts have been obtained utilizing HTSeq-count4. Principal element evaluation (PCA) was carried out in R (v 3.2.0) to judge pattern duplication and organic variability. Differential expression evaluation was performed utilizing DESeq25, with a threshold set at Q worth < 0.05 and fold change > 2 or < 0.5 to determine considerably differentially expressed genes (DEGs). Hierarchical clustering of DEGs was carried out in R to visualise gene expression patterns throughout completely different teams and samples. A radar plot of the highest 30 up- and down-regulated DEGs was generated utilizing the gradar bundle in R. Enrichment evaluation of DEGs was performed for Gene Ontology (GO), KEGG pathways, Reactome, and WikiPathways utilizing hypergeometric distribution, all carried out in R (v 3.2.0). The uncooked RNA sequencing information on this examine is deposited within the public database (the Sequence Learn Archive, SRA) underneath accession quantity PRJNA1143598.
Statistical evaluation
All values have been represented as imply ± SEM. All cell experiments have been repeated 4–6 instances, and animal experiments have been repeated 5–6 instances, apart from the transcriptome which had 3 samples per group. All information have been analyzed with GraphPad Prism software program. Variations between two teams have been carried out utilizing unpaired t-test. Comparisons between a number of teams have been analyzed utilizing ANOVA adopted by the Bonferroni submit hoc take a look at, with p < 0.05 thought-about vital.
