BMP7 ameliorates intervertebral disc degeneration in type 1 diabetic rats by inhibiting pyroptosis of nucleus pulposus cells and NLRP3 inflammasome activity

Establishment of T1DM rat models

All experimental protocols in the present study were approved by the Animal Care Committee at Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. A total of 80 Sprague–Dawley male rats (aged 6–8 weeks, weighing 150–200 g) procured from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) were maintained individually in a pathogen free (SPF) animal laboratory at 22–25 °C, with humidity of 60–65% under a 12-h light/dark cycle. The rats were given ad libitum access to food and water. The experiment was conducted after acclimatization for one week.

The rats were randomly divided into the control (n = 12) and streptozotocin (STZ) (n = 68) groups. T1DM was induced in rats by administering STZ (S0130, Sigma; 50 mg/kg) via a single intraperitoneal injection. Control rats were injected with equal volumes of saline. STZ solution was placed in freshly prepared 0.1 M citrate buffer (pH 4.5). Rats were fasted for 12 h prior to STZ injection. After STZ injection, the rats were provided with standard food and 10% sucrose water for 48 h to prevent lethal hypoglycemia, which were closely monitored every 2 h for a duration of 12 h to observe decreased activity, slow response, or convulsions. At 72 h after STZ injection, blood samples were extracted from the retro-orbital plexus of rats anesthetized with pentobarbital sodium to measure fasting glucose levels using OneTouch Ultra Mini glucose meters. The rats with blood glucose levels of above 250 mg/dL were regarded as T1DM rats. On the 8th week after STZ injection, IDD was observed in 62 STZ-induced T1DM rats (91%) using an X-radiography.

Experimental protocols

The STZ-induced IDD rats were randomly divided into 5 groups (n = 12).

1.

STZ group (untreated STZ-induced rats).

2.

STZ + oe-NC group (STZ-induced rats were injected with over-expression negative control (NC) lentivirus via tail vein).

3.

STZ + oe-BMP7 group (STZ-induced rats were injected with lentivirus over-expressing BMP7 via tail vein).

4.

STZ + oe-BMP7 + dimethyl sulfoxide (DMSO) group (STZ-induced rats were injected with lentivirus over-expressing BMP7 and 1% DMSO via tail vein).

5.

STZ + oe-BMP7 + 4’-Methoxyresveratrol (4’MR) group (STZ-induced rats were injected with lentivirus over-expressing BMP7 and NLRP3 agonist 4’MR (to activate NLRP3) via tail vein).

One week after STZ injection, STZ-induced rats, except for group 1, were injected with 100 μL lentivirus via tail vein with a viral titer of 5 × 108 TU. Two days later, STZ-induced rats in group 4–5 were injected with 100 μL 4’MR (25 mg/kg, HY-N2485, MedChemExpress, Shanghai, China) or 1% DMSO (100 μL, HY-Y0320, MedChemExpress), once every week. Afterwards, the rats were euthanized on the 8th week after STZ injection, and Co6–Co7 discs and nucleus pulposus (NP) tissues were isolated from rats for subsequent experiments.

X-radiographic analysis

On the 2nd, 4th, and 8th week after STZ injection, the SD rats were anesthetized with sodium pentobarbital (at a dosage of 50 mg/kg, P-010, Supelco) intraperitoneally, and an X-ray system (DRX-Evolution, Ruike Medical, China) was utilized for X-ray radiography. Disc height index (DHI) was calculated independently by three researchers who were blinded to the treatment groups (DHI = IVD height/adjacent IVD height) (Chen et al. 2020).

Hematoxylin–eosin (HE) staining and Safranin O-fast green staining

For histological assessment, NP tissues were fixed in 4% paraformaldehyde, paraffin-embedded, and sliced into 4 μm sections. Next, the sections were stained with hematoxylin and then stained with 5% eosin (Liu et al. 2019). Additionally, Safranin O-fast green staining was adopted for bone histopathological observation to reflect the structure of articular cartilage, subchondral bone, and bone tissues. Subsequently, the sections were stained with 0.02% malachite green, and then stained with 0.1% safranin O solution (Zhang and Wang 2014). A total of 10 visual fields of each section were randomly selected under an optical microscope.

Immunohistochemistry (IHC)

Following antigen retrieval, NP tissue sections were blocked with 5% bovine serum albumin (BSA). Next, the sections were immunostained overnight at 4 °C with the following primary antibodies: MMP13 (rabbit, dilution ratio of 1:500, ab219620, Abcam, Cambridge, UK), Aggrecan (rabbit, dilution ratio of 1:200, ab216965, Abcam), COLII (rabbit, dilution ratio of 1:200, 15943-1-AP, Proteintech, Wuhan, China), and ADAMTS5 (rabbit, dilution ratio of 1:200, PA5-32142, Abcam), p-Smad1/5 (rabbit, dilution ratio of 1:100, ab92698, Abcam), Smad1/5 (mouse, dilution ratio of 1:50, ab75273, Abcam) and Ki67 (rabbit, dilution ratio of 1:200, ab16667, Abcam). Subsequently, the sections were re-probed with biotinylated goat anti-rabbit IgG (dilution ratio of 1:2500, ab205718, Abcam) or goat anti-mouse IgG (dilution ratio of 1:2500, ab6788, Abcam) for 20 min, and then incubated with horseradish peroxidase (HRP)-streptavidin reagent (Sigma, Shanghai, China) for 20 min. Finally, the immunoreactivity was detected by means of DAB staining. Images were obtained under a microscope (Leica-DM2500) and quantified using the ImagePro Plus 7.1 software (Media Cybernetics, Silver Spring, MD, USA).

Enzyme linked immunosorbent assay (ELISA)

The ELISA kits utilized for evaluation of supernatant of homogenized NP tissues and cell supernatant included rat NLRP3 (15.63–1000 pg/mL, ab277086, Abcam), rat IL-1β (0.2–0.4 μg/mL, ab9722, Abcam), rat IL-18 (15.6–1000 pg/mL, ab312909, Abcam), rat gasdermin-D (0.156–10 ng/mL, #EKU09199, Biomatik, Delaware), rat BMP-7 (15.63–1000 pg/mL, NBP2-70002, Novus Biologicals).

TUNEL assay

Apoptosis in the NP tissues was assessed with the help of TUNEL Apoptosis detection kits (Sigma) as previously described (Cao et al. 2019). Briefly, paraffin-embedded sections were treated with 20 μg/mL of protease K (Beyotime, Shanghai, China), and subsequently incubated with 3% hydrogen peroxide. Following incubation with 50 μL Streptavidin-HRP working solution for 30 min, the sections were incubated with 0.2–0-0.5 mL DAB solution for 5–30 min, followed by observation under an inverted microscope. Cell apoptosis rate is expressed as apoptotic cells (with brown nuclei)/total cells × 100%.

RNA extraction and sequencing

NP tissues were collected from three T1DM-induced IDD rats and three normal rats. Total RNA content was isolated using the TRIzol reagent (Invitrogen, Shanghai, China). Next, the RNA sample concentration was determined with a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific) through OD260/280. The RNA concentrations were determined by means of Qubit RNA assay kits. Total RNA samples with RNA integrity number (RIN) ≥ 7.0 and a 28S:18S ratio ≥ 1.5 were utilized for subsequent experiments.

A total amount of 5 μg RNA was adopted for each sample. Briefly, ribosomal RNA (rRNA) in the total RNA was removed with the help of Ribo-Zero™ Magnetic Kit (Epicentre Technologies, Madison, WI, USA). To remove linear RNAs, the total RNAs were detached with RNase R (Epicentre Technologies). The libraries for sequencing were constructed according to the manufacturer’s protocols of NEBNext Ultra RNA Library Prep Kit for Illumina (E7760, NEB, MA). Subsequently, the RNA was fragmented into pieces of ~ 300 base pairs (bp) in length in NEBNext First Strand Synthesis Reaction Buffer (5 ×). The first-strand cDNA was synthesized by reverse transcriptase and random hexamer primers, and the second-strand cDNA was synthesized in Second Strand Synthesis Reaction Buffer with dUTP Mix (10 ×). The cDNA fragment was subjected to an end repair process, which included the addition of a ploy (A) tail and ligation of the adapters. Following ligation of Illumina sequencing adaptors, the second strand of cDNA was detached using the USER Enzyme (NEB) to construct a chain specific library. After amplification, the DNA libraries were purified and enriched using AMPure XP beads (Beckman Coulter). Thereafter, the libraries were qualified by Agilent 2100 and quantified with KAPA Library Quantification kits (KAPA Biosystems, South Africa). Finally, paired-end sequencing was performed on an Illumina HiSeq CN500 sequencer.

Sequencing data analysis

The quality of the paired-end reads of the raw sequencing data was checked using the FastQC software v0.11.8. Briefly, the raw data were subjected to pre-processing using the Cutadapt software 1.18, including removal of the Illumina sequencing adaptors and poly (A) tail sequences. The reads with a N content of over 5% were removed with a perl script. The reads with a 70% base mass above 20 were extracted using the FASTX Toolkit software 0.0.13. Next, two-end sequences were repaired using the BBMap software. Finally, the filtered high-quality reads fragment was aligned with rat reference genome using the HISAT2 software (0.7.12).

The gene expression matrices of the sequencing datasets were merged, and the differences between batches were removed using the R software “sva” package. Differentially expressed genes (DEGs) between normal control samples and IDD samples were screened using the R software “limma” package with |logFC|> 1 and P-value < 0.05 serving as the threshold.

Dataset retrieval and differential analysis

The expression profile dataset GSE34000 of dorsal root ganglia in STZ-induced diabetic painful neuropathy rats was retrieved from the GEO database, which included three DM rats and three normal control rats. The three DM rat samples were obtained from L4–L6 dorsal root ganglia 3 weeks after STZ treatment. The gene ID was annotated to the microarray dataset based on the platform information GPL341.

Differential expression analysis was performed using the R software “limma” package with |logFC|> 0.5 and p < 0.05 serving as screening conditions to select the DEGs in microarray data GSE34000. A volcano map was drawn using the R software “ggplot2” package, while a heat map of the expression of DEGs was drawn with the R software “heatmap” package.

Differential genes of sequencing data and GEO microarray data were intersected through the Xiantao Academic website to obtain candidate genes.

Functional enrichment analysis of the selected target genes was performed using the “ClusterProfiler” package in R software, and the Fisher test was adopted to identify significantly enriched GO and KEGG pathways.

GO and KEGG enrichment analysis of transcriptome sequencing data were analyzed using the GSEA database. Next, the selected candidate genes were introduced together into the STRING database for protein interaction analysis with species restricted to rats. The network regulatory relationships were further analyzed with the Cytoscape software (v3.6.0) to screen the top 10 core genes.

The interaction between BMP7 and NLRP3 was analyzed using the GeneMANIA website to identify the target genes.

Gene expression quantitation

Total RNA content was extracted from cells and tissue samples using the TRIzol reagent (Solarbio, Beijing, China). For mRNA expression, the obtained RNA was reverse-transcribed into cDNA using the PrimeScript™ RT-PCR kit (TaKaRa, Tokyo, Japan). RT-qPCR was performed on a LightCycler 480 system (Roche Diagnostics, Pleasanton, CA, USA) using the SYBR Premix Ex Taq™ (TaKaRa). As normalized to GAPDH, the 2−ΔΔCt method was adopted to quantify the relative expression of genes to be tested. The primer sequences (Additional file 2: Table S1) used for amplification were designed and synthesized by Shanghai General Biotechnology (Shanghai, China).

Isolation and culture of NPCs

The NP tissues from control and STZ-induced T1DM rats were rinsed with PBS (Gibco, Grand Island, NY, USA). After being cut into blocks, the samples were detached with 0.25 mg/mL II collagenase (Gibco) for 6 h, and subsequently filtered with a filter mesh size of 70 μm. After washing in PBS and centrifugation, the isolated NPCs were cultured in DMEM/F12 medium containing 15% FBS and 1% penicillin–streptomycin (Gibco). After identification using NPC fluorescence-labeled antibodies (CD24, #BS-4891R, dilution ratio of 1:20, Invitrogen; KRT18, #MA1-06326, dilution ratio of 1:20; Invitrogen) (Additional file 1: Fig. S1A, B), the NPCs at passage 2 were used for subsequent experiments.

NPCs at the logarithmic phase of growth were detached with trypsin and seeded into 6-well plates (density of 1 × 105 cells/well), followed by incubation for 24 h. Upon obtaining 75% confluence, the NPCs were transduced with lentiviruses (Shanghai GeneChem Co., Ltd., Shanghai, China) as instructions described.

1.

Control group (primary NPCs from normal rats).

2.

STZ (primary NPCs from STZ-induced rats).

3.

STZ + oe-NC (primary NPCs from STZ-induced rats transduced with oe-NC).

4.

STZ + oe-BMP7 (primary NPCs from STZ-induced rats transduced with oe-NC).

5.

STZ + oe-BMP7 + DMSO (primary NPCs from STZ-induced rats transduced with oe-BMP7 and treated with DMSO).

6.

STZ + oe-BMP7 + 4’MR (primary NPCs from STZ-induced rats transduced with oe-BMP7 and treated with 4’MR [10 μM, HY-N2485, MedChemExpress]).

Statistical analysis

Data analyses was performed using the SPSS 21.0 software (IBM, Armonk, NY, USA). All quantitative data are presented as mean ± standard deviation. The normality and homogeneity of variance of data were assessed with the Shapiro–Wilk test and the Levene test, respectively. The data of normal distribution and even variance between two groups were analyzed with an unpaired t-test. Welch's correction was adopted for unequal variances, and Mann Whitney rank sum test was utilized for data of skewed distribution. The data of normal distribution and even variance among multiple groups were analyzed by one-way analysis of variance (ANOVA), followed by Tukey’s post-hoc test. Kruscal wallis H rank sum test was adopted for data with skewed distribution and uneven variance. Correlation among indexes was analyzed by Pearson's correlation coefficient. A value of p < 0.05 was regarded statistically significant.

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