Oxidative stress-induced premature senescence and aggravated denervated skeletal muscular atrophy by regulating progerin–p53 interaction

Experimental animals

Male P53 KO mice, aged 6 to 8 weeks and weighing 22–25 g each, were purchased from the Model Organisms Center (Shanghai, China). Male C57 BL/6 mice aged 6 to 8 weeks and weighing 22–25 g were purchased from the Vital River Laboratory Animal Technology Co. (Zhejiang, China). The mice were housed in standard cages in a room at 23°C and 50% relative humidity on a 12-h:12-h light/dark cycle. For qPCR, western and flow cytometry, four mice were used per each experimental group. For histology, six mice were used per experimental group. For ROS or INOS Inhibition, the mice received a sham operation were administered with saline (control group). For other experiments, the mice that received a sham operation were used as controls. For ROS inhibition, the denervated mice were administered with L-NAC (Den+anti-ROS group). For INOS inhibition, the denervated mice were administered 1400W (Den+anti-iNOS group). The mice in both denervated groups were anesthetized and subjected to unilateral sciatic nerve transection [26]. After deep anesthetization, a 0.5-cm-long portion of the sciatic nerve in the right hind leg of each mouse was resected; the two nerve ends were buried in the muscles, and the incision was closed using 4-0 absorbable sutures. The mice were randomly assigned to experimental groups for analysis at specific time points after denervation.

Wet weight

Seven and 14 days after denervation, the mice and control group were anesthetized, and the gastrocnemius muscles of both the left and the right hind legs were removed, washed with saline, and then weighed. The loss of muscle weight ratio was defined as the weight of the contralateral side minus the muscle weight of the side with the nerve injury divided by the weight of the contralateral side. The muscle samples were stored in 4% paraformaldehyde at −80°C until use.

Quantitative real-time PCR (qPCR)

The RNeasy kit (Qiagen, Valencia, CA, USA) was used to extract the total RNA from the gastrocnemius muscle. The cDNA was synthesized using a first-strand cDNA synthesis kit with oligo dT primers (Invitrogen, Carlsbad, CA, USA) and used for quantitative real-time PCR (qPCR) (MJ Research, Waltham, MA, USA). The thermal cycling conditions were as follows: 94°C for 5 min, 35 cycles at 94°C for 30 s, 55°C for 45 s, 72°C for 1 min, and 72°C for 5 min. The relative expression level of the target gene was calculated by using the cycle threshold (Ct) value. The expression levels of iNOS, α-SMA, progerin, and P53 were normalized to GAPDH levels. The primer sequences were as follows: iNOS: R, 5′ GACCTGATGTTGCCATTGT 3′, F, 5′ TTG ACG CTC GGA ACT GTA G 3′; α-SMA: R, 5′ CAC AGC CTG AAT AGC CAC ATA C 3′, F, 5′ CCT GAA GAG CAT CCG ACA CT 3′, progerin: R, 5′ CTC TCG CTG CTT CCC GTT AT 3′, F, 5′ TGG ATG CTG AGA ACA GGC TAC A 3′, P53:R, 5′ ACT CGG AGG GCT TCA CTT 3′, F, 5′ CAG GAG ACA TTT TCA GGC TTA T 3′.

Western blot analysis

The frozen gastrocnemius muscle samples were homogenized in a radioimmunoprecipitation assay buffer containing 1 mM phenylmethylsulfonyl fluoride and the Protease Inhibitor Cocktail (Roche Applied Science). The lysates were centrifuged for 20 min at 12000 × g (4 °C), and the protein level in the supernatant was quantified with a bicinchoninic acid assay kit (Beyotime). The proteins were separated by SDS–PAGE (Beyotime) and transferred to a polyvinylidene difluoride membrane (Beyotime) that was blocked with 5% non-fat dry milk in tris-buffered saline at room temperature, followed by incubation with primary antibodies: mouse anti-progerin (1:200; Santa Cruz Biotechnology, Sc-81611, USA), rabbit anti-α-SMA antibodies (1:1000; Affinity Biosciences, AF1032, USA), rabbit anti-P53 (1:1000; Abcam, Ab32532, UK) and anti-acetyl-P53 (1:1000; Abcam, Ab183544, UK), and rabbit anti-iNOS antibodies (1:1000; Affinity Biosciences, AF0199, USA). After being washed three times, the membrane was incubated with the appropriate secondary antibody (Abcam) at room temperature for 1 h. The enhanced chemiluminescence detection reagent and an X-ray film were used to visualize the proteins.

Immunohistochemistry

The expressions of the P53 and progerin in the gastrocnemius muscle were detected by immunohistochemistry. The sections were deparaffinized with xylene and rehydrated with ethanol, and antigen retrieval was performed in a 0.01-M citrate buffer (pH, 6.0) in a pressure cooker, followed by natural cooling to room temperature. The sections were incubated in 0.3% hydrogen peroxide at room temperature for 10 min; goat serum was used to block the sections for 15 min at room temperature, and they were then incubated overnight at 4°C with mouse anti-progerin (1:50; Santa Cruz Biotechnology, Sc-81611, USA) and rabbit anti-P53 (1:100; Abcam, Ab32532, UK). This was followed by treatment with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (ABclonal, 33301ES60, Wuhan, China) for 30 min at room temperature. Immunodetection was performed by using a diaminobenzidine solution according to the manufacturer’s instructions. After being washed, the sections were counterstained, dehydrated, and then coverslipped by using neutral gum sealant.

Immunofluorescence

To identify the p53 and progerin proteins in the denervated muscles, double immunostaining was performed. The paraformaldehyde-fixed denervated muscles were incubated with the primary antibody followed by the secondary antibody and were subsequently mounted with DAPI. The primary antibodies included mouse anti-progerin (1:25; Cruz Biotechnology, Sc-81611, USA) and rabbit anti-P53 (1:200; Abcam, Ab32532, UK). The number of positive cells was observed by fluorescence microscopy (1 × 71, Olympus, Japan) and quantified by ImageJ software.

Cell preparation and flow cytometryPreparation of single-cell suspension of gastrocnemius muscle

The gastrocnemius muscle tissue was rinsed with phosphate-buffered saline (PBS), placed in a 6-cm Petri dish, and cut into small pieces (2–4 mm). DNAse I at 1 mg/mL was mixed with the 1640 medium, and 5 ml was added to the pieces of tissue. Tissue-block solution was added to a gentleMACS C tube, and 5 μl of type-II collagenase was added as well. The tissue block was then placed in the gentleMACS C tube with the enzyme solution, the tube was installed in the casing of the gentleMACS tissue processor, and the heating module was inserted. The 37C-mr-SMDK-1 program was used for dissociation; after the program had completed execution, the C tube was removed and briefly centrifuged to pellet the tissue debris. The resuspended cells were filtered with a 70-μm filter, and the resulting cell suspension was collected in 50-ml test tubes. The filters were rinsed with 10 ml of the RPMI 1640 medium, the rinse medium with resuspended cells was centrifuged for 5 min, and the supernatant was discarded. The resuspended cells were then counted and stained.

Preparation of peripheral blood monocyte suspension

The red blood cell lysate of mouse was added to 50 μl of an anti-coagulant. The mixture was diluted to 1 ml (BioLegend, Cat # 420301) and incubated at room temperature away from light for 15 min. The lysate was then centrifuged at 4°C at 350 rpm for 5 min, and the sample was observed for the presence of red blood cells. If the red blood cells were observed, 1 ml of red blood cell lysate at room temperature was added while avoiding light and centrifuged at 4°C for 5 min. Following this, 3 ml of PBS was added to the resuspended cells and centrifuged at 4°C for 5 min. Finally, the cells were resuspended and counted and stained.

Detection of ROS

For ROS detection, 3 ml of PBS was added to 1 ml of the single-cell suspension (3×106 cells) to resuspend the cells. The cells were centrifuged at 4°C at 350 rpm for 5 min, and the supernatant was discarded. Following this, 1 ml of the serum-free medium was added to the cell pellet to dilute the 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) solution 1:1000 so that the final concentration was 10 μmol/L. The sample was incubated at 37°C for 30 min through mixing by inverting the tube every 3–5 min so that the probe remained in contact with the cells. The cells were then centrifuged and washed with serum-free cell culture medium three times by centrifugation at 4°C at 350 rpm to fully remove the DCFH-DA that had not entered the cells. A total of 200 μl of serum-free medium was then added to resuspend the cells. The cells were then examined by flow cytometry and the data were analyzed using FlowJo software.

ROS inhibition

The acetylcysteine (L-NAC) (MCE, China) or solvent control group (saline) was injected intravenously at a dose of 1.1 mmol/kg/d [27] before denervation and injected continuously for 14 days after denervation.

INOS inhibition

The iNOS inhibitor 1400W (MCE, China) or solvent control group (saline) was injected intraperitoneally (200 μg/mouse) [28] 1 day before denervation and was injected continuously for 14 days after denervation.

Hematoxylin–eosin (HE) and Masson’s trichrome staining

Gastrocnemius muscle samples from the mice were fixed in 4% paraformaldehyde and embedded in paraffin. Five-μm-thick pieces of the samples were cut, and the sections were stained with hematoxylin–eosin (HE) (Beyotime, Shanghai, China) and Masson’s trichrome (Beyotime) to identify histopathologic changes. The mean area, diameter, and density of the myofibers were determined by blinded analysis using Image-Pro Plus 6.0 software (National Institutes of Health, Bethesda, MD, USA) from six randomly captured images per mouse under each set of experimental conditions.

Statistical analysis

All data are presented as mean ± SEM. Differences between groups were evaluated by using the two-sample t test, Mann–Whitney U test, or the χ2 test. One-way or two-way ANOVA was conducted and was followed by the Bonferroni post hoc test for comparisons among groups. Differences at different time points were evaluated by using the Friedman test. Statistical analyses were performed using SPSS v17.0 software (SPSS Inc., Chicago, IL, USA). P values < 0.05 were considered statistically significant.

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