Establishment and transmission of hspb2 KO mice. Hspb2 knockout mice were produced by Cas9/CRISPR-mediated genome editing (Cyagen Biosciences Inc., Guangzhou, China). The animal background chosen was C57BL/6J. The gDNA vector was first designed (Fig. 1A). The hspb2 gene (NCBI reference sequence: NM_024441; collection: ENSMUSG00000038086) is situated in chromosome 9 of mice, and two exons were identified, with the ATG starting codon in exon 1 and the TGA terminating codon in exon 2 (transcript: ENSMUST00000042790). Exon 2 was chosen as the target site. The gRNA vector and Cas9 vector were then transcribed in vitro and co-injected them into the fertilized eggs, and the mice were identified by PCR and sequencing after birth to obtain positive F0 mice. The F1 heterozygous mice were mated and the toes of the newborn mice were taken for PCR identification. For amplification, the primer sequences are as follows: Forward primer (F1): 5'-GTACTTGGGATCA GGGCTACTAG-3', Reverse primer (R1): 5’-GAATGGGAGATGGTTCACCTCAG-3’ (Targeted allele: 222 bp; Wildtype allele: 253 bp), resulting in KO mice. WT mice show one band at 253bp, while KO mice show one band at 222bp and heterozygous mice show two bands, as shown in Fig. 1B, with mice 3/7 being the hspb2 KO mice.
Animals. Hspb2 KO mice (n = 10, 18–22 g) and age-matched WT mice (n = 10, 18–22 g, purchased from the Animal Experiment Center of Chongqing Medical University) were used in this study. They were housed at an ambient temperature of 23 ± 2°C, 60% relative humidity, and 12/12 h day/night alternation, with free access to feed and water. This study conformed to the ethical criteria set by the Experimental Animal Ethics Committee of the First Affiliated Hospital of Chongqing Medical University.
Preparation of rapamycin mixture. 25 mg of rapamycin powder (cat.no. HY-10219, MCE, USA) was added into 1367 µl of dimethyl sulfoxide (DMSO) to configure a 20 mM master mix. 1.7 µl of the master mix was added 600 µl of pro-solvent (10% DMSO (Beyotime Biotechnology, China), 40% PEG300, 5% Tween-80 (MCE, USA) and 45% saline in that order). Each mouse was administered subcutaneously in the ear at 0.1 mg/kg, and the same volume of pro-solvent was given to the control group.
UVB radiation. Based on the study by Sajo et al.(18), we created a UVB-induced photodamage model of mouse ear skin. In the UVB irradiation group, the ear skin of each mouse was irradiated with a UVB lamp (TL20W/01, Philips, The Netherlands) emitting at 280–311 nm, at a total dose of 2700 mj/cm², once a day for two times.
Quantitative RT-PCR (qRT-PCR) analysis. After 24 h of the last illumination, the mice were executed and the skin tissue of the ears was taken. Total RNA was extracted by RNAiso Plus kit (TaKaRa, Japan) and reverse transcribed into cDNA by RT Master Mix for qPCR (MCE, USA). SYBR Green qPCR Master Mix (MCE, USA) was used to perform qRT-PCR, and GAPDH was amplified and used as an internal control. The primer sequences for the targeted genes are listed as follows:
p62, forward: 5′-ACTACCCCAGAAAGTTCCAGC-3′,reverse: 5′-TTTTCCCGACT CCATCTGTTC-3'; LC3B, forward: 5′-GCTAACCAAGCCTTCTTCCTC-3′,reverse: 5′-TGCTGTCCCGAATGTCTCC-3';GAPDH,forward:5′-GACATCAAGAAGGTGG TGAAGC-3′,reverse:5′-GAAGGTGGAAGAGTGGGAGTT-3';hspb2,forward:5′-CG AGTACGAATTTGCCAACCC-3′,reverse:5′-CAGAAACGCCTGGAACTTGC-3'; ATG5,forward:5′-GTTTTGGCTTTGGTTGAAGGAAGA-3′,reverse: 5′-AATTCGTC CAAACCACACATCTC-3'; CollagenI,forward:5′-CCCGAGGTATGCTTGATCTGT AT-3',reverse:5′-TCCCTCGACTCCTACATCTTCTG-3';Bax,forward:5′-TTTTGCTA CAGGGTTTCATCCAGG-3′,reverse:5′-TCATCCTCTGCAGCTCCATATTG-3';Bcl-2,forward:5′-GGATTGTGGCCTTCTTTGAGTTC-3′,reverse:5′-CTTCAGAGAGAC AGCCAGGAGAAAT-3'; P53, forward: 5′- AGACCGCCGTACAGAAGAAGAAA-3′,reverse:5′-CGGAACATCTCGAAGCGTTTAC-3′;MMP1,forward:5′-GAGTGCCT GATGTGGGTGAATAC-3′,reverse:5′-ATGTCAGCAGTGCCATCATAGAT-3′; MMP3,forward:5′-TGTGCAGCTCTACTTTGTTCTTTG-3′,reverse:5′-CCTCGTATA GCCCAGAACTGATT-3′;MMP9,forward:5′-CCATGTCACTTTCCCTTCACCTT-3′, reverse:5′-TCTCACTAG GGCAGAAACCAAAT-3′
Western blot analysis. Proteins from mouse ear skin were extracted by T-PER™ Tissue Protein Extraction Reagent (Thermo Fisher, USA), and the total protein concentration of each sample was determined using a BCA assay kit (Bimake, China). Protein samples were separated on 12% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and then transferred to nitrocellulose membranes (NC membranes), which were placed at room temperature in 7% skimmed milk for 2 h at room temperature and incubated overnight at 4°C in the following primary antibodies: GAPDH (Bimake, USA), P62 (Bimake, USA), LC3B (Sigma, USA), ATG5 (Bimake, USA), Bax (Proteintech, China), Bcl-2 (ZEN BIO, China), P53 (Proteintech, China), Hspb2 (Abclonal, China), and Collagen I (Abclonal, China). This was followed by incubation in secondary antibodies at room temperature for 1 h. Finally, the protein bands were imaged using the SuperSignal™ West Femto Maximum Sensitivity Substrate Kit (Thermo Fisher, USA).
Histological analysis. Mouse ear skin samples were obtained, fixed in 4% paraformaldehyde for 24 h, dehydrated in ethanol, and then embedded in paraffin and sectioned to a thickness of approximately 3–4 µm. The paraffin was then removed, and hematoxylin-eosin staining (HE staining) and Masson trichrome staining were performed for histological evaluation. Immunohistochemical staining was performed with TGF-β(1:100), Collagen I (1:100) for labelling, p62 (1:50), and LC3B (1:100) for immunofluorescence labelling, and all stained skin samples were observed using a section scanner (Pannoramic DESK, P-MIDI, P250, Hungary).
Statistical analysis. All experiments were repeated at least 3 times and statistical analyses were performed using Graphpad Prism 8.0.2 (GraphPad Software, Inc., USA). All the data are expressed as mean ± standard deviation. Student’s t-test and one-way ANOVA were used to compare the differences between 2 groups as well as between multiple groups, respectively, and p-values < 0.05 were considered statistically significant.