Detection of peripheral IL-17 expression in CGF-treated patients
We recruited 12 patients who were only diagnosed with androgenetic alopecia. Participants who met the following criteria were excluded: pregnancy or lactation, a diagnosis of hypertension, hyperlipidaemia, diabetes, malignancy, thyroid dysfunction, infectious disease, autoimmune disease, bleeding disorder, platelet dysfunction syndrome, a PLT <150,000/µL, a history of drug allergy, anticoagulant therapy, or any acute or chronic medical or laboratory abnormalities that increased the risk of participating in the study. Written informed consent was obtained from all participants. Ethics Committee approval was obtained from the Institutional Ethics Committee of Shanghai East Hospital prior to the commencement of the study. Each patient was given CGF treatment on the scalp 3 times. Peripheral blood was collected before the first and after the third treatment. Sera were separated from venous peripheral blood samples. Then, IL-17A levels were measured by ELISA using an ELISA kit for IL-17A (Shanghai ExCell Biology, Shanghai, China).
CGF preparation
Six milliliters of venous blood was collected from the elbow vein. Blood was prepared with standard CGF extraction protocols (2700 r/min for 2 min, 2400 r/min for 4 min, 2700 r/min for 4 min, and 3000 r/min for 3 min).
Surgical procedure
Scalp disinfection and anaesthesia were achieved using 5.0 g/L iodophor and 2% lidocaine, respectively. After anaesthesia, CGF was injected into the scalp according to previous methods[20].
Statistical analysis
Statistical analysis was performed using Prism 9.0 (GraphPad Software). The results are reported as the mean ± standard deviation (SD). The statistical tests used, number of animals, and P values are described in the legend for each figure or indicated as dots in the figures.
Mouse experiment
The Institutional Animal Care and Use Committee of the Experimental Animal Welfare and Ethics Management Committee of Shanghai East Hospital (Shanghai, China) approved all the animal experiments. Seven-week-old male C57BL/6 mice (obtained from JSJ Experimental Animal Company, Shanghai, China) were divided into four groups (n=3/group). The groups included the control group, CGF group, IMQ group and IMQ+CGF group. The control group and the IMQ group were given saline through MNs every 3 days on the dorsal skin, while the CGF group and the IMQ+CGF group were given CGF treatment through MNs at the same pace. Imiquimod was applied on the dorsal surface of the mice in the IMQ and IMQ+CGF groups on the first week and fourth week. All the treatment were prepared following the induction of anaesthesia with isoflurane. At the end of week 4, the mice were injected with an overdose of ketamine (180 mg/kg) + xylazine (30 mg/kg) immediately for euthanasia. The appearance of the back skin was photographed, and the skin barrier score (GPSKIN) was recorded. Dorsal skin was biopsied, and IL-17 expression was tested through PCR. In addition, pathological examination of the biopsied skin samples was also performed (Fig. 1).
Mouse psoriatic lesion severity assessment
The severity of skin inflammation was assessed using the Psoriasis Severity Index (PSI)[21]. Skin erythema, scaling, and thickness were the indicators, and each was scored independently on a scale of 0 to 4: 0–none, 1–slight, 2–moderate, 3–marked, and 4–very marked. The total PSI was determined by the cumulative score of all indicators.
RNA isolation and library preparation
Total RNA was extracted using TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer’s protocol. RNA purity and quantification were evaluated using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Then, the libraries were constructed using the VAHTS Universal V6 RNA-seq Library Prep Kit according to the manufacturer’s instructions. Transcriptome sequencing and analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China).
RNA Sequencing and Differentially Expressed Genes Analysis
The Illumina NovaSeq 6000 platform was used to sequence the libraries, generating 150 bp paired-end reads. The raw reads in fastq format were processed using fastp[22], and low-quality reads were removed to obtain clean reads. HISAT[23] was used to map the clean reads to the reference genome. FPKM[24] values were calculated for each gene, and the read counts of each gene were obtained using HTSeq-count[25]. To assess the biological duplication of samples, PCA was performed using R (v 3.2.0). DESeq2[26] was used for differential expression analysis, with a Q value < 0.05 and a fold change > 2 or < 0.5 set as the thresholds for significantly differentially expressed genes (DEGs). Hierarchical cluster analysis of DEGs was conducted using R (v 3.2.0) to show the expression patterns of genes in different groups and samples. The expression of upregulated or downregulated DEGs is shown in heat map of the top 10 genes according to the R package ggradar.