The relationship between personality traits and adherence among patients with hypertension

Figure 1.. The expression of CCN1 is significantly upregulated in clinical samples of keratoconus (KC). (A) Immunofluorescence was used to detect the expression of CCN1 in the healthy control group (Ctrl) and KC tissues (KC; scale bar = 100 µm, red = CCN1, blue = DAPI, n = 3). (B, C) Expression levels of CCN1 in Ctrl and KC were determined by Western blot B and related quantitative analysis C (n = 3). (D, E) Western blot analysis (D) and quantification E of CCN1 in primary corneal stromal cells (CSCs) isolated from healthy donor corneas (HCSCs) and CSCs derived from KC (KC-HCSCs; n = 3). **P < 0.01, ****P < 0.0001.

Figure 2.. Transcriptome analysis of healthy CSCs (Ctrl) and CCN1-overexpressing cells (OE CCN1). (A) Volcano plots showing differentially expressed genes (DEGs) between the Ctrl group and the OE CCN1 group. (B) KEGG pathway enrichment of DEGs between the Ctrl group and the OE CCN1 group. (C) Heatmaps of RNA-sequencing data showing the differences in transcription levels of genes in the “cellular senescence” signaling pathway between the Ctrl group and the OE CCN1 group. (D) Gene Ontology (GO) enriched terms of DEGs between the Ctrl group and the OE CCN1 group. (E) The chord diagram illustrates the close interconnections between DEGs and upregulated pathways between the Ctrl group and the OE CCN1 group. (F, G) Gene Set Enrichment Analysis (GSEA) of pathways between the Ctrl group and the OE CCN1 group with indicated KEGG F and GO gene sets G.

Figure 3.. CCN1 upregulation induces senescence of CSCs in vitro. (A) Immunofluorescence was used to detect the expression of γH2AX, P16, P21 and P53 (senescence markers) between the Ctrl group and the OE CCN1 group (scale bar = 50 µm, red = γH2AX/P16/P21/P53, green = Actin, blue = DAPI, n = 3). (B–F) Expression levels of senescence markers (γH2AX, P16, P21, and P53) in the Ctrl group and the OE CCN1 group were determined by Western blot B and related quantitative analysis C to F (n = 3). (G, H) The positive rate of SA-β-Gal in cells of the Ctrl group and the OE CCN1 group was detected G and subjected to related quantitative analysis H. (scale bar = 100 µm, n = 3). *P < 0.05, **P < 0.01, ****P < 0.0001.

Figure 4.. Expression levels of senescence markers are significantly upregulated in clinical samples of KC. (A–E) Expression levels of senescence markers in corneal tissues from healthy controls (Ctrl) and patients with KC were determined by Western blot A and related quantitative analysis B to E (n = 3). (F) The expression of senescence markers in Ctrl and KC was assessed by immunofluorescence (scale bar = 100 µm, red = γH2AX/P16/P21/P53, blue =DAPI, n = 3). **P < 0.01.

Figure 5.. CCN1 upregulation promotes the secretion of senescence-associated secretory phenotype (SASP) in CSCs. (A–I) Expression levels of SASP in the Ctrl group and the OE CCN1 group were determined by Western blot A and related quantitative analysis B to I (n = 3). (J–L) Enzyme-linked immunosorbent assay (ELISA) quantification was performed to analyze the secretion levels of IL-1β, IL-6, and TNF-α (inflammatory cytokines) in cells from the Ctrl group and the OE CCN1 group (n = 3). (M–O) Real-time PCR was used to quantitatively detect the expression of inflammatory cytokines in the Ctrl group and the OE CCN1 group at the gene level (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 6.. Significantly upregulated expression of SASP in KC samples. (A–I) Expression levels of SASP in HCSCs and KC-HCSCs were determined by Western blot analysis A and related quantitative assessment B to I (n = 3). (J–L) ELISA was performed to quantify the secretion levels of inflammatory cytokines in HCSCs and KC-HCSCs (n = 3). (M–O) Real-time PCR was used to detect the expression of inflammatory cytokines at the gene level in HCSCs and KC-HCSCs (n = 3). (P) Immunofluorescence staining was conducted to assess the expression of inflammatory cytokines in HCSCs and KC-HCSCs (scale bar = 50 µm, red = IL-1β/IL-6/TNF-α, green = Actin, blue = DAPI, n = 3). (Q) The expression of inflammatory cytokines in healthy corneal tissues (Ctrl) and KC corneal tissues was assessed by immunofluorescence (scale bar = 100 µm, red = IL-1β/IL-6/TNF-α, green = Actin, blue = DAPI, n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 7.. Analysis of the regulatory effect of CCN1 upregulation on the corneal stromal microenvironment. (A, B) Flow cytometry was used to detect the reactive oxygen species (ROS) expression levels in the Ctrl group and the OE CCN1 group A, with related quantitative analysis B (n = 3). (C, D) Flow cytometry was performed to detect the cell cycle of the Ctrl group and the OE CCN1 group C, and the proportion of each phase was quantitatively analyzed D (n = 3). (E, F) The proportion of apoptotic cells in the Ctrl group and the OE CCN1 group was quantitatively analyzed by flow cytometry F (n = 3). (G) Real-time PCR was applied to quantitatively analyze the expression of matrix metalloproteinases (MMPs) in the Ctrl group and the OE CCN1 group (all data are compared with the Ctrl group, n = 3). (H) The number of lysosomes in the Ctrl group and the OE CCN1 group was observed by transmission electron microscopy (TEM; scale bar = blue: 2 µm; white = 500 nm, n = 3). *P < 0.05, **P < 0.01, ****P < 0.0001, ns, not significant.

Figure 8.. Schematic depiction of CCN1 function during KC progression in this study. Upregulated CCN1 expression in KC stromal cells promotes the accumulation of ROS, which subsequently drives the SASP of CSCs. Secretion of pro-inflammatory cytokines and MMPs further triggers stromal cell death and extracellular matrix (ECM) degradation. These pathological alterations reciprocally elevate intracellular ROS levels and further boost CCN1 expression, thereby establishing a positive feedback loop. Ultimately, this vicious cycle markedly impairs corneal biomechanical strength and accelerates the initiation and progression of KC protrusion.