November 2019
Volume 60, Issue 14
Free
Biochemistry and Molecular Biology  |   November 2019
P38-Mediated Cellular Senescence in Conjunctivochalasis Fibroblasts
Author Affiliations & Notes
  • Minhong Xiang
    Department of Ophthalmology, Putuo Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China
  • Lijuan Mo
    Department of Ophthalmology, Putuo Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China
  • Yueping Zhan
    Department of Ophthalmology, Putuo Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China
  • Hang Wen
    Department of Ophthalmology, Putuo Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China
  • Huanming Zhou
    Department of Ophthalmology, Putuo Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China
  • Wanhong Miao
    Department of Ophthalmology, Putuo Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China
    Department of Ophthalmology, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China
  • Correspondence: Minhong Xiang, Department of Ophthalmology, Putuo Hospital, Shanghai University of Traditional Chinese Medicine, No.164 Lanxi Road, Shanghai 200062, P.R. China; xiangminhong1977@126.com
  • Wanhong Miao, Department of Ophthalmology, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, No. 528 Zhangheng Road, Shanghai 201203, P.R. China; miaowanh@126.com
  • Footnotes
     MX, LM, and YZ contributed equally to the work presented here and therefore should be regarded as equivalent authors.
Investigative Ophthalmology & Visual Science November 2019, Vol.60, 4643-4651. doi:https://doi.org/10.1167/iovs.19-27617
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      Minhong Xiang, Lijuan Mo, Yueping Zhan, Hang Wen, Huanming Zhou, Wanhong Miao; P38-Mediated Cellular Senescence in Conjunctivochalasis Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2019;60(14):4643-4651. doi: https://doi.org/10.1167/iovs.19-27617.

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Abstract

Purpose: Conjunctivochalasis (CCH) is a common ocular disease and has received extensive attention recently. However, its exact pathogenesis remains largely unknown. Owing to the high morbidity of CCH in older people, this study aimed to investigate whether cellular senescence contributes to CCH progression and the underlying mechanism.

Methods: Loose conjunctival tissues from CCH patients (n = 13) and normal conjunctival tissues from age-matched persons (n = 12) were obtained and the fibroblasts were separately induced and obtained. Cellular senescence, and the expression of senescence-associated genes (p53 and p21) and p38 in CCH conjunctival tissues and normal controls, were determined by senescence-associated β-galactosidase (SA-β-Gal) staining and quantitative (q)RT-PCR, respectively. To explore the effects of p38 on cellular senescence in CCH fibroblasts, small interfering RNA (siRNA) targeting p38 (siP38) and p38-specific inhibitor SB203580 was performed in CCH fibroblasts. Then, cellular senescence, cell viability, reactive oxygen species (ROS) production, and gene expression were detected according to the corresponding methods.

Results: CCH conjunctival tissues had significantly more senescent cells, evidenced by more SA-β-Gal–positive cells, and higher expression of senescence-associated genes (p53 and p21) and p38. CCH fibroblasts transfected with siP38 or treated with SB203580 had obviously reduced numbers of senescent cells, decreased ROS production, and increased cell viability, as well as reduced expression of senescence-associated genes. Meanwhile, blocking p38 signaling decreased the expression of p53 and p21.

Conclusions: Therefore, these findings indicate that cellular senescence might be a causative factor for CCH. P38 signaling might play an important role in the progress of cellular senescence in CCH fibroblasts via manipulation of p53/p21 signaling.

Conjunctivochalasis (CCH) is one of the most common ocular diseases and is characterized by loosely redundant conjunctiva folds between the eyeball and the lower eyelid.1,2 CCH occurs in all age groups, especially older people.3 Our previous survey showed that the morbidity of CCH in individuals over 60 years old was up to 44.08%.4 And a hospital-based study in Japan revealed that the morbidity of CCH was more than 98.0% in individuals over 60 years old.3 In recent decades, studies related to CCH have gradually increased.5 Researchers have speculated in a variety of ways in implicating the pathogenesis of CCH. For example, degenerative structural changes, especially elastotic degeneration and collagenolysis by overexpression of matrix metalloproteinases (MMPs), have long been speculated to contribute to the pathogenesis of CCH.69 Additionally, oxidative stress and inflammation have been reported to play a role in the pathogenesis of CCH, and inflammatory cytokines enhanced the expression of MMP-1 and MMP-3 in CCH fibroblasts.10,11 However, the underlying relationship between these reported changes and aging remains unclear. 
Normal human fibroblasts lost the ability to proliferate after a finite number of cell divisions, but still remained viable for a long time.12,13 This progression was termed cellular senescence, which was marked by expression of senescence-associated β-galactosidase (SA-β-Gal).14 It has been reported that cellular senescence plays an important role in aging and age-related diseases,15 such as glaucoma,16 cataracts,17 atherosclerosis,18 diabetes, and osteoarthritis.19,20 Aging is the most important risk factor for CCH.21 Based on these studies, we speculated that cellular senescence may contribute to the development of CCH. However, the role of cellular senescence in CCH is still largely unknown. 
Mitogen-activated protein kinases (MAPKs)—which comprise four subgroups, extracellular signal-regulated kinases (ERKs), c-Jun amino-terminal kinases (JNKs), ERK/big MAP kinase 1 (BMK1), and p38 MAPKs—control various physiological processes, including cellular response to extracellular signals, gene expression, cell growth, and apoptosis.22 Among the three protein kinases, p38 MAPK can be activated by external signals and inflammatory cytokines22 and involved in diverse cellular biological progressions, such as cell multiplication, differentiation, migration, senescence,23 and apoptosis. Activation of p38 MAPK signaling induced cellular senescence and p38 MAPK inhibitor SB203580 inhibited the cellular senescence of human corneal endothelial cells.24 In previous research,25 we found that the inflammatory cytokine TNF-α, which could induce senescence, elevated both the mRNA and protein expression level of p38 MAPK in CCH fibroblasts.25 Therefore, we hypothesized that there is a link between p38 MAPK signaling pathway and CCH cellular senescence. 
To explore the effects of p38 MAPK signaling pathway on CCH cellular senescence, conjunctival fibroblasts were obtained from CCH patients and normal controls. The cellular senescence was assessed by senescence-associated SA-β-Gal staining and the expression of senescence-associated genes. The small interfering RNA (siRNA) targeting p38 (siP38) and p38 specific inhibitor SB203580 were used to investigate the role of p38 MAPK signaling in cellular senescence of CCH fibroblasts. 
Materials and Methods
Ethics Statement
The experiments were approved by the ethics committee of Putuo Hospital Affiliated to Shanghai University of Traditional Chinses Medicine (Ethic code: PTEC-R-25(Y)-1) and followed the tenets of the Declaration of Helsinki. Written informed consent was obtained from all the patients. 
Identification of Cellular Senescence–Associated Genes
All cellular senescence–associated Gene Ontology (GO) terms and involved genes were obtained from the Gene Ontology Resource26,27 (http://geneontology.org; in the public domain). A total of 757 genes that dysregulated in CCH conjunctival tissues compared with normal conjunctival tissues were identified by our previous study.28 The overlapping genes among all genes involved in cellular senescence–associated GO terms and 757 differentially expressed genes (DEGs) were identified as cellular senescence–associated genes that associated with CCH. 
Cell Culture
Loose conjunctival tissues and normal bulbar conjunctival tissues were obtained from patients with CCH (n = 13; 5 males and 8 females, average age: 76.08 ± 3.50) and patients with cataract (n = 12; 5 males and 7 females, average age: 75.50 ± 3.00) who underwent surgery in the Putuo Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, respectively. Firstly, the tissues were cut into small pieces with size of 0.5 to 1 mm2 and tiled in a six-well plate. The fibroblasts were induced in Dulbecco's modified Eagle's medium (DMEM)-H containing 100 mL/L fetal bovine serum (FBS), 1 μL/L fibroblast growth supplement, 100 U/mL penicillin, and 100 g/mL streptomycin in an atmosphere of 37°C and 5% CO2. Then, the collected fibroblasts were maintained in DMEM containing 10% FBS and 1% antibiotic mixture of penicillin and streptomycin. 
Small Interfering RNA
Three siRNAs targeting p38α (siP38α-1, siP38α-2, and siP38α-3) were designed by JRDUN Biotechnology Co., Ltd (Shanghai, China) and synthesized by Guangzhou RiboBio Co., Ltd (Guangzhou, China). The siRNAs were used to induce p38α silence in the CCH fibroblasts. The interference efficiency of the siRNAs was detected by quantitative (q)RT-PCR and Western blot. 
Experimental Design
The CCH fibroblasts were divided into four groups: CCH, CCH+siNC (CCH fibroblasts transfected with pLVX-AcGFP1-C1 vector as negative control [NC]), CCH+siP38α (CCH fibroblasts transfected with p38α shRNA [siP38]), and CCH+SB203580 (CCH fibroblasts treated with 0.01 μM SB203580, the inhibitor of p38). The fibroblasts derived from the normal bulbar conjunctival tissues were used as normal controls. 
Senescence-Associated β-Galactosidase Staining
SA-β-Gal staining of conjunctival tissues and fibroblasts was performed using Senescence β-Galactosidase Staining Kit (C0602; Beyotime, Shanghai, China) according to the manufacturer's instructions. After removal from the patients, the conjunctival tissues were immediately embedded in optimal cutting temperature compound and stored in a −80°C refrigerator. The frozen tissues were cut into slices with a thickness of 5 to 7 μm. The tissue slices or the fibroblasts were fixed with β-galactosidase staining fixative for 15 minutes at room temperature. After washing with PBS three times, the tissue slices or the fibroblasts were incubated with β-galactosidase staining reagents at 37°C overnight. Finally, images were obtained with an optical microscope. 
Quantitative Real-Time PCR
Total RNA was isolated with Trizol (Invitrogen, Carlsbad, CA, USA). RNA (1 ng) was reverse transcribed into complementary DNA using Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). The mRNA levels of p38α, p53, p21, SMP30, MMP-1, and MMP-3 were determined by qRT-PCR, which was performed with Maxima SYBR Green/ROX qPCR Master Mix (2×, Thermo Fisher Scientific) on ABI-7300 (Applied Biosystems, Foster City, CA, USA). Glyceraldegyde-3-phosphate dehydrogenase (GAPDH) was used as the endogenous control. The primer sequences are shown in the Table. The relative expression level was calculated with the 2−ΔΔCT method. 
Table
 
Primer Sequences for RT-PCR
Table
 
Primer Sequences for RT-PCR
Western Blot
Total protein of cells in each group was extracted, respectively. Protein concentration was determined with the bicinchoninic acid protein assay kit (PICPI23223, Thermo, Nanjing, Jiangsu, China). The protein was separated by 10% or 12% polyacrylamide gel (JRDUN Biotechnology Co., Ltd) and electrophoresis and then was put onto a polyvinylidene fluoride membrane after supplementing 5% nonfat dry milk or bovine serum albumin (BSA) into the proteins for blocking for 1 hour at room temperature. After addition with primary antibodies, the sealing fluid was incubated at 4°C overnight. The primary antibodies against P38α (#9218, 1:1000), P53 (#9282, 1:1000), P21 (#2947, 1:1000), and GAPDH (#5174, 1:2000) were bought from Cell Signaling Technology (Danvers, MA, USA). The primary antibody against SMP30 (Ab233007, 1:400) was purchased from Abcam Plc (Cambridge, UK). Then, the membrane was incubated together with horseradish-labeled secondary antibody (Beyotime), including goat anti-rabbit IgG (A0208, 1:1000), donkey anti-goat IgG (A0181, 1:1000), and goat anti-mouse IgG (A0216, 1:1000), at 37°C for 1 hour. Chemiluminescence detection was performed and the protein image was captured by Tanon-5200 (Shanghai Tianneng Technology Co., Ltd, Shanghai, China). 
Cell Counting Kit (CCK-8) Assay
The cell viability was determined with CCK-8 kit (CP002; SAB, Sioux Falls, SD, USA) following the instructions. Total 100 μL cell suspension (3 × 104 cells/mL) was added into 96-well plates followed by various treatments. After maintaining at 37°C for 24, 48, or 72 hours, the cell suspension in each well and 10 μL CCK-8 reagent were mixed and incubated for 1 hour in an atmosphere of 37°C and 5% CO2. Then, the absorbance of the cell culture medium at 450 nm was determined. 
Measurement of Superoxide Dismutase (SOD) Activity and Reactive Oxygen Species (ROS) Production
Total intracellular ROS was measured with the Reactive Oxygen Species Assay Kit (S0033; Beyotime) using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) and dihydroethidium dye following the manufacturer's instructions. The specific activity of SOD was detected spectrophotometrically with a SOD test kit (A0001; Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) following the manufacturer's instructions. 
Statistical Analysis
IBM SPSS Statistics 20 (IBM, Armonk, NY, USA) was used for statistical analysis. Mean ± standard deviation was used to represent the experimental data. Significance was analyzed by 1-way analysis of variance followed by Tukey's test for multiple group comparison. A difference with P < 0.05 (*) was regarded as statistically significant. 
Results
Enhanced Cellular Senescence in CCH Conjunctival Tissues
Here, we compared cellular senescence between normal bulbar conjunctival tissues and CCH tissues by SA-β-Gal staining. As shown in Figure 1, A through D, SA-β-Gal staining was mainly distributed in subepithelial tissues of the conjunctiva. More senescent cells were present in CCH conjunctival tissues than in age-matched normal bulbar conjunctival tissues. Moreover, the expression level of senescence-associated genes, including p53 and p21, was obviously upregulated in CCH tissues (Figs. 1E, 1F). 
Figure 1
 
Senescence-associated β-galactosidase staining and the expression of senescence-associated genes in normal conjunctival tissues (n = 12) and CCH conjunctival tissues (n = 13). (AD) SA-β-Gal staining of normal conjunctival tissues and CCH conjunctival tissues. The expression of p53 (E), p21 (F), and p38α (G) in normal conjunctival tissues and CCH conjunctival tissues. **P < 0.001.
Figure 1
 
Senescence-associated β-galactosidase staining and the expression of senescence-associated genes in normal conjunctival tissues (n = 12) and CCH conjunctival tissues (n = 13). (AD) SA-β-Gal staining of normal conjunctival tissues and CCH conjunctival tissues. The expression of p53 (E), p21 (F), and p38α (G) in normal conjunctival tissues and CCH conjunctival tissues. **P < 0.001.
Additionally, by comparing 757 DEGs with all the genes involved in cellular senescence–associated GO terms, PLA2R1 and TERT, which were involved in the GO terms of negative regulation of cellular senescence, were found significantly dysregulated between CCH conjunctival tissues and age-matched normal conjunctival tissues (Supplementary Fig. S1). All these results indicated that cellular senescence might be a potential factor for the pathogenesis of CCH. 
It has been reported that p38 signaling pathway was associated with cellular senescence and p38 induced cellular senescence by suppressing the transcription of TERT in normal human fibroblasts.29,30 Here, we also found that the expression of MMP-3 in CCH fibroblasts was significantly reduced by p38-specific inhibitor SB203580 (Supplementary Fig. S2), indicating the potential role of p38 in CCH. To determine whether there was a link between p38α and CCH cellular senescence, the expression of p38 was detected. Expression of p38α was notably higher in CCH tissues than in normal tissues (Fig. 1G), suggesting an implication of p38 signaling pathway in the cellular senescence progression of CCH. 
Effect of p38α MAPK Signaling on Cellular Senescence of CCH Fibroblasts
To further investigate whether p38 signaling pathway was involved in cellular senescence progression, CCH fibroblasts were transfected with three siRNAs targeting p38α. Expression of p38α was determined by qRT-PCR and Western blot. Compared with the CCH fibroblasts transfected with or without the scramble siRNA (siNC), CCH fibroblasts transfected with siP38α-1, siP38α-2, and siP38α-3 had significantly decreased expression of p38α (Figs. 2A, 2B). There was no statistical difference in the effects of siP38α-1, siP38α-2, and siP38α-3. Therefore, siP38α-1 was randomly selected for the following experiments. 
Figure 2
 
The expression of p38α affected cellular senescence of CCH conjunctival fibroblasts. The mRNA (A) and protein (B) expression of p38α in CCH fibroblasts transfected with siRNA targeting p38α. (C) SA-β-Gal staining of fibroblasts in different groups. Normal: normal conjunctival fibroblasts; CCH: CCH conjunctival fibroblasts; CCH+siNC: CCH conjunctival fibroblasts transfected with scramble siRNA; CCH+siP38α-1: CCH conjunctival fibroblasts transfected with siP38α-1; CCH+SB203580: CCH conjunctival fibroblasts treated with p38-specific inhibitor, SB203580. **P < 0.001; n = 3.
Figure 2
 
The expression of p38α affected cellular senescence of CCH conjunctival fibroblasts. The mRNA (A) and protein (B) expression of p38α in CCH fibroblasts transfected with siRNA targeting p38α. (C) SA-β-Gal staining of fibroblasts in different groups. Normal: normal conjunctival fibroblasts; CCH: CCH conjunctival fibroblasts; CCH+siNC: CCH conjunctival fibroblasts transfected with scramble siRNA; CCH+siP38α-1: CCH conjunctival fibroblasts transfected with siP38α-1; CCH+SB203580: CCH conjunctival fibroblasts treated with p38-specific inhibitor, SB203580. **P < 0.001; n = 3.
Compared with the normal fibroblasts, CCH fibroblasts showed a higher percentage of SA-β-Gal–positive cells (Fig. 2C). Silence of p38 decreased the number of SA-β-Gal–positive cells in the CCH group. It had similar effects on CCH fibroblasts treated with SB203580, the p38 MAPK-specific inhibitor. All these results indicated that inhibition of p38 MAPK signaling was associated with the prevention of cellular senescence of CCH fibroblasts. 
Effect of p38α MAPK Signaling on Cell Proliferation of CCH Fibroblasts
We found that inhibition of p38 MAPK signaling affected not only cellular senescence, but also cell proliferation. Compared with the normal fibroblasts, cell viability obviously decreased in CCH fibroblasts, evidenced by reduced optical density (OD) value (Fig. 3). SiP38α-1, as well as p38-specific inhibitor SB203580, could notably increase cell viability of CCH fibroblasts. 
Figure 3
 
The cell proliferation of fibroblasts in different groups. *P < 0.05, **P < 0.001; n = 3.
Figure 3
 
The cell proliferation of fibroblasts in different groups. *P < 0.05, **P < 0.001; n = 3.
Effect of p38α MAPK Signaling on the Level of ROS and SOD in CCH Fibroblasts
The results of H2DCF-DA and dihydroethidium dye/staining showed that ROS production was significantly higher in CCH fibroblasts compared with normal fibroblasts (Fig. 4A). The ROS production of CCH fibroblasts showed a noticeable decrease after transfected with siP38α-1 or treated with SB203580. Moreover, CCH fibroblasts had lower SOD activity compared with the normal fibroblasts (Fig. 4B). SiP38α-1 or SB203580 notably elevated the SOD activity of CCH fibroblasts. 
Figure 4
 
Reactive oxygen species (A) level and superoxide dismutase (B) activity of fibroblasts in different groups. *P < 0.05, **P < 0.001; n = 3.
Figure 4
 
Reactive oxygen species (A) level and superoxide dismutase (B) activity of fibroblasts in different groups. *P < 0.05, **P < 0.001; n = 3.
Molecular Mechanism of p38α MAPK Signaling in CCH Fibroblasts
To further investigate the molecular mechanism of p38 MAPK signaling in cellular senescence of CCH fibroblasts, the expression of p38α and senescence-associated genes (p53, p21, and SMP30) in different groups was detected by qRT-PCR and Western blot. Compared with the normal fibroblasts, expression of each of p38α, p53, and p21 was significantly higher in CCH fibroblasts (Fig. 5), which was consistent with the results in CCH conjunctival tissues and normal conjunctival tissues. The p38 MAPK inhibitor SB203580 and siRNA interference fragment could effectively block the p38 MAPK signaling and p53/p21 signaling. Blocking of p38 MAPK signaling also enhanced the expression of anti-senescence protein SMP30, which confirmed that inhibition of p38 MAPK signaling alleviated cellular senescence in CCH fibroblasts. 
Figure 5
 
The mRNA (AD) and protein (E) expression of p38α and senescence-associated genes in different groups. *P < 0.05, **P < 0.001; n = 3.
Figure 5
 
The mRNA (AD) and protein (E) expression of p38α and senescence-associated genes in different groups. *P < 0.05, **P < 0.001; n = 3.
Discussion
Conjunctivochalasis is an age-dependent eye disease3 and can cause a variety of symptoms, such as ocular discomfort and unstable tear film, and have an adverse impact on vision-related quality of life.31 Numerous factors, including aged conjunctiva, ocular inflammation, mechanical friction, tear film instability, and delayed tear clearance, are involved in the pathogenesis of CCH.2,21,32 However, the exact pathogenesis remains largely unknown. In this study, we revealed the important role of cellular senescence in the development of CCH and explored the underlying mechanism. Cellular senescence is characterized by increased levels of senescence-associated biomarkers, such as SA-β-Gal, reduced proliferative activity, and increased expression level of senescence-associated genes, including p53, p21, and p16.33 In the present research, we found that conjunctival tissues and cells from CCH patients had notably more SA-β-Gal–positive cells and higher expression of p53 and p21 compared with conjunctival samples from age-matched normal individuals. Additionally, our previous transcriptome analysis of CCH conjunctival tissues and normal conjunctival tissues showed that the dysregulated genes (fold change ≥ 2, q value < 0.05) between CCH conjunctival tissues and normal conjunctival tissues significantly were enriched in the p53 signaling pathway.28 The dysregulated genes PLA2R1 and TERT, enriched in cellular senescence–associated GO terms, were markedly downregulated in CCH conjunctival tissues. All these results indicated the important role of cellular senescence in CCH. 
It has been reported that p38 MAPK signaling is involved in cellular senescence.29 Notably, in the present study, p38 was expressed highly in CCH conjunctival tissues compared with normal conjunctival tissues. Blocking p38 MAPK signaling in CCH fibroblasts by transfection of siRNA targeting p38, or treatment with the p38-specific inhibitor SB203580, reduced the number of SA-β-Gal–positive cells and increased cell viability, indicating or implying p38 MAPK signaling in the cellular senescence of CCH fibroblasts. Harada et al.30 revealed that p38 induced cellular senescence in normal human fibroblasts by suppressing the transcription of TERT. The p38 MAPK signaling might result in downregulation of TERT in the CCH group. These specific relationships require further experimental data. It has been reported that oxidative stress is present in conjunctival samples of CCH patients compared with conjunctival tissues of normal individuals.10 Mavrogonatou et al.34 reported that there was a link between p38 MAPK pathway and oxidative stress, and both of them were involved in TNF-α–induced premature senescence of human dermal fibroblasts. Both oxidative stress and p38 MAPK pathway participated in metabolic perturbation-triggered senescence of human primary fibroblasts35 and TNF-α–induced premature senescence of human dermal fibroblasts.34 Suppression of p38 MAPK pathway could block the accumulation of ROS in senescent skin fibroblasts.34 High-magnitude compression accelerated cellular senescence by the p38 MAPK-ROS pathway in the nucleus pulposus.36 However, views on the relation of p38 MAPK and ROS are not unanimous. Probin et al.37 demonstrated that ROS acted as an upstream of p38 MAPK pathway in busulfan-induced cellular senescence of normal human diploid WI38 fibroblasts. Here, we found that blocking of p38 MAPK signaling in CCH fibroblasts decreased production of ROS. Whether there is a feedback regulation between p38 MAPK and ROS warrants future study. 
Additionally, the expression level of senescence-associated proteins (p53 and p21) was downregulated and the expression of anti-senescence protein SMP30 was upregulated while p38 MAPK signaling was blocked. P53 triggers cell cycle arrest by activating the cyclin-dependent kinase inhibitor 1A gene p21.33,38 SMP30, a well-known anti-senescence protein, is expressed highly in tissues during the young and adult periods, while it is downregulated in the tissues during senescent periods.39,40 Therefore, p38 MAPK might promote cellular senescence of CCH fibroblasts through p53/p21 signaling. 
Elastotic degeneration and collagenolysis by overexpression of MMPs have long been speculated to contribute to the pathogenesis of CCH.6,8,9 Our previous study revealed that MMP-1 and MMP-3 are expressed highly in CCH conjunctival tissues and mainly distributed in the lamina propria of conjunctiva.8 Here, SA-β-Gal staining was significantly higher in CCH conjunctival tissues than in normal conjunctival tissues, and it was mainly distributed in the subepithelial tissues of conjunctiva, namely lamina propria. Moreover, p38-specific inhibitor SB203580 could significantly decrease the expression of MMP-3 in CCH fibroblasts. Therefore, MMPs might be implicated in the role of p38-mediated cellular senescence in the pathogenesis of CCH. In conclusion, we found that cellular senescence might be a causative factor for the pathogenesis of CCH and p38 MAPK signaling played a vital role in the progression of CCH cellular senescence via manipulation of expression of p53/p21. This study will benefit understanding of the pathogenic mechanism of CCH. 
Acknowledgments
Supported by the Research Project of Health and Family Planning Commission in Shanghai (201840196), Yingcai Program of Putuo Hospital (2017202B), and Yuying Program of Putuo Hospital (2016219A). 
Disclosure: M. Xiang, None; L. Mo, None; Y. Zhan, None; H. Wen, None; H. Zhou, None; W. Miao, None 
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Figure 1
 
Senescence-associated β-galactosidase staining and the expression of senescence-associated genes in normal conjunctival tissues (n = 12) and CCH conjunctival tissues (n = 13). (AD) SA-β-Gal staining of normal conjunctival tissues and CCH conjunctival tissues. The expression of p53 (E), p21 (F), and p38α (G) in normal conjunctival tissues and CCH conjunctival tissues. **P < 0.001.
Figure 1
 
Senescence-associated β-galactosidase staining and the expression of senescence-associated genes in normal conjunctival tissues (n = 12) and CCH conjunctival tissues (n = 13). (AD) SA-β-Gal staining of normal conjunctival tissues and CCH conjunctival tissues. The expression of p53 (E), p21 (F), and p38α (G) in normal conjunctival tissues and CCH conjunctival tissues. **P < 0.001.
Figure 2
 
The expression of p38α affected cellular senescence of CCH conjunctival fibroblasts. The mRNA (A) and protein (B) expression of p38α in CCH fibroblasts transfected with siRNA targeting p38α. (C) SA-β-Gal staining of fibroblasts in different groups. Normal: normal conjunctival fibroblasts; CCH: CCH conjunctival fibroblasts; CCH+siNC: CCH conjunctival fibroblasts transfected with scramble siRNA; CCH+siP38α-1: CCH conjunctival fibroblasts transfected with siP38α-1; CCH+SB203580: CCH conjunctival fibroblasts treated with p38-specific inhibitor, SB203580. **P < 0.001; n = 3.
Figure 2
 
The expression of p38α affected cellular senescence of CCH conjunctival fibroblasts. The mRNA (A) and protein (B) expression of p38α in CCH fibroblasts transfected with siRNA targeting p38α. (C) SA-β-Gal staining of fibroblasts in different groups. Normal: normal conjunctival fibroblasts; CCH: CCH conjunctival fibroblasts; CCH+siNC: CCH conjunctival fibroblasts transfected with scramble siRNA; CCH+siP38α-1: CCH conjunctival fibroblasts transfected with siP38α-1; CCH+SB203580: CCH conjunctival fibroblasts treated with p38-specific inhibitor, SB203580. **P < 0.001; n = 3.
Figure 3
 
The cell proliferation of fibroblasts in different groups. *P < 0.05, **P < 0.001; n = 3.
Figure 3
 
The cell proliferation of fibroblasts in different groups. *P < 0.05, **P < 0.001; n = 3.
Figure 4
 
Reactive oxygen species (A) level and superoxide dismutase (B) activity of fibroblasts in different groups. *P < 0.05, **P < 0.001; n = 3.
Figure 4
 
Reactive oxygen species (A) level and superoxide dismutase (B) activity of fibroblasts in different groups. *P < 0.05, **P < 0.001; n = 3.
Figure 5
 
The mRNA (AD) and protein (E) expression of p38α and senescence-associated genes in different groups. *P < 0.05, **P < 0.001; n = 3.
Figure 5
 
The mRNA (AD) and protein (E) expression of p38α and senescence-associated genes in different groups. *P < 0.05, **P < 0.001; n = 3.
Table
 
Primer Sequences for RT-PCR
Table
 
Primer Sequences for RT-PCR
Supplement 1
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