{"id":59983,"date":"2024-09-30T11:04:47","date_gmt":"2024-09-30T11:04:47","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=59983"},"modified":"2024-10-09T18:22:30","modified_gmt":"2024-10-09T18:22:30","slug":"effect-of-metformin-on-interleukin-6-expression-in-human-fibroblast-cell-aging-model","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol17no3\/effect-of-metformin-on-interleukin-6-expression-in-human-fibroblast-cell-aging-model\/","title":{"rendered":"Effect of Metformin on Interleukin-6 Expression in Human Fibroblast Cell Aging Model"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

The skin is the main medium that plays a\nrole in the interaction of organisms with their environment through\nelectromagnetic irradiation or light, air, skin contact, food, and injections.1,2<\/sup>\nSkin health decreases with age, characterized by changes in skin structure,\nsuch as wrinkles, dryness, and roughness of the skin, reduced skin elasticity,\nand skin pigmentation. Skin aging is based on a molecular fibroblast cell\npopulation mechanism.3,4<\/sup> During skin aging, the number of\nfibroblasts and their functional activity are reduced.5<\/sup> A decrease\nin the number of fibroblasts in the skin leads to a decrease in collagen\nsynthesis and blood vessels, thereby forming wrinkles in the skin.6 <\/sup>In\nthe aging skin layer, cellular senescence occurs, which is characterized by the\ninability of cells to proliferate, maintain viability, and metabolic activity,\ndespite the presence of sufficient nutrients and mitogens.7<\/sup><\/p>\n\n\n\n

Skin aging occurs through two processes;\nintrinsic and extrinsic.8,9<\/sup> Intrinsic processes are closely related\nto an individual’s genetic makeup, such as the production of extracellular\ncollagen and matrix proteins in the skin.10,11<\/sup> Extrinsic aging is\nclosely related to an individual’s lifestyle, including smoking, exposure to UV\nradiation, and intake of large amounts of alcohol.10,11<\/sup> External\nstimuli, such as UV radiation, oxidative stress, and poor nutrition, along with\ninternal stimuli, such as hormonal changes, can trigger inflammatory processes\nin the skin or inflammaging.10,12<\/sup> Several studies have shown that\naging cells and inflammatory cells secrete cytokines and chemokines that can\ntrigger the aging process to become stronger.13,14<\/sup> During skin\naging, several processes occur, such as excessive secretion of TNF-\u03b1, IL-6,\nIL-1\u03b2, IL-2, IL-1, and IL-8, increased levels of metalloproteinases, and\nexcessive production of ROS by mitochondria.12<\/sup><\/p>\n\n\n\n

One of the markers of early inflammatory\nreactions is an increase in IL-6 levels.15<\/sup> IL-6 is a cytokine that\nis involved in the differentiation, activation, and proliferation of\nleukocytes, endothelial cells, keratinocytes, and fibroblasts.16<\/sup> IL-6\nconcentrations increase during aging, particularly in the elderly population.17<\/sup>\nIL-6 plays a role in skin aging and the formation of skin wrinkles.10<\/sup>\nHigh expression of IL-6 can predict increased mortality in the elderly.19,20<\/sup>\n However, low IL-6 expression can cause\nreduced inflammaging.19<\/sup><\/p>\n\n\n\n

Skin aging can affect dermatological\nconditions, which can increase the risk of morbidity and mortality.20,21<\/sup>\nThe consumption of various supplements and anti-aging drugs is one way to\nmaintain skin health.20,22<\/sup> Antiaging agents in topical medications\nor supplements such as vitamin C, vitamin B3, Vitamin E, and polyphenolic\ncompounds have antioxidant and anti-inflammatory activities that can reduce\ncollagen degradation by reducing free radicals in tissues, regulating cell\nmetabolism and regeneration, and inhibiting matrix metalloproteinase.22,23<\/sup>\nResearch on primary fibroblast cells shows that Vitamin E supplementation can\nreduce the number of SA-\u03b2-gal positive cells in vitro and supports reduced cell\nsenescence.24<\/sup> Previous study shows that addition of Vitamin E can\nimprove protective effect on fibroblast cell survival.25<\/sup> Therefore,\nmaintaining skin health by supplementation of antiaging agent has become\nincreasingly important. <\/p>\n\n\n\n

Currently, several drugs with potential as\nanti-aging agents are attracting the attention of many researchers, including\nmetformin.26<\/sup> Metformin is known as an antidiabetic drug for more\nthan 60 years, which is indicated to regulate various aging-related pathways\nsuch as Adenosine monophosphate-activated protein kinase (AMPK), Sirtuin\n(silent mating type information regulation 2 homolog) 1 (SIRT1), Mammalian\ntarget of rapamycin complex 1 (mTORC1), and inhibit pro-inflammatory cytokines.26,27<\/sup>\nMetformin has anti-inflammatory effects by inhibiting the expression of\npro-inflammatory mediators such as IL-6 and IL-17.28<\/sup> Previous\nstudies have shown that metformin can reduce IL-6 secretion from bone\nmarrow-derived macrophages (BMDMs).29<\/sup> In addition, metformin reduced\nthe number of IL-6-induced lung cancer A549 and HCC827 cells.28<\/sup> The\nanti-aging potential of metformin is reinforced by the results of previous\nstudies showing that metformin inhibits MMP-1 and MMP-2, which act as collagen\nand gelatin degradation enzymes, and may be involved in photoaging induced by\nLPS.30,31 <\/sup>Moreover, addition of 100 \u00b5M metformin shows a antiaging\nactivity by extend the lifespan of human fibroblasts and mesenchymal stem\ncells.32<\/sup> However, the antiaging activity of metformin shows various mechanisms.\nLow doses of metformin show the antiaging effect, however at high doses of\nmetformin cause severe mitochondrial dysfunction that damages cells.33<\/sup>\nIn addition to extending the life of diploid fibroblast cells, in its mechanism\nof action, metformin can induce aging activation of Nrf2 target gene, activates\nthe AMPK signaling pathway and increases stress responses, such as superoxide\ndismutase 2 (SOD2), thioredoxin 1 (TrxR1), quinone oxidoreductase 1 (NQO1) and\nquinone oxidoreductase 2 (NQO2).32,34,35<\/sup><\/p>\n\n\n\n

In recent years, many studies have been\nconducted using cell lines and animal models to test the anti-aging activity of\nmetformin.36,37<\/sup> Based on the molecular mechanism of fibroblasts, the\nskin aging process was developed by culturing or subculturing normal human\ndiploid fibroblast cells.38<\/sup> In this regard, cell culture technology\nin the form of cell aging or replication is a widely used way to study cell\naging.39<\/sup> Subculturing or passaging cells is a procedure that allows\nfurther propagation of a cell line or cell strain.40<\/sup> Fibroblast\ncells can be passage between 5 and 10 times. In general, fibroblast cells grown\nin culture media will become more stable and able to proliferate well at\npassage 3 and growth begins to slow down at passage 8.41<\/sup> Therefore,\ntesting the antiaging model of fibroblast cells at passage 3 to passage 8 needs\nto be further analyzed.<\/p>\n\n\n\n

The Targeting Aging with Metformin (TAME)\nclinical trial was conducted to further explore the anti-aging activity of\nmetformin.42,43<\/sup> However, the role of metformin in IL-6 expression in\nthe mechanism of aging in human fibroblasts has not been elucidated. Accordingly,\nthis study aimed to analyze the potential of metformin as an anti-aging agent,\nparticularly in regulating the IL-6 expression pathway in fibroblast cell aging\nmodels. The antiaging model conducted in this study is the process of cell\naging through cell passaging.<\/p>\n\n\n\n

Subjects <\/a><\/strong>and Methods<\/strong><\/p>\n\n\n\n

Human foreskin tissue was collected\nthrough circumcision of male patients aged 1 to 12 years. Protocol procedures\nwere approved by The Research Ethics Committee of the Faculty of Medicine,\nPadjadjaran University. The parents of the study participants took part in the\nstudy by signing an informed consent to participate as respondents for the\ncollection and analysis of foreskin tissue which is usually discarded. The\nspecimens were first collected in 10% povidone-iodine for 5 minutes before\nbeing transferred to 70% ethanol for 15 seconds. The cells were rinsed with 4% antibiotic-antimycotic\n(ABAM) (Sigma-Aldrich, USA) in phosphate-buffered saline (PBS) (Gibco, USA) for\n5 minutes and moved to 4\u00b0C. Before usage, store Dulbecco’s modified eagle\nmedium (DMEM) (Gibco, USA) in a cooler box with 10% Fetal Bovine Serum (FBS)\n(Gibco, USA), 1% penicillin\/streptomycin (Gibco, USA), and 1% HEPES\n(Sigma-Aldrich, USA). To remove any adhering blood, the specimens were washed\nthree times with PBS + 1% ABAM. The adipocyte tissue in the specimen was\ncleaned by cutting it into a 5 mm square segment, incubating it in povidone-iodine\n10% for 5 minutes, and then washing it three times with PBS + ABAM 1%. After\nwashing, the specimen was placed in an empty T25 flask for attachment. Then, 2\nmL of DMEM complete media was added and incubated at 37 degrees Celsius with 5%\nCO2<\/sub>.44,45<\/sup><\/p>\n\n\n\n

Cell\nculture and Treatment with metformin<\/strong><\/p>\n\n\n\n

Human foreskin fibroblasts were cultivated at Padjadjaran University’s Cell Culture and Cytogenetic Laboratory in Bandung, Indonesia. The Research Ethics Committee of the Faculty of Medicine at Padjadjaran University approved this study, which ran from June 2022 to November 2022 (Number: 492\/UN6.KEP\/EC\/2022). Cell culture procedures and treatments were carried out using previously published methods, with minor modifications.46<\/sup> Isolated human foreskin fibroblasts were grown in DMEM media containing 10% FBS, 1% penicillin\/streptomycin, and the minor modification was added 1% HEPES in the media. The cells were cultivated at 37\u2103, 8% CO2<\/sub>, and 100% humidity. The media conditions for the metformin treatment were same; however, 100 \u00b5M metformin (Tocris, UK) was added to nuclease-free H2<\/sub>O. Dosage of 100 \u00b5M metformin has been shown to extend the lifespan of human fibroblasts and mesenchymal stem cells.32<\/sup> The positive control treatment was 50 \u00b5M \u03b1-tocopherol\/Vitamin E (Sigma-Aldrich, USA) in nuclease-free H2<\/sub>O which has been shown the protective effect of Vitamin E by improve fibroblast survival.25<\/sup> Fibroblasts were used at stages 3 through 8. Fibroblast cells with a density of 80-90% were cultured for passage, planted in 12 well microplates at 40,000 cells\/well for treatment, and counted with a hemocytometer. Cells were seeded and grown in the medium determined for each treatment, with the media replenished every 72 hours. RNA was extracted at 80% confluence and isolated for qRT-PCR analysis.<\/p>\n\n\n\n

Gene\nExpression Analysis using qRT-PCR<\/strong><\/p>\n\n\n\n

IL-6 gene expression analysis using qRT-PCR was performed at the Laboratory of Molecular and Genetics of Padjadjaran University, Bandung, Indonesia. The Promega GoTaq 1-Step RT-qPCR System was used to isolate total RNA and execute qRT-PCR in accordance with the manufacturer’s instructions. After 72 hours, the cells were washed twice with PBS and homogenized with 1 mL TRI reagent per 10 cm2<\/sup> area (Life Technologies, Gaithersburg, MD). The cells were scraped to remove them, and the cell lysate and TRI reagent were combined in a 1.5 mL Eppendorf tube and left to stand for 5 minutes at room temperature (25\u00b0C). The recovered RNA was rinsed with 1 mL of 75% ethanol in DEPC-treated water and then centrifuged at 7500-9500 rpm for 5 minutes at 2 \u2013 8\u2103. The dried RNA pellet was reconstituted using DEPC-treated water (1:40). The Nano Drop 2000 spectrophotometer (Thermo Scientific\u2122, USA) was used to measure total RNA quantity and quality. <\/p>\n\n\n\n

Human IL-6 gene expression was measured using the following primers that are shown in Table 1. Quantitative RT-PCR was performed in a total volume of 50 \u03bcL and was carried out in triplet for each measurement. Each reaction had a primer concentration of 01-0.5 \u00b5M. The following cycling conditions were used: denaturation at 95\u00b0C for 2 min, followed by 31-35 cycles of 95\u00b0C for 15 s, primer annealing at 50-60\u00b0C for 20 s, extension at 72\u00b0C for 1 min\/1 kb, and 5 min.<\/strong> Threshold cycle (Ct) values from each trial were used to compute the fold change in household gene expression using Livak-Schmittgen method.47,48<\/sup><\/p>\n\n\n\n

Table 1: Primer sequence for RT- PCR<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n
\n

Gene<\/strong><\/p>\n<\/td>\n

\n

Primer sequence<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

IL-6<\/p>\n<\/td>\n

\n

Forward<\/p>\n<\/td>\n

\n

5\u2032-GAACTCCTTCCACCAGCGCCTT-3\u2032<\/p>\n<\/td>\n<\/tr>\n

\n

 <\/p>\n<\/td>\n

\n

Reverse<\/p>\n<\/td>\n

\n

5\u2032-CAAAAGACCAGTGATGATTTTCACCAGG-3\u2019<\/p>\n<\/td>\n<\/tr>\n

\n

GAPDH<\/p>\n<\/td>\n

\n

Forward<\/p>\n<\/td>\n

\n

5\u2032-AGAAGGCTGGGGCTCATTTG-3\u2032<\/p>\n<\/td>\n<\/tr>\n

\n

 <\/p>\n<\/td>\n

\n

Reverse<\/p>\n<\/td>\n

\n

5\u2032-AGGGGCCATCCACAGTTTTC-3\u2019<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n

Data\nmanagement and Statistical Analysis<\/strong><\/p>\n\n\n\n

Statistical\nanalysis was performed using SPSS software 26 version (SPSS Inc., USA). The\nobtained data were analyzed using the Shapiro-Wilk test for normality, to\ndecide whether the mean can be used as a representative value of the data or\nnot. If applicable, then means were compared using parametric tests, otherwise,\nmedians were used to compare groups, using nonparametric methods.49<\/sup> The\nIL-6 gene expression is presented as mean \u00b1 standard deviation. The difference of\nIL-6 expression among treatment and passage were evaluated using one-way ANOVA\ntest was performed followed by Games Howell post hoc. A Games Howell post hoc was\nused to make multiple comparisons among means of groups with unequal variances\nand unequal sample sizes.50<\/sup> A p-value less than 0.05 was considered\nstatistically significant. The data of IL-6 gene expression was reported using\nGraphPad Prism 8.0.1 (GraphPad, USA). <\/p>\n\n\n\n

Result<\/strong><\/p>\n\n\n\n

To analyze the effect of metformin\ntreatment in reducing aging, cytokine IL-6 was measured using the RT-PCR\nmethod, and the measurement results were analyzed using the Livak method. IL-6\nexpression for NC, Metformin, PC, and histogram of metformin treatment results\nare shown in Figure 1. <\/p>\n\n\n\n

IL-6\nrelative gene expression among cell passage<\/strong><\/p>\n\n\n\n

IL-6\ngene expression in negative control had the lowest value compared to metformin\ntreatment and positive control. In the negative control, the IL-6 expression in\npassage 6 showed the smallest value (0.048 \u00b1 0.04) and passage 3 showed the\nhighest expression (0.166 \u00b1 0.04). On the other hand, negative control IL-6\nexpression showed the highest value at passage 4 (5.590 \u00b1 3.34), continued to\ndecrease until passage 7 (0.000 \u00b1 0.00), and increased again at passage 8 (2.988\n\u00b1 4.78). Based on Figure 1(b), metformin-treated IL-6 expression increased from\npassage 3 with the lowest expression (0.078 \u00b1 0.02) until passage 5 (0.836 \u00b1\n0.15) and decreased again at passage 6 (0.123 \u00b1 0.03). Figure 1(a) shows that\nIL-6 expression of metformin treatment did not show lower results than the\nnegative control, but showed low values compared to the positive control. <\/p>\n\n\n\n

In this study, the results of homogeneity testing of data variance between groups were not homogeneous (data not shown), hence the next analysis was data analysis using One way ANOVA test (parametric test) and followed by post hoc analysis using Games Howell test. Based on one way ANOVA analysis, only Metformin treatment showed a difference between the passages (p = 0.000). To determine the difference between the passages, the Games Howell test was conducted and the p value for each passage is shown in Table 2. However, in the negative control and positive control, no significant difference was found between cell passages. Based on the results of statistical analysis shown in Figure 1(b) and Table 2, metformin treatment showed significant differences between passage 5 and other passages (p<0.05).<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 1: IL-6 gene expression (a) each treatment in each cell passage, (b) metformin treatment in each cell passage.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Table 2: p-value Games Howell post hoc of IL-6 comparison among passages<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

p-value of comparison IL-6 relative expression among passages<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

 <\/p>\n<\/td>\n

\n

Passage 3<\/strong><\/p>\n<\/td>\n

\n

Passage 4<\/strong><\/p>\n<\/td>\n

\n

Passage 5<\/strong><\/p>\n<\/td>\n

\n

Passage 6<\/strong><\/p>\n<\/td>\n

\n

Passage 7<\/strong><\/p>\n<\/td>\n

\n

Passage 8<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Passage 3<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

0.339<\/p>\n<\/td>\n

\n

0.043<\/p>\n<\/td>\n

\n

0.481<\/p>\n<\/td>\n

\n

0.742<\/p>\n<\/td>\n

\n

0.903<\/p>\n<\/td>\n<\/tr>\n

\n

Passage 4<\/p>\n<\/td>\n

\n

0.339<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

0.039<\/p>\n<\/td>\n

\n

0.559<\/p>\n<\/td>\n

\n

0.995<\/p>\n<\/td>\n

\n

0.459<\/p>\n<\/td>\n<\/tr>\n

\n

Passage 5<\/p>\n<\/td>\n

\n

0.043<\/p>\n<\/td>\n

\n

0.039<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

0.042<\/p>\n<\/td>\n

\n

0.032<\/p>\n<\/td>\n

\n

0.042<\/p>\n<\/td>\n<\/tr>\n

\n

Passage 6<\/p>\n<\/td>\n

\n

0.481<\/p>\n<\/td>\n

\n

0.559<\/p>\n<\/td>\n

\n

0.042<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

0.944<\/p>\n<\/td>\n

\n

0.975<\/p>\n<\/td>\n<\/tr>\n

\n

Passage 7<\/p>\n<\/td>\n

\n

0.742<\/p>\n<\/td>\n

\n

0.995<\/p>\n<\/td>\n

\n

0.032<\/p>\n<\/td>\n

\n

0.944<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

0.878<\/p>\n<\/td>\n<\/tr>\n

\n

Passage 8<\/p>\n<\/td>\n

\n

0.903<\/p>\n<\/td>\n

\n

0.459<\/p>\n<\/td>\n

\n

0.042<\/p>\n<\/td>\n

\n

0.975<\/p>\n<\/td>\n

\n

0.878<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

The statistical method used is ANOVA following by Games Howell post hoc with p < 0.05. p-value < 0.05 = there is a difference between groups and p-value> 0.05 = there is no difference between groups<\/p>\n\n\n

IL-6\ngene expression among treatments<\/strong><\/p>\n\n\n\n

To analyze the differences in each treatment between cell passages, a statistical test was carried out using the ANOVA method. The p-value results for each passage determine the differences in IL-6 expression in the three treatments are shown in Table 3.<\/p>\n\n\n\n

Table 3: Statistical analysis of IL-6 gene expression against treatment in each cell passage<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Cell Passage<\/strong><\/p>\n<\/td>\n

\n

IL-6 expression<\/strong><\/p>\n<\/td>\n

\n

p-value<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

NC<\/strong><\/p>\n<\/td>\n

\n

Met<\/strong><\/p>\n<\/td>\n

\n

PC<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

3<\/p>\n<\/td>\n

\n

0.116 \u00b1 0.04<\/p>\n<\/td>\n

\n

0.078 \u00b1 0.02<\/p>\n<\/td>\n

\n

1.214 \u00b1 1.04<\/p>\n<\/td>\n

\n

0.110<\/p>\n<\/td>\n<\/tr>\n

\n

4<\/p>\n<\/td>\n

\n

0.149 \u00b1 0.03<\/p>\n<\/td>\n

\n

0.023 \u00b1 0.09<\/p>\n<\/td>\n

\n

5.590 \u00b1 3.45<\/p>\n<\/td>\n

\n

0.025<\/p>\n<\/td>\n<\/tr>\n

\n

5<\/p>\n<\/td>\n

\n

0.158 \u00b1 0.05<\/p>\n<\/td>\n

\n

0.836 \u00b1 0.15<\/p>\n<\/td>\n

\n

0.284 \u00b1 0.14<\/p>\n<\/td>\n

\n

0.001<\/p>\n<\/td>\n<\/tr>\n

\n

6<\/p>\n<\/td>\n

\n

0.048 \u00b1 0.04<\/p>\n<\/td>\n

\n

0.123 \u00b1 0.03<\/p>\n<\/td>\n

\n

0.589 \u00b1 0.85<\/p>\n<\/td>\n

\n

0.408<\/p>\n<\/td>\n<\/tr>\n

\n

7<\/p>\n<\/td>\n

\n

0.095 \u00b1 0.01<\/p>\n<\/td>\n

\n

0.187 \u00b1 0.13<\/p>\n<\/td>\n

\n

0.000 \u00b1 0.00<\/p>\n<\/td>\n

\n

0.070<\/p>\n<\/td>\n<\/tr>\n

\n

8<\/p>\n<\/td>\n

\n

0.072 \u00b1 0.02<\/p>\n<\/td>\n

\n

0.104 \u00b1 0.03<\/p>\n<\/td>\n

\n

2.988 \u00b1 4.75<\/p>\n<\/td>\n

\n

0.378<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

NC= negative control, normal cell; Met= Metformin, cell + metformin 100\u00b5M; PC= positive control, cell + Vitamin E 50\u00b5M. n = 4, the statistical method used is ANOVA with p < 0.05. p-value < 0.05 = there is a difference between groups and p-value> 0.05 = there is no difference between groups.<\/p>\n\n\n

According to\nTable 3, significant variations in IL-6 expression were observed among\ntreatments, particularly in passages 4 (p=0.025) and 5 (p=0.001). Further test subsequently\nanalyzed the expression of IL-6 in passages 4 and 5, as depicted in Figure 2. <\/p>\n\n\n\n

We observed that the positive control group exhibited the highest IL-6 expression, showing an increase of up to 24 times compared to metformin-treated cells. However, the difference was not found to be statistically significant, as indicated by Games Howell’s further test. In passage 5, metformin IL-6 expression showed the highest value with a difference of 5 times compared to the negative control and 3 times compared to the positive control.  Interestingly, a significant difference in IL-6 expression was observed between the metformin treatment group and both the negative control and positive control groups (Figure 2).<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 2: Differences in IL-6 gene expression with treatment at passages 4 and 5.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Discussion<\/strong><\/p>\n\n\n\n

Inflammaging refers to the chronic,\nlow-grade inflammation associated with aging, leading to a diminished capacity\nto regulate persistent inflammatory processes. This phenomenon may be linked to\nskin aging, given that the skin, as the outermost organ, is consistently\nexposed to external stressors such as UV radiation, airborne particles, and the\nhuman microbiome.51<\/sup> Cellular senescence is a program of cessation of\ncell proliferation, one of which is initiated in response to replicative\nsenescence (RS).52<\/sup> With age, fibroblast cells produce SASP rich in\nproinflammatory cytokines, Interleukin-6 (IL-6), Interferon-gamma (IFN-\u03b3), and\nTumor necrosis factor alpha (TNF-\u03b1).51,53<\/sup> Aging fibroblast culture\nresearch has various terms, one of which is cumulative population doubling\n(CPD), which is the number of times the number of cells doubles. Cells that are\nconsidered senescent are cells that cannot complete one procurement\/doubling\nfor 4 weeks by giving fresh media for 3 consecutive weeks.38<\/sup> To\nprevent skin aging, several studies have been conducted on natural and\nsynthesized compounds that have a relationship with cell senescence.54<\/sup>\nAs an anti-diabetic drug, is known to have good anti-inflammatory activity, and\nresearch on its anti-aging activity has been conducted.54,55<\/sup> This\nstudy hypothesized that IL-6 gene expression would be lower in the\nmetformin-treated group than in the negative control group in an in vitro human\nfibroblast cell aging model. However, the result warrant rejection of the null\nhypothesis that treatment of metformin does not lower the IL-6 gene expression\nthan negative control.<\/p>\n\n\n\n

In this study, IL-6 gene expression was\nmeasured in 3 treatments (negative control, metformin, and positive\ncontrol\/vitamin E) and 6 cell passages (passages 3, 4, 5, 6, 7, and 8). The\nresults showed that the negative control group produced the lowest IL-6 gene\nexpression value compared to the metformin treatment group and the positive\ncontrol group. IL-6 expression in negative control cells showed a very small\nexpression value, where the highest result was shown in passage 5 with a value\nof 0.158 \u00b1 0.05. The present results are in line with earlier research\nindicating the highest expression of IL-6 in fibroblast cells reached 0.17 in\npassage 1, and in the following passage, IL-6 expression was at a value of\n0.05.56,57<\/sup> By statistical analysis, the difference in IL-6\nexpression in the metformin treatment group compared to the negative control\ngroup is significant. This can be seen in the descriptive picture of IL-6\nexpression data (Table 3), especially in passage 4 and 5, it was found that the\nincrease in IL-6 expression in the metformin treatment group and the positive\ncontrol group was higher when compared to other passage stages. This finding\nprovides new information that metformin has an anti-inflammatory effect on cellular\nreprogramming related to cell immunosenescence through increased IL-6\nexpression, although the process of subculture or passage that is carried out\nrepeatedly cannot be ignored in the induction of inflammatory factors in this\nstudy.<\/p>\n\n\n\n

IL-6 is one of the most prominent\ncytokines involved in several inflammaging-related diseases.58<\/sup>\nProinflammatory cytokines such as IL-6 are one of the characteristics of the\naging-related secretory phenotype (SASP) and are upregulated in cells\nundergoing replicative senescence (RS).52<\/sup> Senescent cells represent\nonly a fraction of cells within organ tissues.59<\/sup> A previous study\nfound that healthy fibroblasts expressed pro-inflammatory cytokines such as\nIL-6, TNF-\u03b1, and IL-8 during passage.56 <\/sup>Therefore, IL-6 secretion in\nsenescent cells only results in very low concentrations.60<\/sup><\/p>\n\n\n\n

The results of this study showed that IL-6 expression after metformin treatment was higher than in the negative group. This result is not comparable to some previous studies, where metformin can reduce the expression of IL-6 and IL-8 in human vascular smooth muscle cells (SMCs), macrophages (M\u03c6s), and endothelial cells (ECs).61<\/sup> In addition, metformin treatment of liposaccharide (LPS)-induced rabbit annulus fibrosus stem cells (AFSCs) showed that metformin decreased the expression of pro-inflammatory cytokines (IL-1\u03b2, IL-6, TNF-\u03b1) and MMPs by blocking the translocation of HMGB1 from the cell nucleus to the cytoplasm.62<\/sup> By contrast, treatment with 0.5 mM metformin in primary human fibroblasts promotes IL-g gene than compared to without the addition of metformin,46<\/sup> which is in line with present study funding. The higher expression of IL-6 in Metformin treatment may be attributed to the reparative function of IL-6 involved in fibroblast cell differentiation, activation, and proliferation.16<\/sup> Cytokines such as IL-6 are often referred to as pro-inflammatory, however increased IL-6 can stimulate the production of IL-1 receptor antagonists (anti-inflammatory cytokines).63<\/sup> In addition, IL-6 knockout mice show reduced insulin sensitivity, glucose intolerance64<\/sup> and late-onset obesity65<\/sup>; it has also been shown to be important in the regeneration and protection of some tissues (e.g. intestinal epithelial cells).66,67<\/sup> Therefore, pro-inflammatory cytokines upregulated in response to metformin may be involved in a more complicated role in maintaining pro- and anti-inflammatory cytokine homeostasis.46<\/sup><\/p>\n\n\n\n

This study shows that there are\ndifferences in IL-6 gene expression at each passage, which can be observed in\nall treatments. Previous research showed similar results, where there were\ndifferences in IL-6 expression between Synovial fibroblast passage 6, 7, and 8\ncells.68<\/sup> Although IL-6 gene expression between passages showed\ndifferent values, the difference in IL-6 gene expression between cell passages\nin the negative and positive control treatments did not show significant\ndifferences (p>0.05). This study is in line with previous research, that is,\nthere was no significant difference in IL-6 expression between old and young\nfibroblasts.53<\/sup> Moreover, earlier research found that changes in IL-6\nexpression in passages 3 and 4 were not statistically different, which supports\nthe findings of our study.57<\/sup> Similarly to Adam Rolt et al., IL-6\nexpression in human male neonatal foreskin primary fibroblasts (HF043) was not\nsignificantly different at passage ‘young’ cells (CPD 36.6) and ‘middle aged’\n(CPD 65.5).60<\/sup> Based on previous studies, antiaging\ntesting of inflammatory cytokine expression in fibroblast cells is carried out\nat passages 10 to 20 because the fibroblast cell population is known to\nexperience aging at passage 50.46,69<\/sup> This\nmay be the cause of no significant difference between all passages, especially\nin the negative control and positive control treatments. However, there were\ndifferences in IL-6 expression after metformin treatment, which increased at\npassage 5 and then decreased until passage 8. <\/p>\n\n\n\n

The difference in IL-6 expression at each\ncell passage may be due to differences in the inflammatory responses regulated\nby NF-\u03baB. NF-\u03baB plays a role in inducing the expression of various proteins\ninvolved in cell proliferation and migration and increases collagen synthesis\nand fibroblast differentiation.70<\/sup> In aging skin, increased NF-\u03baB\nactivity decreases the expression of type 1 collagen (COL1A1 and COL1A2) by\nincreasing the expression of collagenase (MMP-1).55,71<\/sup> In addition,\nthere are differences in the response of fibroblasts to double stress during\npassage. Passaging can result in selection pressure on parts of the cell\npopulation, such as adherent cells and trypsin-sensitive cells, which are\nreleased at different flash levels. This can alter the overall gene expression\nprofile at higher passages. Thus, the expression profiles of inflammatory genes\nin the same cell type can vary between cell passages.72<\/sup><\/p>\n\n\n\n

As\nwe already know, metformin exhibits antiaging activity at the cellular and\norganismal levels.73<\/sup> Metformin reduces several effects that are\nclosely associated with signs of aging, such as inflammation,26<\/sup>\nautophagy,74<\/sup> and cellular aging.75<\/sup> Metformin has\nanti-inflammatory activity by decreasing the expression of multiple\nproinflammatory cytokines including IL-6, IL-1\u03b2, and TNF-\u03b1.76-78<\/sup> The\naddition of metformin can modulate the inflammatory process in skin fibroblasts\nby decreasing the expression of mTOR and STAT3.54<\/sup> The decrease in\nmTOR and STAT3 expression results in the inhibition of proinflammatory cytokine\nexpression mediated by mTOR-STAT3 signaling, and can further reduce aging.54,79<\/sup>\nThe antiaging potential of metformin is reinforced by the results of previous\nstudies, which can increase the antioxidant capacity of cells and increase the\nproduction of type I and type III collagen, thus inhibiting the level of\nintracellular ROS, MMP expression and NF-\u03baB (p65) activity in aging fibroblast\ncells.55,80<\/sup> Based on this, metformin shows a cell aging inhibitory\neffect comparable to vitamin E. Comparison of the antiaging effect of metformin\nand vitamin E which is not significantly different is shown in the results of\nthe present study which shows insignificant differences in some cell passages,\nnamely passages 3, 6, 7, and 8 (p>0.05). However, this finding needs to be\nconfirmed by further research.<\/p>\n\n\n\n

The\nevaluation of metformin as an anti-aging agent by determining IL-6 gene\nexpression has several limitations. The first limitation lies in the method\nperformed that shows the results of gene expression. Measurement of IL-6 gene\nexpression using RT-PCR does not reflect functional IL-6 protein levels, as\ncellular regulation can impact several steps between mRNA production and\nfunctional protein secretion, including mRNA stability, translational\nefficiency, post-translational processing, and control of intracellular\ntransport and secretion pathways.60<\/sup> <\/sup>In this study, phenotyping of each group and\npassage was not performed because it considers time and cost factors. In\nfurther research, a cell morphological phenotype, including cell shape, size,\nintensity, and texture of cellular compartments, is required to determine\nchanges in cell morphological features with changes in cellular function due to\nthe addition of metformin. Moreover, the evaluation of metformin as an anti-aging\nagent can be examined in larger passages, and more appropriate proteins such as\nAMPK, mTOR, p53, IGF-1, EGF, and c-fos, and the activity of\nsenescence-associated-\u03b2-galactosidase (SA-\u03b2-GAL) can be added for more\nreasonable statistical power and more reliable results. To complete the\nresearch information, it is recommended to conduct further research with\nvariations in metformin concentration on fibroblast cell treatment or on aging\ncells with higher passage to analyze the anti-aging potential of metformin. In\naddition, it is expected that in future studies, experiments or in vivo\nclinical trials can be conducted so that the potential of metformin as an\nanti-aging agent can be better explored.<\/p>\n\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

The absence of a statistically significant decrease in IL-6 expression compared to the negative control indicates that the metformin treatment did not influence the expression of the IL-6 gene in human fibroblasts. However, a statistically significant difference in IL-6 expression between the treatment groups under the negative control and the positive control suggests potential anti-aging effects of metformin. These findings may contribute to understanding how metformin impacts IL-6 gene expression in aging models of human fibroblast cells and lay the groundwork for further exploration into the anti-aging properties of metformin.       <\/p>\n\n\n\n

Acknowledgments<\/strong><\/p>\n\n\n\n

The authors are grateful to Drs Astrid Feinisa\nKhairani, Reti Hindritiani, Oki Suwarsa, and Dr Putri Tessa for fruitful\ndiscussions. We would also like to express our thanks to all the staff at the\nCell Culture and Cytogenetic Laboratory, Laboratory of Molecular and Genetics,\nPadjadjaran University, who have assisted in this study.<\/p>\n\n\n\n

Conflict of\nInterest<\/strong><\/p>\n\n\n\n

All authors\ndeclare no conflict of interest in the publication of this research.<\/strong><\/p>\n\n\n\n

Funding Sources<\/strong><\/p>\n\n\n\n

This study received no additional funding.<\/p>\n\n\n\n

Data Availability<\/strong><\/p>\n\n\n\n

The data used to support the findings are available from the corresponding author upon request.<\/p>\n\n\n\n

Reference<\/strong><\/p>\n\n\n\n

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