Vol.:(0123456789)1 3 Molecular Biology Reports (2022) 49:73–83 https://doi.org/10.1007/s11033-021-06843-7 ORIGINAL ARTICLE Topical application of metformin accelerates cutaneous wound healing in streptozotocin‑induced diabetic rats Fatma Kubra Tombulturk1,2   · Zeynep Gizem Todurga‑Seven3,4 · Onder Huseyinbas5 · Sibel Ozyazgan3 · Turgut Ulutin2 · Gonul Kanigur‑Sultuybek2  Received: 25 April 2021 / Accepted: 14 October 2021 / Published online: 31 October 2021 © The Author(s), under exclusive licence to Springer Nature B.V. 2021 Abstract Background  Diabetic chronic wound, which is one of the diabetic complications caused by hyperglycemia, characterized by prolonged inflammation has become one of the most serious challenges in the clinic. Hyperglycemia during diabetes not only causes prolonged inflammation and delayed wound healing but also modulates the activation of nuclear factor-kappa B (NF-κB) and the expression of matrix metalloproteinases (MMPs). Although metformin is the oldest oral antihyperglycemic drug commonly used for treating type 2 diabetes, few studies have explored the molecular mechanism of its topical effect on wound healing. Therefore, we aimed to investigate the molecular effects of topical metformin application on delayed wound healing, which’s common in diabetes. Methods and results  In this context, we created a full-thickness excisional wound model in Wistar albino rats and, investi- gated NF-κB p65 DNA-binding activity and expression levels of RELA (p65), MMP2 and MMP9 in wound samples taken on days 0, 3, 7, and 14 from diabetic/non-diabetic rats treated with metformin and saline. As a result of our study, we showed that topically applied metformin accelerates wound healing by suppressing NF-κB p65 activity and diminishing the expres- sion of MMP2 and MMP9. Conclusions  Diabetic wounds treated with metformin healed even faster than those in the control group that mimicked standard wound healing. Keywords  Diabetes mellitus · Metformin · MMP · NF-kB · Wound healing Introduction Diabetes mellitus is considered to be associated with a series of changes in the metabolism of connective tissue, as a result of which diabetics face the problem of delayed wound heal- ing, which is stalled in the persistent inflammatory phase with elevated levels of pro-inflammatory cytokines and pro- teases. The wound healing process does not proceed toward the next phases to heal. A frequent and severe problem in patients with diabetes is delayed or poor wound healing, affecting 15–20 per cent of all people with diabetes [1–3]. The wound healing process is dominated by the coordina- tion of numerous cell-signalling events, growth factors, pro- inflammatory cytokines, NF-κB signalling pathway, is one of a key regulator of the inflammation [4, 5]. Increased inflam- matory cytokines, and activated NF-κB pathway in diabetic wounds subsequently leads to the suppression of some growth factors signalling impairing wound healing and cause a pro- longed inflammation process, creating a result that negatively affects wound healing [6, 7]. Besides, hyperglycaemia in dia- betes increases MMP activity directly or indirectly through oxidative stress or advanced glycation end products (AGEs). The wounds need a certain number of these enzymes for an efficient healing process, contrarily, may be damaging at high levels, causing disrupted wound healing [8–10]. It is known * Gonul Kanigur‑Sultuybek kanigur@istanbul.edu.tr 1 Medical Laboratory Techniques, Vocational School of Health Services, Istinye University, Istanbul, Turkey 2 Department of Medical Biology, Cerrahpasa Medical Faculty, Istanbul University-Cerrahpasa, Istanbul, Turkey 3 Department of Medical Pharmacology, Cerrahpasa Medical Faculty, Istanbul University-Cerrahpasa, Istanbul, Turkey 4 Department of Medical Pharmacology, Medical Faculty, Halic University, Istanbul, Turkey 5 Research Centre, Medical Faculty, Bezmialem University, Istanbul, Turkey http://orcid.org/0000-0002-4358-2309 http://orcid.org/0000-0001-9029-1910 http://crossmark.crossref.org/dialog/?doi=10.1007/s11033-021-06843-7&domain=pdf 74 Molecular Biology Reports (2022) 49:73–83 1 3 that the expression of MMPs is regulated by NF-κB until now most of the researchers have focussed on the expression pat- terns of MMP-9 and -2. There are reports showing that MMP2 and MMP9 levels are higher in diabetic wounds, thus causing delayed wound healing and risked repair. Furthermore, MMP9 and MMP2 are the best known pro-inflammatory members of proteases that degrade ECM components. Disruption of the optimization of inflammation due to high MMP levels in the scar tissue delays wound healing process [1, 9–12]. Metformin is one of the most constantly prescribed first- line oral anti-hyperglycemic drug for the management of type 2 DM for many years [13, 14]. Although metformin is one of the oldest and most effective agents in the treatment of patients with diabetes, there are few studies on the potential effect of its on wound healing. Recently, it has been reported in studies that topically administered drugs are known to be effective in faster wound contraction, wound closure, and overall heal- ing due to the desired local effect directly at the wound site. Few studies have reported that metformin showed accelerated wound-healing effects in healthy animals diminished activa- tion of NF-κB, inhibited the expression of pro-inflammatory mediators such as MMP [15–19]. As a result, considering drug interactions and systemic side effects of pharmacological agents, due to the known anti-inflammatory effect of metformin, it’s likely that apply- ing it topically will show its effect faster than systemic cir- culation. Also, agents that decrease inflammatory molecules expression such as inhibiting the NF-κB signalling pathway and decreasing the MMP levels may be a useful effective therapeutic method to cure the impaired wound healing in diabetic patients. Thus, wound healing can pass from the prolonged inflammatory phase to the proliferative phase in an optimal time. The preventive efficacy of metformin against wound healing has been supported by several inves- tigations, but there are many unanswered questions to illu- minate the underlying impaired wound healing in diabetes. Therefore, the target of this study is to evaluate the effect of metformin in the STZ-induced diabetic rat wound model and to figure out the role of NF-κB, MMP2 and MMP9. Materials and methods Animals The experimental protocols of this study were reviewed and approved by the Laboratory Animals Local Ethics Commit- tee of Bezmialem University (2019/216). Adult, male, with an average weight of 250–300 g Wistar albino rats were obtained. The rats included in the study were housed in a temperature-controlled (22–24 °C) room within individually polycarbonate cages on a 12-h light/dark cycle. Standard pellet and water were provided ad libitum. Six rats were randomly distributed to each group, and these rats were divided into two main groups as diabetic and non-diabetic rats. Each group was again divided into two groups as metformin (treatment groups) and saline applied groups (control groups) once daily for 14 days. The four groups were as follows: Non-diabetic control group-NC: Only sterile saline was applied to the wounds. Non-diabetic Metformin treatment group-NT: Rats were treated topically with 3 mM metformin. Diabetic control group-DC: Only sterile saline was applied to the wounds. Diabetic Metformin treatment group-DT: Rats were treated topically with 3 mM metformin. Induction of diabetes At the beginning of the study, blood glucose levels and body weights of all animals were measured. Diabetes was induced in rats constituting the experimental group with a single dose of intraperitoneal (IP) 60 mg/kg STZ (Cayman) in citrate buffer solution (0.1 M, pH 4.5), which is immediately once prepared. After 72 h, blood samples drawn from the tail vein of rats were measured for fasting glucose levels using a glu- cometer (Accu-Check, Roche). Those higher than ~ 250 mg/ dL were considered diabetes and selected for further experi- ment. Rats with blood glucose levels below this level were excluded from the study [20]. Creation of excisional wound model Rats were administered intraperitoneally 50 mg/kg pento- thal sodium for anaesthesia before each operation. After the dorsal region of each rat undergoing anaesthesia was shaved off with an electric razor, the wound site was made ready for operation by disinfection with povidone-iodine and 70% alcohol. Three circular full-thickness excisional wounds were created in the back region of the animals by 12 mm2 diameter sterile skin biopsy punch on each rat in each group. This first operation was accepted as 0 days and the experi- mental process was started. The biopsy days were planned for the 3rd, 7th, and 14th days. The biopsy procedure was performed according to the clock position, sequentially in the wounds. The wound tissue upper left on the 3rd day, the wound tissue upper right on the 7th day, and wound tis- sue lower midline on the 14th day were taken under sterile conditions. The wound area was traced on days 0, 3, 7, and 14 post-wounding by taking photos from a stable height. Wound size measurements were made with the Autocad® program. 75Molecular Biology Reports (2022) 49:73–83 1 3 Application of the treatment Sterile surgical sponges were absorbed with saline and applied on the wound to control groups once daily through- out 14 days. Metformin-HCl (Aarti Drugs) prepared at a concentration of 3 mM was absorbed into sterile surgical sponges and topically applied on the wound to treatment groups once daily throughout 14 days. The blood glucose levels of the rats were checked before the surgical proce- dure on each operation day, and their blood glucose lev- els of the diabetic rats were continuously monitored to be above ~ 250 mg/dL. After the wound biopsy was taken on the 14th day, the experiment was terminated. Rats were sacri- ficed with a high dose anaesthetic on day 14 post-wounding. Total RNA isolation and RT‑qPCR Total RNA was isolated from all snap-frozen wound tis- sues using High Pure RNA Tissue Kit (Roche) according to the manufacturer’s protocol and stored at − 80 °C until the experiment day. The RNA quality was determined via Nan- oDrop-One (Thermo-Scientific) by measuring the OD260/ OD280 ratio (> 2.0). mRNA expression levels of RELA, MMP2 and MMP9 in wound tissues were determined by quantitative reverse transcriptase PCR. RT-qPCR analy- sis was accomplished using LightCycler 480 System 1.5 (Roche) by LightCycler® EvoScript RNA SYBR® Green I Master Mix. For each sample, RT-qPCR assay was done at least in duplicate. The final reaction volume was 20 μl. All quantifications were normalized to the β-actin as housekeep- ing gene. 2−ΔCt results of relative mRNA expression levels were calculated using Lightcycler 480 Software Release 1.5.0 SP4 system compared to ACTB. Nuclear extraction and ELISA Separation of nuclear fractions from tissue was performed to the manufacturer’s instructions (Abcam). NF-kB (p65) activ- ity in the nuclear extracts was determined using NF-κB p65 Transcription Factor Assay Kit according to the manufac- turer's instructions (Abcam). The optical density of the per- oxidase product was read using an ELISA reader at 450 nm. The results were expressed as OD. Statistical analyses The number of animals in this study was determined by power analysis (power of 0.8 with alpha value 0.05) (G Power 3.1). The sample size was estimated from data of a similar previous study [21] to detect a 50% difference in the average wound healing, with an α of 0.05 and β of 0.20. Statistical analyses were performed using Graphpad Prism 8.0.2. Results were expressed as mean ± SEM, where “n” is the number of individual animals per group. A p-value of ≤ 0.05 was considered statistically significant. The differ- ences between the groups were examined using ANOVA test followed by Tukey test to compare groups with each other for meaningful results. Results Effect of topically applied metformin on wound healing The improvement in DC is much behind on the 14th day compared to the other groups. In the NT, almost all animals had closed wounds on day 14, while a faster wound healing was observed in the DT than the NC, which represents the spontaneous treatment process (Fig. 1A, B). Each group was evaluated in comparison to the days within itself (Fig. 1A). On the 7th day and the 14th day, there was a significant decrease compared to the 0th day (p < 0.0001) in the NC group. In the NT group, it was shown that metformin at a statistically significant level (p < 0.0001) accelerated wound healing on the 3rd, 7th, and 14th days compared to the 0th day. In the DC group, the speed of wound healing was prolonged, and no significant difference was found on the 3rd day. But, on the 7th and 14th days, wound healing showed a significant decrease (p < 0.0001). Because of metformin administration in the DT group, a sig- nificant decrease was observed on the 3rd, 7th, and 14th days compared to the 0th day (p = 0.0049, p < 0.0001, p < 0.0001; respectively). When comparing the groups on the 3rd, 7th, and 14th days of healing; when the wound measurement results on the 7th day are evaluated; there is a significance between the DC group and NC group (p = 0.0119), this finding shows the negative effect of diabetes on wound healing. The DT group also showed a significant wound healing result (p = 0.0001) compared to DC. Also, there is a significant difference between treatment groups (p = 0.0038). On the 14th day; The NT group showed a dramatic decrease (p = 0.0005) com- pared to the NC, and a significant improvement was also found in the DT group compared to the DC (p < 0.0001). Also, a significant difference was found when the two con- trols were compared (p = 0.0083). Effect of metformin on NF‑κB p65 activity According to these results, when each group is compared within itself at the level of days (Fig.  2A); in the NC group, p65 activity on day 3 increased significantly com- pared to day 0 (p < 0.0001). On the 14th day, it showed a significant decrease compared to the 0th day (p = 0.0026, respectively). In addition, a significant decrease was seen 76 Molecular Biology Reports (2022) 49:73–83 1 3 on days 7 and 14 compared to day 3 (p < 0.0001). It was observed that there was a significant increase on the 3rd day and 7th day in the DC group compared to the 0th day (p < 0.0001, p = 0.0005, respectively). In the NT and DT groups, expression values on the 3rd, 7th, and 14th days were significantly decreased compared to the 0th day (p < 0.0001). When the comparison between groups was made on the 3rd, 7th, and 14th days of wound healing (Table 1A); On the 3rd day of wound healing, the NT and DT groups decreased dramatically compared to their control groups and statistically significant (p < 0.0001). According to the evaluation results on the 7th day; No significance was found between the NT group and its control (NC). In the DT group, compared to the control (DC), the sig- nificant decrease continued (p < 0.0001). According to the evaluation on the 14th day of wound healing; a sig- nificant decrease was seen in the DT group compared to DC (p < 0.0001). On 3., 7., and 14. days of the healing, whereas no significant difference was observed between the treatment groups, among the control groups, the DC group was significantly higher than the NC group (p < 0.0001). Effect of metformin on RELA (p65) mRNA expression level When comparing within the group according to days (Fig. 2B) in the NC group, it showed a significant increase on the 3rd day compared to the 0th day (p < 0.0001). A sig- nificant decrease was observed on the 7th and 14th days compared to the 0th day (p = 0.001, p = 0.0259, respec- tively). A dramatic decrease was observed on the 7th and 14th days compared to the 3rd day (p < 0.0001). A statis- tically significant increase was found in the DC group on day 3 and day 7 compared to day 0 (p = 0.0005, p = 0.0155, respectively). When the NT and DT groups were evaluated compared to days, there was no difference on day 3 com- pared to day 0. However, the dramatically lower levels on the 7th and 14th days compared to the 0th day showed sta- tistical significance (p < 0.0001). A significant decrease was also observed on days 7 and 14 compared to day 3 of wound healing (p = 0.0003, p = 0.0004, p < 0.0001, p < 0.0001, respectively). According to the intergroup evaluation results according to the days (Table 1B); Results on day 3 showed a signifi- cant decrease in the NT and DT groups compared to their control groups (p = 0.036, p = 0.0007, respectively). Among the control groups, it was observed that the DC group had a significantly higher expression level than the NC group (p = 0.0016). According to the results on the 7th day; It was Fig. 1   A Graphical evaluation of the healing effect of topically applied metformin on cutaneous wound healing in the experimen- tal groups. Data shown are the mean ± SEM (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; compared to day 0. + p < 0.05; +  +  p < 0.01; +  +  + p < 0.001; +  +  +  + p < 0.0001; compared to day 3. #p < 0.05; ##p < 0.01; ###p < 0.001; ####p < 0.0001; compared to day 7- within-group significance level is indicated). B Photo images of wound contraction of metformin-treated (3 mM) rats on days 0, 3, 7, and 14 77Molecular Biology Reports (2022) 49:73–83 1 3 found that the expression level in the DT group was signifi- cantly reduced (p < 0.0001) compared to the control (DC). No significance was found between the NT group and NC. There was also a significant difference between the NC and DC groups due to the increase in the expression level of diabetes (p < 0.0001). The RELA expression level results on day 14 of wound healing showed a significant decrease in the DT group compared to the DC (p < 0.0001). However, no significant change was observed in the NT group compared with NC. The DC group was found to be statistically signifi- cantly higher than the NC group (p < 0.0001). Effect of metformin on MMP2 mRNA expression level When the comparison results of the experimental groups according to the days are evaluated (Fig. 3A); Results on days 7 and 14 in the NC group showed a dramatic signifi- cant decrease compared to day 0 (p = 0.0012, p = 0.0003, respectively) and day 3 (p < 0.0001). Although there was an increase on the 3rd day and a decrease on the 7th day in the DC group compared to the 0th day, no significance was found. However, a significant decrease was observed on day 14 compared to day 0 (p = 0.0003). When the results of the NT and DT groups were evaluated, a dramatic decrease was observed on the 3rd, 7th, and 14th days compared to the 0th day and was statistically significant (p < 0.0001). When comparing the groups on the 3rd, 7th, and 14th days of the healing (Table 1C); On day 3, the NT group showed a significant decrease compared to the NC (p = 0.0051). In the DT group, it was found that MMP2 gene expression level was considerably decreased compared to DC and was considered significant (p < 0.0001). The expres- sion levels of NC and DC groups, which are the control groups, were found to be significantly increased compared to the NC group in the DC group (p < 0.0001). According to the results on the 7th day; there was a significant difference in the expression level of the DT group compared to the DC (p < 0.0001). There was no significant effect of metformin on MMP2 expression in the NT group compared to its control. Also, a significant difference was found between the con- trol groups (p < 0.0001). Looking at the results of the 14th Fig. 2   A Graphical evalu- ation of the NF-κB (p65) activity results detected by ELISA method on days 0, 3, 7, and 14 of wound healing. B Graphical evaluation of the RELA mRNA expression level determined by RT-qPCR method on days 0, 3, 7, and 14 of wound healing. Data shown are the mean ± SEM (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; compared to day 0. + p < 0.05; +  + p <  0.01; +  +  + p < 0.001; +  +   +  + p < 0.0001; compared to day 3. #p < 0.05; ##p < 0.01; ###p < 0.001; ####p < 0.0001; compared to day 7-within-group significance level is indicated) 78 Molecular Biology Reports (2022) 49:73–83 1 3 day; the decrease in the DT group was significant compared to the DC (p = 0.0005). While no significant change was observed in the NT group compared to the control, a sig- nificant difference was again observed between the control groups (p = 0.0014). Effect of metformin on MMP9 mRNA expression level Within-group evaluation results of MMP9 expression lev- els compared to days (Fig. 3B); it showed that the increase on day 3 and the decrease on days 7 and 14 did not have a significant effect compared to day 0 in the NC group. However, it was found that the decrease observed on the 7th and 14th days compared to the 3rd day was a sig- nificant difference (p = 0.0066, p = 0.0192, respectively). The decrease in the 7th and 14th days in the NT group was considered to be statistically significant (p < 0.0001). Similarly, expression levels on the 7th and 14th days were found to be significantly decreased compared to the 3rd day (p < 0.0001). Results of the DC group; It showed that the expression levels on day 3 increased significantly to day 0 (p = 0.0011). On the 14th day, it was determined that the decrease observed compared to the 0th day was also significant (p = 0.0169). In the DT group; The results of the 7th and 14th days decreased significantly compared to the 0th day (p = 0.0004, p = 0.001, respectively). The decrease observed on the 7th and 14th days compared to the 3rd day was found to be significant (p = 0.0083, p = 0.0218; respectively). According to the evaluation results between groups compared to days (Table  1D); On the 3rd day, the decreased expression level of the DT group compared to the DC was found to be statistically significant (p = 0.0012). In the NT group, although there was a sig- nificant decrease compared to the control (NC), there was no significant difference. The results of the control groups were found to be significantly increased in the DC group compared to the NC group (p = 0.0058). While the MMP9 expression level did not show a significant change in the NT group compared to the NC on the 7th day similar to the 3rd day, it was found that the DT group showed a dramatic decrease compared to the DC (p < 0.0001). The difference in the control groups was found that the DC group showed a significant increase compared to the NC (p = 0.0004). Expression results on day 14 showed a significant change, showing a decrease (NC) compared to the control of the NT group. It was also found that the DT group showed a statistically significant decrease compared to its control (p = 0.0427). Table 1   A NF-κB (p65) activation results on the 0, 3, 7, and 14 days of wound healing. RELA (B), MMP2 (C), MMP9 D mRNA expression levels on the 0, 3, 7, and 14 days of wound healing Results are expressed as mean ± standard error values (Mean ± SEM) †Compared to NC group ‡Compared to DC group §Compared to NT group- The level of significance between groups is indicated (p < 0,05) NC NT DC DT p value A) NF-κB (p65) activity Day 0 0,106 ± 0,007 0,159 ± 0,006† 0,0001 Day 3 0,186 ± 0,007 0,071 ± 0,004† 0,4164 ± 0,028†, § 0,1089 ± 0,007†, §, ‡  < 0,0001 Day 7 0,083 ± 0,007 0,063 ± 0,003 0,3031 ± 0,028†, § 0,08,843 ± 0,004‡  < 0,0001 Day 14 0,07 ± 0,002 0,0596 ± 0,001 0,149 ± 0,016†, § 0,084 ± 0,004‡  < 0,0001 B) RELA mRNA expression levels Day 0 1,159 ± 0,098 2,148 ± 0,236† 0,0007 Day 3 2,436 ± 0,253 1,052 ± 0,199† 4,471 ± 0,481†, § 2,257 ± 0,322‡  < 0,0001 Day 7 0,327 ± 0,051 0,232 ± 0,026 3,751 ± 0,259†, § 0,508 ± 0,059‡  < 0,0001 Day 14 0,585 ± 0,085 0,243 ± 0,030 2,998 ± 0,321†, § 0,421 ± 0,071‡  < 0,0001 C) MMP2 mRNA expression levels Day 0 0,240 ± 0,043 0,582 ± 0,104† 0,0124 Day 3 0,333 ± 0,039 0,050 ± 0,013† 0,773 ± 0,093†, § 0,065 ± 0,018†, ‡  < 0,0001 Day 7 0,049 ± 0,014 0,049 ± 0,007 0,393 ± 0,040†, § 0,070 ± 0,021‡  < 0,0001 Day 14 0,024 ± 0,002 0,020 ± 0,004 0,061 ± 0,011†, § 0,020 ± 0,002‡ 0,0002 D) MMP9 mRNA expression levels Day 0 1,507 ± 0,121 2,494 ± 0,242† 0,0045 Day 3 2,284 ± 0,485 1,404 ± 0,079 4,064 ± 0,197†, § 1,950 ± 0,401‡ 0,0001 Day 7 0,839 ± 0,164 0,477 ± 0,040 2,373 ± 0,373†, § 0,510 ± 0,146‡  < 0,0001 Day 14 1,026 ± 0,151 0,362 ± 0,054† 1,343 ± 0,071§ 0,683 ± 0,273‡ 0,0023 79Molecular Biology Reports (2022) 49:73–83 1 3 Discussion Hyperglycemia associated with diabetes induces proinflam- matory cytokines and an increase in inflammatory cells that disrupt components of the ECM and growth factors neces- sary for wound healing. As a result, the inflammatory phase of healing is prolonged, granulation tissues cannot develop, and chronic wounds eventually occur. These findings suggest that hyperglycemia is responsible for delayed wound healing process in diabetic patients [22–25]. In present study, we showed that wound healing in STZ diabetic rats was delayed due to hyperglycemia compared to the non-diabetic group. In vitro and in vivo studies have shown that metformin has a protective effect against oxidative damage induced by hyperglycemia and inhibits the expression of pro-inflamma- tory cytokines [17–19]. Recently, interest in metformin has been increasing due to its anti-inflammatory properties. It is suggested that metformin achieves its effect on wound healing by changing cytokine and chemokine expression patterns and eliciting some pleiotropic effects [14, 17, 26]. In light of this information, we created a full-thickness exci- sional wound model in STZ diabetic and non-diabetic rats, and applied 3 mM metformin topically on the wounds for 14 days. It was observed that the improvement in metformin treatment groups was statistically significantly increased. Our striking finding was that diabetic wounds improved in a shorter time with metformin administration, even than the control group. We demonstrated an accelerated wound healing significantly at day 7 and day 14, while there was an acceleration in wound healing from day 3 compared to controls in the diabetic group treated with metformin (Fig. 1A, B). We found that metformin treatment signifi- cantly decreased wound diameters in both diabetic and non- diabetic groups. In Lee et al.'s studies, metformin was shown to cause an 85% versus 67% wound closure at day 7 and an 85% versus 97% wound closure at day 14 compared to con- trol wounds [15]. This study supports our findings. In the study of Qing et al., topically applied Metformin (2 mM) treatment in a pluronic gel formulation in wounds created in diabetic rats was shown to accelerate the healing of excisional wounds in rat skin compared to the control group [27]. In another study, the effects of locally applied metformin on wound healing in elderly skin were investi- gated. Regarding cutaneous wound healing rates, metformin Fig. 3   A and B Graphical evaluation of the MMP2 and MMP9 mRNA expression levels determined by RT-qPCR method on days 0, 3, 7, and 14 of wound healing. Data shown are the mean ± SEM (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; compared to day 0. + p < 0.05; +  + p <  0.01; +  +  + p < 0.001; +  +   +  + p < 0.0001; compared to day 3. #p < 0.05; ##p < 0.01; ###p < 0.001; ####p < 0.0001; compared to day 7- within- group significance level is indicated) 80 Molecular Biology Reports (2022) 49:73–83 1 3 significantly accelerated wound healing in aged rats [18]. Although there are few studies showing the potential effect of metformin on wound healing, the data are consistent with the data we obtained as a result of our study. Consistent with our study with STZ diabetic rats, studies show that metformin leads to faster wound healing [15, 18, 28–32]. In addition to these studies, it is shown that administered orally metformin to show its effect accelerates wound heal- ing in different diabetes models compared to diabetic con- trol groups, but when metformin is applied topically on the wound, it is more effective than oral use [28, 33]. Hyperglycemia activates various transcription factors. NF-κB, is a biomarker of inflammation, and can lead to chronic inflammation and hence tissue damage. In this case, it is clear that NF-κB signals have directly critical impor- tance in cutaneous wound healing. The low level of NF-κB in the diabetic wound can help prevent progression to sys- temic inflammation that initiates the development of diabetic complications [34–37]. In our study, we investigated the NF-κB p65 DNA binding activity and RELA gene expres- sion to show whether it is related to the delays in wound healing seen in STZ-induced diabetes. NF-κB p65 activ- ity was found to be significantly higher on the 0.3, 7 and 14 days in wounds with diabetes compared to the wounds in the non-diabetic group. As a result of metformin treat- ment, it was observed that NF-κB p65 activity decreased statistically significantly in both groups compared to con- trols (on day 3: p < 0.0001 (NC/NT), p < 0.0001 (DC/DT); on day 7: p < 0.0001 (DC/DT); on day 14: p < 0.0001 (DC/ DT)). The NF-κB p65 activity that significantly increased on the 3rd day in the NC group and the 3rd and 7th day in the DC group was quite low in the treatment groups. The significant decrease not only by days but also within the group compared to the starting day every 3 days is note- worthy. Moreover, the activity values in the DT group were even lower than the NC group on the 3rd day, almost the same as the spontaneous wound healing on days 7 and 14. After metformin treatment in RELA gene expression, a sig- nificant decrease was observed in the NT group on the 3rd day compared to the first day, while a statistically signifi- cant decrease was observed in the DT group on the 7th and 14th days. Such that the expression level of the DT group on the 3rd day was lower than the RELA expression level in the NC group. These results suggest that treatment with metformin resulted in a faster improvement in the diabetic treatment group not only compared to the control but even than the normal control group, which mimics spontaneous healing. In the study conducted by Cam et al., the NF-κB level was significantly lower in the treatment groups where metformin was administered in groups, compared to the con- trol and, showed better anti-inflammatory effects than the other groups [31]. In addition, in the study conducted and in recent studies using different active substances in line with our study, it has been shown that these substances reduce inflammation and accelerate wound healing via an NF-κB inhibitor, as in the use of metformin [31, 36–38]. NF-κB also regulates the expression of MMPs, it has been reported that NF-κB is one of the main components of the intracellular signalling pathways specifically responsible for MMP-2 and MMP-9 [39, 40]. Studies have confirmed that uncontrolled MMP activity causes tissue damage and func- tional changes, and that increased MMP levels (especially MMP-2 and MMP-9) lead to negative effects on wound healing [9, 41, 42]. Topical application of MMP inhibitors accelerate diabetic wound healing, reduces inflammation, increases fibroblast tissue formation [10, 43]. In our study, as a result of topical metformin administration in diabetic and non-diabetic groups, a significant decrease was observed in MMP2 mRNA expression levels on all days and, in MMP9 mRNA expression levels on the 7th and 14th days com- pared to the first day. In light of this information, MMPs are important mediators regardless of acute or chronic wounds. Thus, MMP levels are critical biomarkers when analyzing the therapeutic efficacy of wound healing processes. There are many studies specifically aimed at inhibiting MMPs [44, 45]. These results are in line with our results. The healing effect of topically applied metformin in a full-thickness excisional wound model created on both dia- betic rats and non-diabetic rats is the first study in the world that we present to what extent MMP2, MMP9 and RELA expression changes relative to NF-κB activity. Therefore, since there is no study showing that the topical application of metformin affects NF-κB and MMPs together, studies, where different substances were applied, were compared. According to Singh et al. study, the icariin molecule was tested on a wound model created in rats. The results show that NF-κB protein expression is decreased in tissue samples taken from wounds, especially in the icariin-treated groups compared to the control group, and also, it decreases both MMP-2 and MMP-9 activities. Thus, icariin is thought to cause the acceleration of wound healing compared to con- trol [46]. In the study investigating the effects of topically applied mangiferin molecule on excisional wound model in rats with type 2 diabetes model, it was shown that NF-κB p65 protein level was lower and MMP expression was decreased after mangiferin treatment. This suggests that it was sped up the wound healing process [47]. In the cutane- ous wound model created by Durmuş et al., as a result of topical application of arginine silicate inositol, the amount of both MMP-2 and MMP-9 gradually decreased on the 5th, 10th, and 15th days, and in parallel, the amount of NF-κB was significantly decreased, hence, they demonstrated that wound healing was accelerated accordingly [48]. A study parallel with our study shows that topical application of syringic acid effectively enhances the wound healing process in diabetic rats. According to the data of the current study; 81Molecular Biology Reports (2022) 49:73–83 1 3 showed that following administration of syringic acid to dia- betic wounds for 14 days, decreased NF-κB p65 activity and levels of MMP-2, MMP-9 mRNA expression compared to control [49]. In line with the results of our study; metformin acceler- ates wound healing by suppressing NF-κB activation and inflammatory response to improve the wound healing pro- cess in hyperglycemic rats (Fig. 4). This may be related to anti-inflammatory properties (reduction of NF-κB p65 activity) and inhibition of MMPs (down-regulation in MMP-2 and MMP-9 mRNA). As a result; although the topical application of metformin on wounds is much more effective in diabetic wounds, we have also shown that non- diabetic wounds increase wound healing rate by inhibiting NF-κB p65 activity and decreasing RELA mRNA expres- sion level, and in addition, reducing MMP2 and MMP9 expression levels (Table 2). In the future, used topically metformin may be an effective, inexpensive and reliable potential therapeutic agent applied not only to diabetic wounds but also to all wounds. Fig. 4   Topically applied metformin in the treatment of non-healing or delayed-healing wounds because of hypergly- cemia affects transcriptional regulation by decreasing NF-kB p65 activity. This leads to decreased expression levels of RELA, MMP2 and MMP9. Thus, it has positive effects on wound healing by passing the inflammation phase, which is a critical stage for wound healing, and accelerating the transition to the proliferation phase 82 Molecular Biology Reports (2022) 49:73–83 1 3 Authors’ contributions  FKT and GKS designed the research study con- cept. FKT, ZTS, OH carried out the animal experiments. Laboratory analyzes were studied by FKT. TU and SO participated in the design. FKT, GKS interpreted the data and drafted the final version of the manuscript. 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Life Sci 233:116728 Publisher's Note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. https://doi.org/10.1155/2019/3182627 https://doi.org/10.1155/2019/3182627 https://doi.org/10.1080/09546634.2020.1721419 Topical application of metformin accelerates cutaneous wound healing in streptozotocin-induced diabetic rats Abstract Background Methods and results Conclusions Introduction Materials and methods Animals Induction of diabetes Creation of excisional wound model Application of the treatment Total RNA isolation and RT-qPCR Nuclear extraction and ELISA Statistical analyses Results Effect of topically applied metformin on wound healing Effect of metformin on NF-κB p65 activity Effect of metformin on RELA (p65) mRNA expression level Effect of metformin on MMP2 mRNA expression level Effect of metformin on MMP9 mRNA expression level Discussion References