INTRODUCTION:

Wounds are defined as a break or opening in the skin that results in a change in the anatomy and function of the skin. Tissue regeneration is a process that begins with trauma and is invariably accompanied by the formation of scar tissue.² The healing process is divided into four distinct phases: coagulation, epithelialization/inflammation, granulation, and collagen tissue remodelling, as well as an orderly organisation of newly formed tissue within the granulation tissue.

Inflammation is associated with mononuclear infiltration, macrophage and myelocyte migration, neutrophil and monocyte emigration, as well as increased leukocyte permeability, whereas acute inflammation is associated with increased monocyte and neutrophil migration.¹ Interleukin 6 (IL-6), interleukin 8 (IL-8), and vascular endothelial growth factor (VEGF) are three molecules involved in wound healing that can be distinguished as having the greatest influence on this type of cell growth. Additionally, IL-6 has anti-inflammatory and pro-inflammatory properties. As with tumour necrosis factor (TNF), IL-6 plays a physiological role in interleukin gene expression stimulation.³ Even after the functional and anatomical integrity of unhealed wounds is restored, inflammatory mediators continue to be produced, resulting in inflammation persisting beyond the normal period of healing. That is, when an inflammatory response occurs.⁴

SGLT2 inhibitors are suffixed as gliflozins which have a basic physiology in the urinary tract especially nephrons and also modulates the sodium and glucose channels present in the mucosa of the intestinal tract. The activities of the drugs belonging to this class cause the inhibition of glucose reabsorption in the kidneys and so lowers the blood glucose levels.⁵ The activity is due to the inhibition of sodium glucose transport protein 2 (SGLT2)⁶. They are basically used in the treatment of type 2 DM and also exhibit cardiovascular effects.^7,8 Many medications of this class of drugs including Dapagliflozin, Canagliflozin and Remogliflozin are used to control the blood sugar levels and to lower the body weight. These are also used to control the blood pressure (systolic and diastolic).³ Gliflozins also possess protective effects on various organs like heart, liver and kidneys and exhibit anti-hyperlipidemic, anti-obesity, anti-neoplastic activities. They are also reasonably proven to cause homeostasis, glycosuria, and antioxidant activities.^9,10 Canagliflozin is a SGLT2 inhibitor which was approved by the US in 2013 with a brand name invokana and also marketed all over the world.¹¹ Dapagliflozin was also approved by the US in 2012 under the brand name farxiga by FDA in 2014. which is used in the treatment of type 2 DM. Remogliflozin was also approved for type 2 DM treatment in 2019 manufactured by Glenmark.¹² Extensive research was done on the above drugs, Dapagliflozin^13,14,15, Canagliflozin^16,17 and Remogliflozin¹⁸ to determine the efficacy using advanced quantitative estimation methods in vitro and in vivo.

Surprisingly, SGLT2 inhibitors have been shown to play a significant role in the inflammatory response. Inhibiting SGLT2 has been reported to reduce inflammation and slow the progression of inflammatory-mediated disorders.^19,20 Dapagliflozin triggers the anti-inflammatory mechanisms by reducing the synthesis of collagen and mobilizing the macrophages into the tissue when tested in rats.²¹In a diabetes rat model, Empagliflozin caused the reduction in the inflammatory cytokines, TNFα and IL-6 and also the apoptotic mediators.²² When the cytotoxicity of the gliflozins like Canagliﬂozin, Dapagliﬂozin and Empagliﬂozin were compared on human RPTEC cells, Canagliﬂozin showed a SGLT2 dependant cytotoxicity that is mediated by inhibiton of glutamate dehydrogenase and mitochondrial electron transport. Other studies showed that Dapagliflozin and Canagliflozin displayed significant cytotoxicity activity against oral cancer using MTT assay.²³Considering previous researches, there had been a significant correlation between the wound healing and anti-inflammatory property and glifozins were proven effective against reducing inflammation. So in this research the anti-inflammatory property of the SGLT2 inhibitors, Dapagliflozin, Canagliflozin and Remogliflozin was determined and its effect on wound healing was compared on VERO cells lines and cytotoxicity of three gliflozins were evaluated.

MATERIALS AND METHODS:

Methodology:

MTT assay kit, Dimethyl sulfoxide and Dulbecco’s modified eagles (DME) media were purchased from HiMedia, Mumbai, Fetal Bovine Serum (FBS), Trypsin in Phosphate Buffered Saline (PBS) and EDTA were supplied from Invitrogen, India. All the reagents used in the research otherwise specified were procured from SD Fine Chem, India and were of analytical grade.

Cell Lines:

VERO cell lines were purchased from NCCS Pune were maintained in DMEM enriched with FBS (10%w/w) along with 1% streptomycin and incubated in a CO₂incubator at 37°C in 5% CO₂. The cultures were centrifuged at 1000rpm for 5min and re-suspended in culture medium. This cell cultures in log growth phase were used in further study.

Determination of Anti-inflammatory activity using Protease Inhibition Assay:

The anti-inflammatory activity of the drugs was evaluated using the method developed by Oyedepo and Femure (1965)^24,25 for trypsin inhibition. Solutions of standard concentration (200, 400, 600, 800 and 1000 μg/ml) of drugs were prepared and 100 µL of the solution was mixed with 100µL of bovine serum albumin. The mixture was allowed to react at room temperature and after 5mins 250µL of trypsin was added to stop the reaction to continue. The mixture was centrifuged at 4000rpm for 5mins and supernatant was collected for measuring the absorbance at 210nm by taking Acetyl salicylic acid as a standard drug. The experiments were conducted to get triplicate readings and % activity was determined using following equation.

% Inhibition=100− ((A_s –A_c)/A₀)*100)

Where A_s=absorbance of sample drugs, A_c=absorbance of the control and A₀=absorbance of the positive control.

Cytotoxicity Assay using MTT:

VERO cell lines (1x10⁴) were seeded into 96-well plate with 100𝜇L of DMEM medium per well. The cells were incubated for 24hrs and medium was treated with different concentration of sample drugs (15, 30, 60, 120, and 250𝜇g/ml). After 24 hrs of incubation 10𝜇L of 5mg/mL MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl Tetrazolium Bromide ) reagent was added to the cells and allowed incubation for another 4hrs. 100 𝜇L dimethyl sulfoxide was added to all the wells and gently mixed. This mixture was allowed to react in a dark place at ambient temperature for 30mins after which the absorbance was measured using a microplate reader at 540nm. The % cell viability was calculated by comparing to the control.

% cell viability =

(A540 of treated cells/A540 of control cells) × 100%

Where A540=absorbance of the well at 540 nm

Invitro Wound Healing Assay (Scratch Method):

The migration of VERO cell lines were determined to investigate the wound healing activity of the drugs in-vitro.²⁶ The cells (2x10⁵) were seeded into six wells and incubated (37^oC and 5% CO₂) for 24 hours. The confluent cells were gently scrapped in a pattern with sterile pipette and washed successively with PBS. The collected cells were treated with sample drugs (100 𝜇g/mL) and Phenytoin (standard drug- 50 𝜇g/mL) by diluting with DMEM (serum free). The starch was induced in the medium and was photographed using phase contrast microscopy (x40) before incubation which was considered as 0hr. The migration of cells was again photographed after 6hrs of incubation and the growth of cells was calculated in triplicate readings.

% Wound closure = [(Measurement at 0 h −Measurement at 6 h)/Measurement at 0 h]×100.

DATA ANALYSIS:

The results were represented as the Mean±SD and the consistency of the results was analysed using ANOVA in Graph pad prism version 5.1. Statistical significance of the results was estimated by subjecting the values to student’s t-test. Differences in the values were considered as significant at p<0.05 in comparison to control group.

RESULTS:

Anti-inflammatory activity:

Anti-inflammatory activity of Dapagliflozin, Canagliflozin and Remogliflozin is investigated at an increasing concentration of the drugs of 200, 400, 600, 800 and 1000𝜇g/ml by estimating the protease inhibition capacity of the drugs in-vitro. Results displayed a clear dose dependant activity of all the drugs where in drugs at 200𝜇g/ml showed a minimal inhibition of about 36, 90 and 63%. Out of the three drugs Canagliflozin showed best activity of inhibition of 90% at minimum dose. At highest dose 1mg/ml Canagliflozin showed a 99% inhibition. Even though other two drugs, Dapagliflozin and Remogliflozin showed least activities at 36% and 63% at lower dose, at 1mg/ml these drugs showed a similar activity of 95% inhibition. Overall it can be advocated that the drugs showed an effective anti-inflammatory activity estimated via protease inhibition as shown in figure 1 and the results were tabulated in table 1.

Table 1: Anti-inflammatory activity of Dapagliflozin, Canagliflozin and Remogliflozin using Protease inhibition Assay

Con-centration

(𝜇g/ml)

%Inhibition

Dapagliflozin

Canagliflozin

Remogliflozin

Positive control

1000

95.23±

4.65

99.04±

3.51

95.23±

4.61

101.51±

6.85

800

88.88±

2.08*

95.55±

5.24

83.33±

5.23*

95.36±

5.14

600

62.50±

1.69*

93.00±

4.06

75.00±

3.26*

92.61±

5.92

400

53.84±

2.03*

92.30±

3.65

69.23±

3.07*

89.32±

3.99

200

36.36±

1.55*

90.90±

3.85

63.63±

2.66*

85.45±

4.06

* Indicates p<0.05 statistically significant compared to control

Figure 1: Anti-inflammatory activity of Dapagliflozin, Canagliflozin and Remogliflozin

Using Protease inhibition Assay:

Cell viability assay:

The effect of Dapagliflozin, Canagliflozin and Remogliflozin on cell viability on VERO cells has been investigated in DMEM medium using MTT assay. The drugs showed a similar viability control at dose of 250 𝜇g/ml of nearly 120% so it can be stated that the drugs did not show any toxicity on the growth of VERO cells at that particular dose. Further, drugs at 120 𝜇g/ml showed a highest viability of around130% which is similar in all the drugs as shown in figure 2. There is no significant difference between the viability of the drugs. The results were tabulated in table 2.

Table 2: Cytotoxicity Assay using MTT of Dapagliflozin, Canagliflozin and Remogliflozin

Concentration (𝜇g/ml)	% control of cell viability
Concentration (𝜇g/ml)	Dapagliflozin	Canagliflozin	Remogliflozin
15	84.00±3.61	89.71±3.85	86.11±3.70
30	92.67±3.06	98.97±3.26*	95.00±3.13
60	103.33±3.06*	110.36±3.26*	105.93±3.13*
120	124.67±4.16*	133.14±4.45*	127.80±4.27*
250	121.67±1.53*	129.94±1.63*	124.72±1.57*

* indicates P<0.05 statistically significant compared to control

* Indicates P<0.05 statistically significant compared to control

Figure 2: Effect of Dapagliflozin, Canagliflozin and Remogliflozin on cell viability

*p<0.05 considered significant compared to control; p<0.05 considered significant compared to standard

Figure 3: Wound healing activity invitro of Dapagliflozin, Canagliflozin and Remogliflozin

Dapagliflozin showed a highest wound healing activity of 60% which was similar to the standard drug of 65%. Remogliflozin and Canagliflozin showed a better activity than control of around 47 and 49% respectively. The results were depicted in figure 3. The pictures of VERO cell wound closure were clearly shown in figure 4 indicate the migration of cells into the wound cavity. The closure of wound in control group was not rapid compared to the drug treatment groups. The photographs clearly support the data represented in Figure 3.

DISCUSSION:

During Wound healing process expanding of the connective tissue and the extracellular matrix formation helps in remodelling of the blood vessels and cell activation lead to matrix growth. The presence of metaplastic cells may not yet be a known, however, have an important function in the healing of tissue wounds, alongside other cells outside the sheath, the tissue is supported by active mesenchymal stem cells which have already been identified. Wound healing in further analysis, more specifically, is a complex process that relies on the ability of fibroblasts to proliferate and migrate. Inflammation as well as wound healing capacity was determined using the VERO cell line's well-established scratch assay, which is also popular with researchers.²⁰

It was in our research, as shown in the figure 4, that when VERO cells were administered the drugs, they migrated more than that is observed in control group. This indicates that fibroblasts tend to migrate in response to the treatment with gliflozins. Additional testing was also performed on the anti-proliferative properties of the medications using an MTT test. The first test's intended use is to identify potential cytotoxicity so that it can identify both the potential and/the underlying reason for toxicity in an early assessment of a possible metabolites that are cytotoxic to cells. The dose can be considered as half of the minimal inhibitory concentration (IC50) and is used for this study. A high IC50 means that drug concentration is toxic at low doses, whereas a low IC50 indicates low toxicity.²⁷ In our study, we treated VERO cells with drug concentrations far above the recommended thresholds, and the cell's percentage viability was unaffected. This supports our claim that certain gliflozins can be used safely in wound healing.

The site of action of the SGLT2 inhibitors determines the base of the anti-inflammatory activity or cytotoxicity. There are many investigations in this area to estimate the invitro cytotoxicity and to determine the gliflozin's mechanism of action.²⁸ One study showed cytotoxicity of Canagliflozin at low concentration in the tubular epithelial cells tested invitro.²⁹ In the present study the cytotoxicity of the gliflozins was tested and all the drugs showed the cytotoxicity at a dose around 250 𝜇g/ml out of which Canagliflozin showed more activity which supports the previous studies suggesting the cytotoxicity of the drug. Others described a Canagliflozin-induced cytotoxicity at particularly low concentrations in proliferating immortalized tubular epithelial cells in vitro.³⁰ Wound healing is further expansion, more specifically, is a complex process that relies on the ability of fibroblasts to proliferate and migrate. Inflammation as well as wound healing capacity was determined using the VERO cell line's well-established scratch assay, which is also popular with researchers.³¹

Diabetes patient's elevated glucose levels cause physiological dysfunctions, resulting in a slower healing process than non-diabetic individuals. As a result, the wound healing process stays in the inflammatory phase for an abnormally long time, never progressing to the next healing phase and thus delaying healing.^32,33 Because a prolonged inflammatory phase is one of the most significant problems in the wound healing process, because of which an effective wound healing therapeutic candidate should be rich in anti-inflammatory properties. Anti-inflammatory properties are of great interest in the treatment of a wide range of dermatological disorders. Thus, treatment with selected gliflozins that have multiple mechanisms of action may be a promising therapeutic strategy for promoting wound healing and preventing inflammation in a chronic inflammatory condition. Moreover, high glucose content in the blood and the inflammation are major causes of the tissue damage and hold as major reasons for slow healing in diabetic individuals. Acute oxidative stress in the region is also an underlying cause for delayed healing of wounds.³⁴ The antioxidant activity of the drugs also tends to help in lowering of inflammation.³⁵ In such cases it can be advocated that Diabetes Mellitus itself is a pro inflammatory disorder that causes deterioration of organ systems in the human body.³⁶ Few studies have shown that the SGLT2 inhibitors also cause reduction in inflammation by lowering the fibrotic factors that are triggered by elevated glucose levels. Thus the SGLT2 inhibitors tested in this study can be appreciated as significantly effective in controlling the inflammation which is evident from protease inhibition assay.

CONCLUSION:

The anti-inflammatory and wound-closure properties of various SGLT2 inhibitors were convincingly proven in this investigation. Which lends credence to the processes of sodium and glucose channel blockage, which are implicated in the manifestation of behaviours other than diabetes. Further it opens up a new avenue for investigating other SGLT2 inhibitors for the aforementioned activities and helps in discovering alternative therapeutic uses for this class of drugs apart from its approved Pharmacological uses. This also enables the avoidance and restriction of the use of various medications for diverse pharmacological actions.

ACKNOWLEDGEMENTS:

Authors thank everyone those who supported for the conduction and success of the work

CONFLICT OF INTEREST:

Authors declare no conflict of interest that could affect the research.

FUNDING SUPPORT:

Authors did not receive any funding support for the conduction of the research work.

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Received on 16.06.2021 Modified on 11.10.2021

Research J. Pharm. and Tech 2022; 15(10):4457-4462.

DOI: 10.52711/0974-360X.2022.00747