Effect of spirulina and chlorella alone and combined on the healing process of diabetic wounds: an experimental model of diabetic rats

Abstract

Background

Using chemical agents to cure diabetes mellitus and its complications may be accompanied by complications. New natural agents, such as spirulina and chlorella, could be used as alternative choices in this case.

Methods

65 male Wistar rats were allocated to 5 groups: A (healthy control), B (diabetic rats with a normal diet), C (diabetic rats supplemented with 50 g/kg/day spirulina), D (diabetic rats supplemented with 50 g/kg/day chlorella) and E (diabetic rats supplemented with 25 g/kg/day chlorella and 25 g/kg/day spirulina). After 21 days, wounds were inflicted on the back of rats. Assessment of blood sugar (BS), high-sensitivity C-reactive protein (hs-CRP), vascular endothelial growth factor (VEGF), granulation tissue formation, vascularization, epithelialization, and percentage of wound healing were determined along with macroscopic examinations.

Results

The microscopic changes at days 3, 7, 14, and 21 showed significant evidence of improved angiogenesis, epithelial proliferation, and granulation tissue formation in the spirulina and chlorella treated rats compared with the controls (p˂0.05). Both spirulina and chlorella treatments of diabetic rats resulted in a significant reduction in BS and weight (p˂0.05), but VEGF and hs-CRP levels did not significantly change (p > 0.05). Percentage of wound healing was 100% on day 21 in all groups, except the control group B (97.8 ± 1.15%).

Conclusions

The results of this study showed that supplementation with spirulina and chlorella alone and combined could improve wound healing indices in diabetic rats and could therefore be recommended for the management of diabetic ulcer.

Introduction

Diabetes is a common and costly disease that annually puts many people in danger of cardiovascular disease, blindness, chronic ulcer, kidney disease, and other comorbidities [1]. International Diabetes Federation’s (IDF) estimates indicate that >96,000 new cases of diabetes are diagnosed globally per year [2]. Chronic ulcers are one of the most important long-term complications of diabetes [3]. They are defined as spontaneous or traumatic lesions usually induced in the foot and end limbs which can lead to serious infection, amputation, and death [4]. Several factors cause or delay the healing process of diabetic wounds, including impaired regulation of inflammatory and angiogenic responses, formation of glycation products, and peripheral neuropathy [5]. Increased expression of proinflammatory cytokines such as high-sensitivity C-reactive protein (hs-CRP), elevated blood sugar (BS), decreased expression of endothelial nitric oxide synthase (eNOS) activity, and suppression of vascular endothelial growth factor (VEGF) and hypoxia inducible factor (HIF) cause endothelial dysfunction and delayed wound healing during proliferative phase in diabetes [6, 7].

Recently, new therapeutic approaches have been introduced to accelerate the process of diabetic wound healing. There is evidence that supplementation with Spirulina and Chlorella algae may be effective in this matter [8, 9].

Spirulina, a blue-green alga and a member of the cyanobacterial family, is rich in bioactive compounds such as proteins, vitamins and vitamin precursors such as carotenoids, and linoleic acid [8, 10]. Chlorella is also a green alga that contains protein, fiber, essential fatty acids, and a full range of vitamins, minerals and phytonutrients [11]. These algae have recently been considered not only as a nutritional compound, but also as a bioactive source with various therapeutic effects on diabetes. Evidence suggests that spirulina and chlorella can ameliorate insulin sensitivity, increase secretion of insulin from pancreatic β cells, and reduce the level of blood glucose [11, 12]. They also exert anti-inflammatory properties due to their antioxidant and vitamin content [13, 14].

There is evidence that the flavonoids in spirulina increase the survival of collagen fibrils by enhancing DNA synthesis and stimulating keratinocytes and hence, they enhance wound healing processes [15]. Similar effects have been observed following the administration of chlorella in the cell culture. Chlorella increased the expression of proteins involved in the connections of the skin layers, including collagen and laminin in fibroblasts and keratinocytes [16]. It could also prevent the breakdown of collagen and elastin by inhibiting metalloproteinase enzymes [17].

Given the lack of sufficient evidence on the efficacy of spirulina and chlorella alone and in combination in healing diabetic wounds, this study aimed to evaluate the effect of these algae on the recovery process of diabetic wounds in an animal model.

Materials and methods

Animals

This experimental study was performed on 65 male Wistar rats weighing 200 ± 20 g and 3 to 4 months of age. They were purchased from the Center of Experimental Studies (Baqiyatallah University of Medical Sciences, Tehran, Iran) and brought to the Center for Experimental Studies of the Institute of Nutritional Research and Food Industry, Shahid Beheshti University of Medical Sciences, and housed in standard cages for rodent (1 rat in a cage). Animals were kept and treated in accordance with internationally accepted ethical guidelines for the care of laboratory Animals. They were kept under the stable condition (20–24 °C, 50 ± 5% humidity and 12 h light/ 12 h dark). Sufficient water and food were made available to the animals for one week during acclimatization without any restrictions. This study was approved by the ethics committee of the Iran University of Medical Sciences (IR.IUMS.FMD.REC.1396.9411468010).

Induction of diabetes

To prepare citrate buffer (PH = 4.5), 47 ml of citric acid monohydrate solution was slowly added to 50 ml of 3-sodium dihydrate solution. The prepared solution was autoclaved and then stored in the refrigerator. Streptozotocin (STZ) was purchased from Sigma-Aldrich (USA). 60 mg/kg STZ + 1 cc citrate buffer were injected to rats in order to induced diabetes by destroying β cells in pancreas. Rats with blood glucose above 250 mg/ dl after 72 h of STZ injection were considered diabetic.

Preparation of diets

To make a 5% spirulina diet, 50 g of spirulina in 1000 g of standard chow, to make a 5% chlorella diet, 50 g of chlorella in 1000 g of standard chow, and to make a diet of 5% spirulina and chlorella in combination, 25 g of spirulina and 25 g of chlorella in 1000 g of standard chow was prepared. The mixture was then homogenized in distilled water, dried, and sliced into pellets. Spirulina and Chlorella were manufactured by Fe Ni Company, Taiwan (manufacturer of green algae) in form of powder. The prescribed dose was determined according to previous studies which showed that 5% of spirulina significantly improved the glycemic index in diabetic rats, while diarrhea and growth retardation were observed in rats taking higher doses [18, 19]. The control group received regular chow in the form of pellets as well. Food intake and body weights were measured and recorded weekly throughout the study. The amount of spirulina and chlorella taken by the rats was calculated by weighing the remaining amount of food in their cages.

Experimental procedure

After acclimation, animals were assigned to one of five groups for 1 month before the induction of wounds and 21 days after that: group A) healthy rats that received normal diet, group B) diabetic rats who received normal diet, group C) diabetic rats supplemented with 25 g/kg of the diet/day of spirulina, group D) diabetic rats supplemented with 25 g/kg of the diet/day of chlorella, and group E) diabetic rats receiving 50 g/kg of the diet/day spirulina and chlorella. Stratified randomization and allocation of rats into the study groups were performed using the codes derived from EDA website (https://eda.nc3rs.org.uk/). The allocation sequence was concealed from the researcher assigning the animals to intervention groups, until the moment of assignment. Lab technicians who administered the treatment to the rats and took care of the animals afterwards, were blinded to the treatment each animal had received.

Wound induction

Each rat was anesthetized with an intramuscular injection of 2% xylazine at a dose of 5 mg/kg body weight and 5% ketamine at a dose of 35 mg/kg via peritoneum. Following anesthesia, the dorsum (back) of each rat was prepared for aseptic surgery by shaving and sterilization. Skin wounds were induced by an 8 mm puncture in the vicinity of the spine, the skin was completely removed so that the depth of the wound included the dermis and epidermis. The day of wound induction was considered as day 0. The rats were given analgesics after wound induction to minimize their pain and distress.

Biochemical assessment

Biochemical assessment was performed using the peripheral blood and the blood taken from the heart of the animals on day 21. After taking the blood samples, they were incubated for 2 h at room temperature, and then centrifuged for 15 min at 1500 round/ min. Serum was stored in refrigerator at −80 °C until testing. The blood concentration of glucose was measured by glucometer (Glucocard 01-mini, Arkray Factory, Inc., Japan). VEGF and hs-CRP were measured using ELISA method (Zellbio, Germany).

Wound healing assessment

Histomorphologic parameters including granulation tissue, vascularization, and epithelialization were estimated and scored, as described in Table 1, using the semi-quantitative four-point scale scoring system [20]. Macroscopic examination of wound surface was performed on days 0, 3, 7, 14 and 21 using Digimazer software. A digital camera mounted at a certain distance from the wound (maintaining the same distance for all rats) took photographs of the wound’s surface, which were further transferred to a computer to measure the area of ​​the wound.

$$\%\text{of wound healing}=\frac{\mathit{\text{wound area}}\mathit{on}\mathit{day}\left(\mathit{\text{zero}}\right)-\mathit{\text{wound area in}}\mathit{day}\left(n\right)}{\mathit{\text{wound}}a\mathrm{r}\mathit{ea}\mathit{on}\mathit{day}\left(\mathit{\text{zero}}\right)}x100$$

Table 1.

Scoring of histomorphologic parameters

Score
	0	1	2	3	4
Epithelialization	Lack of thickening of the edges and proliferation of barbed cells	Observing proliferation of barbed cells, prominent integumentary tissue at the edges of the wound	migration of barbed cells on the wound	Complete bridging of barbed cells on the wound	Observing creatine on the wound
Vascularization	Observing no blood vessels	Observing 4–8 vein cross sections	Observing 12–15 vein cross sections	Observing 15–20 vein cross sections	Observing more than 20 vein cross sections
Granulation tissue formation	Observing 70% exudative inflammation	More than 60% exudative inflammation with the tissue	More than 40% granulation tissue	Extensive granulation tissue along with the formation of collagen filaments and vessels perpendicular to them	More than 80% of fleshy granulation tissue

For microscopic examination of the wound, 3 rats were randomly selected from each group on days 3, 7, 14 and 21. Excisional biopsies of the rats’ wounds were obtained following animals’ anesthesia using ketamine (70 mg/kg) and xylazine (15 mg/kg) injections. The entire wound region was excised in-depth with liberal margins of the surrounding skin and the underlying connective tissues above the external fascia of the dorsal muscles and fixed in 10% formalin solution. The tissue sections were then prepared as slides and stained with haematoxylin and eosin stain and Masson’s trichrome methods. The slides were then evaluated by an expert pathologist.

Statistical methods

Statistical analysis was performed using SPSS software version 22. One-way ANOVA was used to investigate the mean difference of granulation tissue, vascularization, epithelialization, wound healing percentage, hs-CRP, VEGF, BS, and weight. Post-hoc Tukey test was used for two-by-two comparisons. Data are represented as mean ± standard deviation (SD) and p˂ 0.05 is considered significant.

Results

Wound healing indices

Figure 1 summarizes the changes of wound healing indices, and Fig. 3 represents the surface of the wounds on the day 7th of the study period in the 5 studied groups. The results of this study showed that granulation tissue formation in groups treated with Spirulina and Chlorella alone and combined (C, D, and E) happened more than the diabetic control group (B) on days 3, 7, 14, and 21 (p˂0.05). To determine which groups were significantly different from each other, the post-hoc Tukey test was performed (data not represented in the tables). It showed that a better granulation tissue formation occurred in the chlorella group (D) in comparison to the diabetic control group (B) on day 21 (p = 0.03). A similar supremacy was observed between the combined spirulina and chlorella group (E) and control group B on days 14 (p = 0.02) and 21 (p = 0.003) (Table 2).

Fig. 1.

Mean score of wound healing indices. A: healthy control, B: diabetic rats with a normal diet, C: diabetic rats supplemented with 50 g/kg/day spirulina, D: diabetic rats supplemented with 50 g/kg/day chlorella, and E: diabetic rats supplemented with 25 g/kg/day chlorella and 25 g/kg/day spirulina

Fig. 3.

Comparison of granulation tissue, vascularization, and epithelialization on day 7 of the study. Photomicrographs showing histologic section of all the groups on day 7 showing the wound healing and epithelialization across the groups. a creatine with more than 20 vein cross sections are observed. Extensive granulation tissue along with the formation of collagen filaments and vessels perpendicular to the collagen filaments in the healthy control group. b Proliferation of barbed cells, prominent integumentary tissue at the edges of the wound is observed with more than 60% exudative inflammation and 15 to 20 vein cross sections in the diabetic control group. c Complete bridging of barbed cells on the wound with more than 20 vein cross sections and more than 40% of granulation tissue formation in the group receiving spirulina. d More than 40% of granulation tissue formation with complete bridging of barbed cells on the wound and more than 20 vein cross sections in the chlorella receiving group. e Complete bridging of barbed cells on the wound with more than 20 vein cross sections along with extensive granulation tissue formation, formation of collagen and vascular filaments perpendicular to the collagen filaments in the recipients of spirulina and chlorella in combination

Table 2.

Changes of wound healing indices during the study period

Variable	Groups	Day3	Day 7	Day 14	Day 21
granulation tissue formation	A	2	3.33 ± 0.33	4.66 ± 0.33	5
	B	0.66 ± 0.33	1.33 ± 0.33	2.66 ± 0.33	4
	C	1.66 ± 0.33	3	4	4.75 ± 0.25
	D	1.33 ± 0.33	2.66 ± 0.33	3.66 ± 0.33	4.75 ± 0.25
	E	2	2.66 ± 0.33	4	5
	P value	0.022*	0.008*	0.004*	0.003*
vascularization	A	2.66 ± 0.33	4	4.66 ± 0.33	5
	B	1	2.33 ± 0.33	3	4.5 ± 0.58
	C	2.66 ± 0.33	4	4	5
	D	2.66 ± 0.33	3.66 ± 0.33	4	5
	E	2.33 ± 0.33	4	4.33 ± 0.33	5
	P value	0.011*	0.001*	0.003*	0.053
epithelialization	A	2.33 ± 0.33	3.66 ± 0.33	4.66 ± 0.33	5
	B	0.66 ± 0.33	1.66 ± 0.33	2.66 ± 0.33	4.5 ± 2.28
	C	2	3.33 ± 0.33	4.66 ± 0.33	5
	D	1.66 ± 0.33	3.33 ± 0.33	4.33 ± 0.33	5
	E	2	3	4.66 ± 0.33	5
	P value	0.009*	0.006*	0.007*	0.053
Percentage of wound healing (%)	A	25 ± 0.98	66.043 ± 3.9	98.17 ± 0.18	100
	B	18.62 ± 1.01	42.82 ± 2.52	85.03 ± 3.02	97.8 ± 1.15
	C	22.16 ± 1.19	59.34 ± 2.43	95.54 ± 1.2	100
	D	21.66 ± 1.03	60.81 ± 2.34	93.71 ± 1.54	100
	E	22.03 ± 0.982	58.17 ± 3.9	94.75 ± 2.06	100
	P value	0.021*	0.004*	0.005*	0.001*

A: healthy control, B: diabetic rats with a normal diet, C: diabetic rats supplemented with 50 g/kg/day spirulina, D: diabetic rats supplemented with 50 g/kg/day chlorella, and E: diabetic rats supplemented with 25 g/kg/day chlorella and 25 g/kg/day spirulina

*p value˂0.05, significant

Data presented as mean ± SD, One-way ANOVA

Vascularization score was also significantly different between the studied groups. Tuckey test showed higher vascularization score for the spirulina and chlorella alone and combined (C, D, and E) in comparison to diabetic controls (B) on days 3, 7 and 14 (p < 0.05). Group A could also increase vascularization more successfully than the control group B on days 3 (p = 0.01) and 7 (p = 0.008).

There was a significant difference between the studied groups regarding the mean epithelialization score (p < 0.05). Diabetic recipients of regular diet (B) showed a lower epithelialization score in comparison with groups treated with Spirulina and Chlorella alone and combined (C, D, E) on days 7 and 14 r (p < 0.05). The epithelialization also increased with a slower slope in group B in comparison to control group A on days 7 (p = 0.05) and 21 (p = 0.01).

Wound healing had a growing trend throughout the study period in all groups. Spirulina (C), combined spirulina and chlorella (E), and chlorella (D) had the greatest effect on the process of wound healing, respectively. The wounds were 100% healed on the day 21 in all groups, except for diabetic control group (B), in which ˷ 97% recovery was achieved on day 21.

Weight and biochemical indices

Table 3 and Fig. 2 show the changes in weight and biochemical indices. A significant weight reduction was observed in all groups throughout the study period (p < 0.05). two-by-two comparisons by Tukey test showed that the diabetic control group receiving normal diet (B) had the highest weight reduction compared with groups receiving spirulina (C) and chlorella (D) alone or in combination (E) (p < 0.05). Also, the blood sugar significantly reduced over time in all groups, with the highest reduction being observed in combined chlorella and spirulina (E) (mean change: 40.5 ± 39.56) and chlorella group (D) (mean change: 36.33 ± 28.71). Tukey test revealed a significant difference between the control group B and groups C, D, and E in terms of blood sugar on days 7, 14 and 21 (Fig. 3).

Table 3.

Changes in weight, BS, hs-CRP and VEGF during the study period

Variable	Groups	Day 3	Day 7	Day 14	Day 21
Weight (g)	A	277 ± 3.78	279 ± 6.80	299.66 ± 8.45	315.25 ± 14.41
	B	276 ± 12.5	227.66 ± 19.96	212 ± 3.78	221 ± 7.61
	C	252.33 ± 17.83	255.66 ± 17.47	261.66 ± 15.38	269.5 ± 4.29
	D	244 ± 3.21	249.33 ± 3.84	258.33 ± 3.17	275.5 ± 5.78
	E	241.66 ± 8.81	256.33 ± 7.12	267.66 ± 17.13	274 ± 5
	P value	0.104	0.161	0.004*	0.001*
hs-CRP (mg/L)	A	10.33 ± 0.371	7.93 ± 0.48	9.7 ± 1.17	8.45 ± 0.81
	B	12.36 ± 0.82	11.2 ± 1.21	10.23 ± 0.79	10.82 ± 0.56
	C	10.9 ± 0.50	10.6 ± 1.03	10.26 ± 1.16	9.55 ± 0.57
	D	12.13 ± 1.58	10.7 ± 0.86	10.23 ± 0.68	10.15 ± 0.89
	E	11.53 ± 0.93	9.5 ± 0.25	10.03 ± 0.29	9.25 ± 0.15
	P value	0.551	0.120	0.989	0.162
VEGF (pg/ml)	A	225.23 ± 6.70	218.93 ± 2.22	192.06 ± 7.2	193.22 ± 8.64
	B	199 ± 11.20	200.70 ± 6.40	181.56 ± 10.1	191.47 ± 5.18
	C	241.4 ± 9.27	221.5 ± 13.11	193.16 ± 7.65	202.3 ± 5.93
	D	235.56 ± 36.11	218.6 ± 8.61	225.66 ± 14.56	205.9 ± 12.27
	E	207.56 ± 9.82	201.96 ± 8.34	183.56 ± 6.52	192.17 ± 7.1
	P value	0.458	0.298	0.054	0.636
Blood Sugar (mg/dl)	A	88.66 ± 2.6	80.66 ± 1.85	86.33 ± 2.60	83.5 ± 3.66
	B	449.66 ± 28.42	451.66 ± 27.53	463 ± 35.02	405.5 ± 17.33
	C	352 ± 32.04	367 ± 31.62	320.66 ± 27.87	329.5 ± 5.48
	D	378.33 ± 48.31	346.66 ± 19.20	334.66 ± 28.60	342 ± 9.42
	E	367 ± 50.06	351.33 ± 8.37	325.33 ± 7.12	326.5 ± 10.5
	P value	0.001*	0.001*	0.001*	0.001*

A: healthy control, B: diabetic rats with a normal diet, C: diabetic rats supplemented with 50 g/kg/day spirulina, D: diabetic rats supplemented with 50 g/kg/day chlorella, and E: diabetic rats supplemented with 25 g/kg/day chlorella and 25 g/kg/day spirulina

*p value˂0.05, significant

hs-CRP high-sensitivity C-reactive protein, VEGF vascular endothelial growth factor

Data presented as mean ± SD, One-way ANOVA

Fig. 2.

Mean weight, BS, hs-CRP and VEGF. A: healthy control, B: diabetic rats with a normal diet, C: diabetic rats supplemented with 50 g/kg/day spirulina, D: diabetic rats supplemented with 50 g/kg/day chlorella, and E: diabetic rats supplemented with 25 g/kg/day chlorella and 25 g/kg/day spirulina

Although a declining trend was observed for hs-CRP and VEGF in all the 5 groups, there were no significant differences between the studied groups on days 0, 7, 14, and 21 (p > 0.05). Hence, intervention with Spirulina and Chlorella alone and in combination could not effectively lower hs-CRP and VEGF levels in rats.

Discussion

In this study we investigated the effect of spirulina and chlorella alone and combined on the healing process of diabetic wounds in rats. It was found that the combination of spirulina and chlorella was more effective than spirulina or chlorella alone in augmenting granulation tissue formation, vascularization, and epithelialization of the diabetic wound, and percentage of wound healing. The algae could also significantly reduce weight and blood sugar in rats, yet the level of hs-CRP and VEGF did not significantly change across the groups during the study period.

Wound healing process occurred significantly faster in groups A, C, D, and E than in diabetic controls (B), which is consistent with the results of previous research. Canan et al. assessed the anti-ulcerogenic activity of phycobiliproteins in spirulina. They showed that spirulina extract stimulates the proliferation and growth of keratinocytes in the vitro, which may enhance the process of wound healing [21]. Kim et al. found that 10–1000 mg/g body weight of spirulina ameliorated healing process of wounds and tissue regeneration in rats [22]. Hidalgo-Lucas et al. also reported that oral administration of 250–500 mg/kg chlorella for 14 days had significant positive effects on the wound healing process [23].

There are plenty of mechanisms, through which chlorella and spirulina could boost the wound healing process in the studied diabetic rats. Both of the mentioned algae are good sources of flavonoids and antioxidants. Flavonoids not only reduce lipid peroxidation, but also increase the survival of collagen fibrils by enhancing DNA synthesis [24]. Flavonoids also can promote wound healing process, due to their antibacterial properties [25, 26]. Besides, plenty of antioxidants in chlorella and spirulina can suppress the inflammatory pathways involved in induction and development of wounds [13, 14, 27]. Chlorella and spirulina are also great sources of necessary fatty acids too. It has been proven that gamma linoleic acid, alfa linolenic acid, and eicosapentaenoic acid (EPA) increase the migration of cells towards the wounded area and can improve epithelialization [28]. In addition, fatty acids and their derivatives enhance wound healing through angiogenic activity [28]. Chlorella has been reported to inhibit the matrix of metalloproteinase in skin cells, and indirectly prevent the breakdown of collagen and elastin [17]. It also prevents the reduction of flexibility of collagen and elastin [29]. The same protective effect has been observed for spirulina as well [21]. Hence, spirulina and chlorella can enhance wound healing by their anti-ulcerogenic activity.

Our results also showed that the mean blood glucose level significantly reduced on days 14 and 21. Senthilkumar et al. showed that administration of 100 mg/ kg chlorella for 90 days in STZ-induced diabetic rats caused a significant decrease in plasma glucose and elevated plasma insulin levels [11]. Gargouri et al. also showed that receiving a 5% spirulina-enriched diet for 21 days could reduce serum glucose level [30]. Spirulina and Chlorella alone and in combination exerted antihyperglycemic effect, which has been reported by previous researches as well [11, 18]. It is suggested that cyanobacteria act like insulin or stimulate the β cells of islets of Langerhans to increase the output of insulin, and therefore, lowers blood sugar level [12]. Chlorella has also been reported to enhance glucose uptake in the liver and skeletal muscles [31], and besides its ability to increase insulin sensitivity, chlorella might also induce regeneration of insulin secreting from β cells, that results in increased levels of insulin [32].

The effect of chlorella and spirulina on insulin secretion, in addition to their role in improving lipid metabolism justify the reduction of weight observed in groups C, D and E. Diabetes mellitus may be accompanied by significant increase in triglyceride (TG) levels, which leads to lipid breakdown and obesity [33, 34]. Evidence suggests that consumption of spirulina as a dietary supplement prevents hypercholesterolemia due to the large amount of cysteine found in the Cphycocyanin protein in spirulina [35]. Some authors suggest that the addition of this alga into the diet diminishes the intestinal absorption of cholesterol as well as the re-absorption of bile acids in the ileum. Thus, they suggest that spirulina can be considered a functional food capable of reducing weight [18, 36]. Chlorella is also shown to reduce TG levels by inhibiting hepatic fatty acid synthesis [37].

Our results showed no significant effect of spirulina and chlorella on hs-CRP and VEGF, which could indicate that these algae could improve vascularization and wound healing independently of anti-inflammatory pathways or VEGF. Our results are inconsistent with that of Yousefi et al. who reported that consumption of 2 g/day of spirulina for 12 weeks in overweight and obese people could significantly reduce hs-CRP levels [38]. Although the result of this study is not comparable to that of ours, for it being a human study and different dosage and duration of treatment with spirulina. On the other hand, Pang et al. reported that using 2.50 g/ kg flavonoid extract of Blumea balsamifera locally for 10 days on wounded skin in rats, increased the expression of VEGF gene [39]. This inconsistent result could be due to different flavonoid content of chlorella and spirulina in comparison to Blumea balsamifera. As there is not enough studies in this area, more investigations are needed to assess the effect of algae on VEGF and inflammatory markers.

It should be noted that keeping rats in separate cages and feeding them with spirulina and chlorella orally (a non-invasive method) instead of force-feeding methods such as gavage, were the strengths of the current study. Among the limitations of this study was budget limitation due to which we were unable to assess other relevant parameters such as insulin and inflammatory cytokines. Another limitation of this study could be the small sample size. After injection of STZ, some of the rats did not develop diabetes and some died, therefore, they had to be removed from the study.

Conclusion

Taken together, feeding diabetic rats with spirulina, chlorella and a combination of the two may have some beneficial impact on wound healing as indicated by the improvement in granulation tissue formation, vascularization, and epithelialization. Spirulina and chlorella could be recommended for the treatment of various types of wounds in humans and animals especially in low-income communities where these algae are affordable and available. Further studies are recommended to find out the potential anti-diabetic benefits of the studied algae in human population.

Authors’ contribution

Naeim Mehdinezhad, Naheed Aryaeian, and Mohammadreza Vafa designed this research, Atoosa Saeedpour and Naeim Mehdinezhad conducted research, Reza Fahimi provided essential materials and visualization, Abdolali Ebrahimi and Tofigh Mobaderi performed statistical analysis, Zohreh Sajadi Hezaveh did the original draft preparation, and Naheed Aryaeian had primary responsibility for final content. All authors have revised and approved the final manuscript.

Compliance with ethical standards

Competing interests

None.