Research Article |
Corresponding author: Monir Doudi ( doudimonir@yahoo.com ) Academic editor: Mikhail Korokin
© 2022 Soheila Rajaei, Monir Doudi, Mahbubeh Setorki, Ali Mohammad Ahadi.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Rajaei S, Doudi M, Setorki M, Ahadi AM (2022) Clinical and histopathological effects of ointment prepared from kombucha floating cellulose layer on wound healing and the activity of matrix metalloproteinase 1 in diabetic rats. Research Results in Pharmacology 8(3): 9-18. https://doi.org/10.3897/rrpharmacology.8.81288
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Introduction: High blood glucose results in high levels of matrix metalloproteinases. Clinical and histopathological effects of the kombucha ointment on the healing of diabetic wounds were evaluated.
Materials and methods: This study was conducted at research Lab, Department of Micobiology, Falavarjan of Branch Islamic Azad University, Isfahan, Iran from October 2019 to September 2020. A 6 mm diameter ulcer was aseptically created on the back of forty-eight rats with streptozotocin-induced diabetes. The animals were randomly divided into 4 groups: the group that was treated with base ointment, the group that was treated with 10% kombucha ointment, the group that was treated with 20% kombucha ointment, and the group that received no ointment treatment. Then the rats in each group were divided to 4 sampling groups that were sampled on the second, fifth, tenth, and fourteenth days. Microscopic features, inflammation and vasculature and fibroblast infiltration, as well as the matrix metalloproteinase 1(MMP1) were evaluated on days 2, 5, 10, 14 after wound healing.
Results: 20% kombucha ointment let to inflammation and an angiogenesis decrease compared to those in the basic group and 10%-kombucha-ointment group. Also 20% kombucha ointment led to an increase in vascular remodeling and penetration of fibroblasts. MMP1 levels increased on the second (p < 0.001) and fifth days after wounding when treatrd with 10% and 20% kombucha ointment (p > 0.05). The expression of MMP1 decreased on the ten and fourteenth days when using 20% kombucha ointment compared to the control, placebo, and 10% kombucha ointment groups (p > 0.05).
Discussion: The histopatological finding indicated that both quantity and time duration of the treatment had significant effects on a degree of inflammation and angiogenesis.
Сonclusion: Ointment prepared from 20% scoby improved the healing of diabetic ulcers within 14 days.
kombucha, matrix metalloproteinase, ointment
Skin wounds caused by various disorders, such as burns and special diseases like diabetes, as well as severe skin damage can be life threatening (
The protocol of the study was approved by the Ethics Committee of Flavarjan University Branch Islamic Azad University (IR.IAU.FALA.REC.1397.036).
In order to prepare 10% and 20% ointments, kombucha cellulose layers equal to 10 g and 20 g of its dry mass were added to 80 g and 90 g of pre-prepared base ointment, respectively. The base ointment was prepared from a combination of deionized water, paraffin, stearic acid, propylene glycol, glycerol, vaseline, cetyl alcohol, glycerolmonostearate, triethanolamine, bee wax, methyl paraben and propyl paraben (
48 adult Wistar rats weighing (150 g to 250 g) were used in this study. The animals were held on 12 hours light/dark cycle. Water and food were available all the time, and the animals were assigned into diabetic groups. Diabetes was induced by a single intraperitoneal injection of 50 mg/kg b.w. of streptozotocin (Sigma-Aldrich, St. Louis, MO, USA). A week after the induction, a glucose level was assessedThe blood-glucose level was >300 mg/dl in all STZ injected animals (
On the day of surgery (day zero), the rats were anesthetized with ether. The hair on the back of the body was shaved with a sterile surgical blade and disinfected. Subsequently, using a 6 mm punch, wounds with 6 mm in diameter were created between the ducts at the back of the animals. Then the rats were randomly divided into 4 groups of 12, including a control group, the treated groups that received 10% and 20% kombucha ointments, respectively, and a basic group. Each group was divided into 4 groups of 3 rats each, which were sampling groups on days 2, 5, 10, and 14 (
Angiogenesis, fibroplasias, inflammatory cell infiltration were evaluated in H&E-stained sections and scored semi quantitatively as follows: no or minimal angiogenesis, fibroplasias, inflammatory cells (1); mild angiogenesis, fibroplasias, inflammatory cells (2); inflammatory cells form extent and moderate to angiogenesis, fibroplasias (3); inflammatory cells form extent and severity to sever angiogenesis, fibroplasias (4) (
On days 2, 5, 10, and 14 after wound healing, tissues were kept in normal saline for at least 1–2 hrs. Then the tissues were washed several times with normal saline to remove excess material and kept at -80 °C in normal saline with the volumes 4 times of tissue weight. For assessment of MMP1 activity, the collected tissues were removed from -80 °C and suspended for 15 minutes using a homogenizer. Finally, each tissue was centrifuged at 250 rpm for 15 minutes, and the supernatant was collected. The MMP1 levels were measured using the ELISA kit (HANGZHOU EASTBIOPHARM), according to the manufacturer proposed method (
The data were analyzed using the One-way analysis of variance (ANOVA) to determine the significant differences between the groups, and Dunnett’s multiple comparison test as well as Tukey’s post hoc test was used to compare the means. The data analyses were performed using Statistical Package for Social Sciences (SPSS) software version 16 at a P value equal to or less than 0.05.
Statistical assessment of matrix metalloproteinase 1 expression in diabetic rats
The expression levels of MMP1 were compared among the experimental groups on day 2 after wounding. The groups included: base ointment receiving diabetic group (control group), no ointment diabetic group (basic), diabetic treatment group receiving 10% kombucha ointment, and diabetic treatment group receiving 20% kombucha ointment. The expression of MMP1 was significantly increased by using 10% and 20% kombucha ointments when compared to the control and basic groups on day 2 (p < 0.001) (Fig.
Comparison of matrix metalloproteinase (MMP1) levels between different experimental groups on the second day after wounding. The expression level of MMP1 was significantly increased by using 10% and 20% kombucha ointments when compared to control and basic groups (### and *** indicate p < 0.001).
The expression of MMP1 was increased by using 10% kombucha ointments when compared to the basic group and decreased when compared to the control group. Also, the expression of MMP1 was increased by using 20% kombucha ointments when compared to other groups, but no significant difference was observed between all the different experimental groups on day 5 (p > 0.05) (Fig.
Comparison of matrix metalloproteinase (MMP1) levels between different experimental groups on day 5 after wounding. Also the expression of MMP1 was decreased by using 10% kombucha ointments and was increased by using 20% kombucha ointments when compared to the control group, no significant differences were seen among the experimental groups (p > 0.05).
The expression of MMP1 was decreased by using 20% kombucha ointment when compared to the basic group and increased by using 10% and 20% kombucha ointments when compared to the control group, but no significant difference was observed between the different experimental groups on day 10 (Fig.
Comparison of matrix metalloproteinase (MMP1) levels between different experimental groups on day 10 after wounding. The expression of MMP1 was increased by using 10% and 20% kombucha ointments when compared to the control group, but no significant differences were seen among the experimental groups (р > 0.05).
The expression levels of MMP1 were different among the experimental groups on the 14th day after wounding. There was a decrease in the expression of MMP1 by using 10% and 20% kombucha ointments when compared to the control and basic groups, but the differences between different experimental groups on the 14th day were not significant (Fig.
According to Table
Changes in effective parameters in skin wound healing in the study groups as well as at different times are presented in Table
Comparison of changes in effective parameters on skin wound healing in the studied animals at different times
Treatment group | Inflammation | Angiogenesis | Fibroplasia |
---|---|---|---|
A: | |||
2nd day | severe | severe | absent |
5th day | moderate | moderate | mild |
10th day | mild | mild | moderate |
14th day | mild | mild | moderate |
B: | |||
2nd day | severe | severe | Absent |
5th day | moderate | moderate | mild |
10th day | mild | mild | moderate |
14th day | absent | absent | severe |
C: | |||
2nd day | severe | severe | absent |
5th day | moderate | moderate | mild |
10th day | mild | mild | moderate |
14th day | mild | mild | moderate |
Z: | |||
2nd day | severe | Severe | absent |
5th day | moderate | moderate | mild |
10th day | moderate | mild | mild |
14th day | mild | mild | moderate |
Microscopic images of the skin cross section with 40× magnification. Row A: Investigation of the inflammatory factor on the 2nd day in all studied groups. Severe inflammation can be seen on the second day in all treatment groups, as well as in groups treated with base ointment, and without the use ointment. Row B: Evaluation of inflammatory factor on the 14th day of wound formation (end of treatment period). As can be seen, a small inflammation is still observed in the 10% ointment treatment group (A). There are no signs of inflammation in 20% ointment treatment group (B). In the group treated with base ointment (C), inflammation is still seen in the tissue samples. As can be seen in the group without the use of ointment (Z), inflammation and angiogenesis of blood vessels are still observed.
Microscopic images of the skin cross section with 10× magnification. Row A: Evaluation of angiogenesis on the 2nd day in all studied groups. As can be seen, in both treatment groups (10% and 20% kombucha ointment), the base ointment group, and the group without the use of ointment, angiogenesis is observed. Row B: Examination of angiogenesis on the 14th day from the wound formation (end of treatment). As can be seen, small angiogenesis is still observed in the group that used 10% kombucha ointment treatment (A). In the group that used 20% kombucha ointment treatment (B), no angiogenesis is observed. In the base group (C), small angiogenesis is still observed. In the group without the use of ointment (Z), angiogenesis is clearly observed.
Microscopic images of the skin cross section with 10× magnification. Row A: Evaluation of fibroplasia on the 2nd day in all studied groups. As can be seen, in both treatment groups (10% and 20% kombucha ointment), the base ointment group, and the group without the use of ointment, no fibroblast cells are observed. Row B: Examination of fibroplasia on the 14th day from the wound formation (end of treatment). As can be seen, fibroblasts are observed in the group that used 10% kombucha ointment treatment (A). In the group that used 20% kombucha ointment treatment (B), the presence of fibroblasts on the tissue surface is quite evident. In the base group (C), slight fibroblasts are observed. In the group without the use of ointment (Z), fibroblasts are not seen. Comparing the above results, it was shown that the function of 10% kombucha drug, the control group and the base ointment treatment group were the same, but by using 20% kombucha treatment, an increase in spindle-shaped cells and fibroblasts was observed.
Microscopic examination of angiogenesis
See Fig.
See Fig.
Following wound occurrence, the planned healing process begins and eventually results in the cellular and molecular status of the damaged tissue returning to its normal state. Enzymatic activity in the wound area has an important role in cellular interactions, tissue vascular remodeling as well as the formation or final arrangement of collagen fibers to restore the normal skin structure. The most important of these enzymes are from the matrix metalloproteinases family, which are classified into four groups of collagenases, gelatinases, stromelysins, and metalloenzymes based on their specific substrate and structural similarity (
In the wound healing process, collagenase 1 breaks down collagen types 1 and 2, which are the major matrix of the epidermis. This enzyme, which is secreted by skin fibroblasts, is turned on or off during different stages of wound healing (
Traditional medicine has used kombucha to treat many diseases, including different kinds of wounds. Kombucha consists of a floating cellulose layer and a sour taste solution under it (
In addition to cellulose, Acetobacter xylinum is capable of producing other polysaccharides, such as N-acetylglucosamine containing polymers. These polysaccharides are decomposed by lysozyme in body fluids, producing N-acetyl glucosamine that accelerates the healing process (Lahiri et al. 2021). In addition, N-acetyl glucoseamine, along with glucuronic acid, produce one of the hyaluronic acid - forming disaccharides found in the cartilage gland tissue. In this regard, Mghsoudi et al. (2009) showed the effect of kombucha in removing intraperitoneal adhesions after surgery and attributed it to oxidized restored cellulose and hyaluronic acid membranes (Mghsoudi et al. 2009). In another study,
According to the results of this study, it is concluded that ointment prepared from kombucha cellulose floating disk was able to improve the healing process of diabetes wounds, as well as the induction of matrix metalloproteinases expression, which are some of the effective factors in wound healing histopathological process. Therefore, kombucha nanocellulose layer is proposed for the production in scaffold areas, such as the production of dental implants, vascular grafts, catheter coatings, cardiovascular and cranial stents coatings, and controlled drug delivery carriers. Also the ointment prepared from kombucha floating cellulose layer) scoby (is proposed for manufacturing medicinal dressings.
The authors declare no conflict of interests.
Soheila Rajaei, PhD Candidate of the Department of Microbiology, e-mail: et.mars72@yahoo.com, ORCID ID http://orcid.org/0000-0002-2621-829X. Planned and conducted the experiment, conceived and designed the analysis, and wrote the paper.
Monir Doudi, Assistant Professor of the Department of Microbiology, e-mail: doudimonir@yahoo.com, ORCID ID http://orcid.org/0000-0002-0895-1586. Conceived and designed the analysis, performed the analysis, and supervised the project.
Mahbubeh Setorki, Assistant Professor of the Department of Biology, e-mail: doctor.setorgi@gmail.com, ORCID ID http://orcid.org/0000-0001-6983-9929. Carried out the experiment, collected the data, and contributed the data and analysis tools.
Ali Mohammad Ahadi, Assistant Professor of the Department of Genetics, e-mail: ahadi52@gmail.com, ORCID ID https://orcid.org/0000-0001-9580-2740. Provided a critical analysis of study design, results and the text of the final manuscript.