Glycyrrhizic acid ameliorates submandibular gland oxidative stress, autophagy and vascular dysfunction in rat model of type 1 diabetes
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OPEN Glycyrrhizic acid ameliorates
submandibular gland oxidative
stress, autophagy and vascular
dysfunction in rat model of type 1
diabetes
Saad Mohamed Asseri1, Nehal M. Elsherbiny2,3*, Mohamed El‑Sherbiny4,5, Iman O. Sherif6,
Alsamman M. Alsamman7,8, Nadia M. Maysarah9 & Amira M. Elsherbini10*
The burden of diabetes mellitus (DM) and associated complications is increasing worldwide,
affecting many organ functionalities including submandibular glands (SMG). The present study aims
to investigate the potential ameliorative effect of glycyrrhizic acid (GA) on diabetes-induced SMG
damage. Experimental evaluation of GA treatment was conducted on a rat model of type I diabetes.
Animals were assigned to three groups; control, diabetic and GA treated diabetic groups. After
8 weeks, the SMG was processed for assessment of oxidative stress markers, autophagy related
proteins; LC3, Beclin-1 and P62, vascular regulator ET-1, aquaporins (AQPs 1.4 and 5), SIRT1 protein
expressions in addition to LC3 and AQP5 mRNA expressions. Also, parenchymal structures of the
SMG were examined. GA alleviated the diabetes-induced SMG damage via restoring the SMG levels
of oxidative stress markers and ET-1 almost near to the normal levels most probably via regulation
of SIRT1, AQPs and accordingly LC-3, P62 and Beclin-1levels. GA could be a promising candidate
for the treatment of diabetes-induced SMG damage via regulating oxidative stress, autophagy and
angiogenesis.
Diabetes mellitus (DM) is a metabolic disorder characterized by disturbance in insulin secretion and action1.
Subsequent diabetic complications that affect many organs and systems including oral tissue and salivary glands
were reported2. Salivary glands play crucial role in maintaining oral homeostasis depending largely on salivary
secretions3. Parotid gland, sublingual gland, and submandibular gland (SMG) are the major salivary glands
that are responsible for synthesis of saliva, which is an enriched milieu composed mainly of water, electrolytes,
and biologically active proteins, including growth factors and cytokines in addition to transporting water and
electrolytes3. Specifically, SMG is responsible for production of more than 60% saliva and altering its capacity
of secretion will lead to a significant disruption of oral health. It has been reported that during diabetic course,
salivary glands are negatively affected, resulting in reduced salivary secretions, periodontal destruction, salivary
hypofunction and oral mucosal lesions4.
Though controversial, a relationship was established between diabetic complications and salivary dysfunction
in which xerostomia (dry mouth due to reduced salivary flow rate) and polydipsia (pathological thirst) were
prevalent in diabetic subjects due to the reduced ability of the salivary gland to secrete saliva2. A recent study
1
Department of Clinical Medical Sciences, College of Medicine, AlMaarefa University, P.O. Box 71666, Riyadh,
Saudi Arabia. 2Department of Biochemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516,
Egypt. 3Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Tabuk, Tabuk 71491,
Saudi Arabia. 4Department of Basic Medical Sciences, College of Medicine, 11597, AlMaarefa University, Riyadh
P.O. Box 71666, Saudi Arabia. 5Department of Anatomy, Faculty of Medicine, Mansoura University, Mansoura,
Egypt. 6Emergency Hospital, Faculty of Medicine, Mansoura University, Mansoura 35516, Egypt. 7African Genome
Center, Mohammed VI Polytechnic University, Ben Guerir, Morocco. 8Agricultural Genetic Engineering Research
Institute, PO Box 12619, Giza, Egypt. 9Department of Pharmacology and Toxicology, College of Pharmacy, Qassim
University, Buraydah, Saudi Arabia. 10Department of Oral Biology, Faculty of Dentistry, Mansoura University,
Mansoura 35516, Egypt. *email: drnehal@hotmail.com; amiraelsherbini@mans.edu.eg
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showed that diabetes was associated with characteristic histopathological alterations as well as decreased secre-
tion of the SMG without affecting either the sublingual gland or parotid gland3,5.
The pathophysiological mechanism of oral diabetic complications is still not fully elucidated, however the
implication of reactive oxygen species (ROS) generation was documented and resulted in oxidative damage of
the DNA, proteins, and lipids causing cellular d ysfunction6. Moreover, the two processes of oxidative stress and
autophagy were reported to be interrelated. Autophagy is considered as a major survival mechanism implied by
adaptation of the cells to stress as well as a vital cellular consequence to prevent stem cells damage by extrinsic
influences7. Additionally, it is proposed that autophagy may play a crucial role in maintaining irradiated salivary
glands8,9.
The central regulator of autophagy is the microtubule-associated protein 1 light chain 3 (LC3) which is
involved in autophagosomes formation and has been identified as a biomarker of a utophagy10. Interestingly,
silent information regulator of transcription1 (SIRT1), a member of sirtuin family, can deacetylate LC3 to initiate
autophagosomes formation implying a role of SIRT1 in autophagy m odulation11. In aging, SIRT1 is recognized
as autophagy substrate and is degraded in cytoplasmic autophagosome–lysosome, via LC11. On the other hand,
endothelines (ETs) are family of peptides that are composed of 3 identified isoforms and are well-known for their
vasoconstrictive action. ET-1 might be present in many body fluids including saliva, thus the determination of
salivary ET-1 might be a useful tool in examining the oral pathological conditions12. Moreover, SIRT1 overexpres-
sion has been reported to modulate endothelin (ET-1)13 and oxidative stress in diabetic complications14. Based on
these reports, autophagy and SIRT1/ET axis represent valuable targets in diabetes associated oral complications.
Herbal remedies have been used as an effective treatment for chronic diseases, such as diabetes by traditional
medicines or even through advanced pharmacological protocols15. Glycyrrhizic acid (GA) is the major active
constituent of a Chinese herbal medicine Glycyrrhiza glabra and possesses a wide spectrum of pharmacologi-
cal actions as antioxidant, anti-inflammatory, antiviral, anticancer, and antidiabetic activities16. Additionally,
our team has recently reported its ameliorating effect on salivary gland toxicity induced by sodium n itrite17. In
18–20
diabetes, much evidence verified the ameliorative effects of GA on associated c omplications .
Several evaluation protocols should be followed to assess the potentiality of GA in diabetes treatment. Experi-
mental evaluation of GA using various molecular techniques that quantify changes in diabetes-related biological
markers is part of these p rotocols16. Therefore, our goal was to investigate the molecular pathophysiological
mechanisms of SMG damage in diabetic rats, as well as the possible molecular protective mechanism of GA
activity via detecting oxidative stress, SIRT1, autophagy signaling, water channel proteins aquaporins (AQPs)
in addition to ET-1 expressions in SMG tissue.
Results
Histopathological analysis. H&E staining revealed almost normal structure of SMG in control group.
Acinar cells with basal nuclei and interlobular ducts with prominent granular convoluted ducts (GCD) were
found in control group (Fig. 1A). On the other hand, notable atrophy, and degeneration in the diabetic SMG,
including vacuolization of acinar cells, pyknotic nuclei and lysis of entire acini and granular convoluted tubules
were observed (Fig. 1B,C). These histopathological changes were markedly reduced and almost reversed by
the GA treatment (Fig. 1D). Similarly, in semithin sections of diabetic SMG (Fig. 1F), obvious loss of secretory
granules in acinar cells and disruption of acinar and ductal structures were found in addition to vacuolation and
nuclear changes. These changes were reduced by GA application (Fig. 1G) that restored almost normal structure
resembling control group (Fig. 1E).
GA ameliorated oxidative stress damage in diabetic SMG. Diabetic group showed an oxidative
damage of SMG with statistically significant increase in oxidative stress markers MDA (P < 0.001) (Fig. 2A),
and statistically significant reduction in GSH (P < 0.001) and SOD (P < 0.001) compared to the control group
(Fig. 2B,C). GA application significantly attenuated MDA (P < 0.001) and restored GSH and SOD (P < 0.001,
P < 0.05 respectively) compared to the DM group.
GA restored autophagy related markers LC3, P62 and Beclin‑1 levels in diabetic SMG. Com-
pared to control group, diabetic group showed a statistically significant increase in brown dot-like staining, indi-
cating both cytoplasmic and nuclear positive reaction especially in the acini for LC3 (P < 0.0001) (Fig. 3A–D,G).
Moreover, diabetic group showed statistically significant increase of P62, both cytoplasmic and nuclear positive
reaction especially in the ducts, compared to control group (Fig. 3K,J,M) (P < 0.0001). However, GA applica-
tion significantly ameliorated LC3 (Fig. 3E,F,G) and P62 expression compared to diabetic group (P < 0.0001)
(Fig. 3L,M). Similarly, the gene expression of LC3 in diabetic group showed a statistically significant fold
increase (P < 0.0001) compared to control group (Fig. 3H). However, GA application significantly decreased
LC3 gene expression compared to diabetic group (P < 0.0001) (Fig. 3H). Moreover, Quantitative results of ELISA
showed statistically significant increased LC3II and Beclin-1 expression in DM group compared to control group
(P < 0.0001 for both), while GA treated group showed a significant reduction in both LC3II and Beclin-1 com-
pared to DM group (P < 0.001, P < 0.0001 respectively) (Fig. 3I,N).
GA ameliorated SIRT1 expression and its associated vascular marker. There was a nuclear posi-
tive expression of SIRT1 in SMG ducts of both control group and GA treated group (P < 0.0001) (Fig. 4A,C,D),
which was not the case in DM group where there was a statistically significant reduction in SIRT1 expression
(P < 0.0001) (Fig. 4B,D).
Moreover, our results revealed ET-1 expression around interlobular ducts and blood vessel in control group
(Fig. 5A). DM group showed a statistically significant upregulation in ET-1 expression compared to control
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Figure 1. Haematoxylin and eosin-stained SMG showing parenchymatous structure with acini (asterisks) and
interlobular ducts (arrows) with prominent granular convoluted ducts (GCD) in control group (A). Diabetic
group showed vacuolation in both acini and GCD, with stagnation of secretions (curved arrows) (B,C). GA
treated group (D) revealed an ameliorative effect by reversing the diabetes impact with minimal atrophy and
vacuolations. Similarly, in semithin sections of diabetic SMG (F) obvious loss of secretory granules in acinar
cells and disruption of acinar and ductal structures were found in addition to vacuolation and nuclear changes,
the changes were reduced by GA application (G) that restored almost normal structure resembling control
group (E) (H&E, × 20; Semithin sections TB stain, × 20).
group (P < 0.001) (Fig. 5A,B,J), while GA significantly downregulated the ET-1 expression compared to DM
group (P < 0.0001) (Fig. 5C,J). Interestingly, AQP1 showed a similar pattern of expression. DM showed increased
expression of AQP1 (P < 0.0001) compared to control group (Fig. 5D,E,K), while its expression was significantly
downregulated in GA group compared to DM group (P < 0.0001) (Fig. 5F,K). However, the expression of AQP4
showed different pattern, where DM group revealed the least expression (Fig. 5H,L) and both control and GA
treated group revealed a higher expression related to the basal part of acini and blood vessels (P value < 0.001)
(Fig. 5G,I,L).
GA restored AQP5 gene and protein expressions. Immunohistochemically, the AQP5 was expressed
in the control group on the apicolateral membrane of acinar cells (Fig. 6A). However, a cytoplasmic transloca-
tion with a statistically significant reduction of apicolateral AQP5 expression was found in DM group (Fig. 6B,D)
(P < 0.0001). GA application restored the expression of acinar protein AQP5 significantly when compared with
the diabetic group (P value < 0.0001), (Fig. 6C,D). Similarly, GA administration markedly upregulated SMG
AQP5 gene expression in comparison to the diabetic group (P value < 0.0001), (Fig. 6E).
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Figure 2. Effect of GA treatment on oxidative stress markers in SMG of diabetic rats. (A) MDA, (B) GSH
and (C) SOD. Data were expressed as mean ± SE. P < 0.05 was considered significant. ***Significant compared
to control group at P < 0.001, #Significant compared to diabetic group at P < 0.05, ###Significant compared to
diabetic group at P < 0.001.
Discussion
The present study reported for the first time the potential protective effect of GA on diabetes-induced SMG
disorder. Herein, we demonstrated marked structural and functional alterations of the SMG in the experimen-
tally induced diabetes on histopathological examination. In agreement with our results, de Souza et al. reported
marked structural, functional, and biochemical alterations in salivary glands including SMG in STZ diabetic
rats and theses alterations were reversed by ameliorating the hyperglycemic s tatus21.
Our study showed suppressed SIRT1 immunostaining in diabetic SMG accompanied by triggered oxidative
stress as evident by increased MDA and reduced cellular antioxidative moieties represented by GSH and SOD.
Concurrently, Lee et al. demonstrated a cytoprotective role of SIRT1 against cytokine induced pancreatic LGR-
cells injury22. Naderi et al. reported improvement of STZ-induced pancreatic apoptosis by SIRT1 activation23.
Moreover, Huang et al. reported the protective role of SIRT1 in diabetic nephropathy via activation of cellular
antioxidative mechanisms24. This might be attributed to SIRT1 ability to prevent endothelial cells senescence
and vascular injury in addition to ET-1 activation11,23.
Our results showed a restoration of the SMG levels of SIRT1, oxidative stress, ET-1 near to the normal levels
following GA administration to diabetic rat. In line, various studies reported valuable pharmacological effects of
GA via activation of SIRT125. In this context, Huo et al. explained the reno-protective effect of GA against high
glucose-induced tubular proliferation and oxidative stress via SIRT1 dependent mechanism26. Moreover, in the
present study, ET-1 expression was increased in the SMG of diabetic rats, suggesting its role in diabetes-induced
SMG injury. Interestingly, Ventimiglia et al. showed existence of an endothelinergic system in the SMG and its
participation in central and peripheral regulation of SMG secretion. Moreover, ET-1-induced reduction of blood
flow suppress the SMG secretory responses to parasympathetic stimulation27. Of note, a recent study reported a
suppressive effect of GA on ET-1 in hepatic ischemia–reperfusion injury20.
Our results also showed implication of autophagy dysregulation in the development of DM related changes in
the SMG, and associated decline in AQP5 proteins. Concomitantly, Huang et al. reported activation of autophagy
in SMGs of both diabetic patients and mice28. Moreover, Zhou et al. reported increased basal level of autophagy
with overexpression of P62 by disrupting the association between Beclin-1 and Bcl-2, resulting in Beclin-1
activation29. Of note, Yang et al. documented an efficient anti-tumor activity of GA against hepatocellular car-
cinoma though inhibition of autophagy25. Moreover, blocking autophagy was linked to the enhancement of GA
anti-tumor action against human sarcoma30.
Aquaporins (AQPs) are transmembrane water channel proteins that permit water passage across membrane.
Thus, they are widely distributed among water handling organs and exocrine glands as salivary gland. AQP1 is
found in myoepithelial and endothelial cells. Therefore, it is not surprising that it was reported to play a crucial
role in angiogenesis31. AQP4 is located in the ductal cells and at the basal region of acinar cells32. AQP5 is located
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Figure 3. LC3 immunostained SMG sections showing positive nuclear and cytoplasmic reaction in intralobular
ducts and GCD (arrows) in control group (A,B), while diabetic group showed strong positive cytoplasmic and
nuclear reaction in both acini and ducts (arrows) (C,D). GA treated group (E,F) revealed a positive reaction
in ducts only. (LC3, × 10, inserts × 20). Quantitative analysis of LC3 immunostaining (%area) (G) and fold
change of LC3 gene expression (H). Quantitative analysis of ELISA results, LC3 II expression (I). Data were
expressed as mean ± SE. P < 0.05 was considered significant. ****Significant compared to control group at
P < 0.0001, ###Significant compared to diabetic group at P < 0.001and ####Significant compared to diabetic group
at P < 0.0001. P62 immunostained SMG sections showing both cytoplasmic and nuclear positive reaction
especially in the ducts in DM group (K). While, little mild cytoplasmic reaction and nuclear ductal reaction in
both control (J) and GA group (L) (arrows) (P62 IHC, × 20). (M) Quantitative analysis of P62 immunostaining
(%area). (N) Effect of GA treatment on Beclin-1expression. Data were expressed as mean ± SE. P < 0.05 was
considered significant. ****Significant compared to control group at P < 0.0001, ####Significant compared to
diabetic group at P < 0.0001.
in apicolateral membrane of acinar cells and is important for production of the primary saliva in the acini and
is proposed to play a critical role in both development and r egeneration32. A previous study attributed the SMG
dysfunction in diabetes to autophagy activation and associated degradation of A QP528. Herein, our data dem-
onstrated that diabetes was associated with increased expression of AQP1 as well as suppressed expression of
AQP4 and AQP5 in SMG. These effects were reversed by GA administration.
In conclusion, the present study clearly indicated that diabetes affected both parenchymatous (acini and
ducts) as well as connective tissue of the SMG. The acini, which are responsible for synthesis of saliva, were
mostly affected as depicted with AQP5 degradation and autophagy activation. The vasculature supporting the
acini and ducts revealed altered expression of AQP1, AQP4 and ET1. On the other hand, GA treatment exhibited
an ameliorative effect against aforementioned features of diabetes-induced SMG dysfunction possibly through
SIRT1 activation. Further studies are recommended to validate our findings.
Methodology
Ethical statement, study design and allocation. Ethical statement. Approval was obtained from the
ethical committee of Faculty of Medicine, Mansoura University (No. R21.05.1328) in accordance with “prin-
ciples of laboratory animal care NIH publication revised 1985” (Code number: 2020–107). Reporting of all
experimental procedures complied with recommendations in ARRIVE guidelines.
Study design and allocation. Randomized, placebo-controlled, blinded animal study was conducted. The sam-
ple size was calculated using G power 3.9.1.4 software, to detect a 0.7 effect size between the null hypothesis and
the alternative hypothesis with significance level of 0.05 and a power of 0.85, using a one-way ANOVA F-test.
Twenty seven male Wistar rats, 100–120 g, were maintained in a controlled temperature (24–26 °C), relative
humidity of 60–80% and on a 12-h light–dark cycle for one week acclimatization. Rats were randomly allocated
using list randomizer (https://www.random.org/lists) into 3 groups with 9 rats/group as follow; Group1: served
as a control, Group 2: represented diabetic rats, and Group 3: denoted as the treated group in which the diabetic
rats received intraperitoneal (IP) injection of 100 mg/kg/3 times a week GA (Sigma-Aldrich, St Louis, MO, USA)
for 8 weeks33,34.
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Figure 4. Immunostained SMG sections with SIRT1 showing positive nuclear expression of SIRT1 in both
control and GA group (arrows) (A,C). Diabetic group (B) showed reduction of ductal expression (arrow)
(SIRT1, IHC, × 20). (D) Quantitative analysis of SIRT1 immunostaining (%area). Data were expressed
in mean ± SE. P < 0.05 is considered significant. ****Significant compared to control group at P < 0.0001,
####
Significant compared to diabetic group at P < 0.0001.
Diabetes induction. After overnight fasting, rats assigned to groups 2 and 3 were injected with (50 mg/
kg/ip) of freshly prepared streptozotocin (STZ) dissolved in citrate buffer, pH 4.5 (STZ, Sigma Chemical Co.,
St. Louis, MO, USA) while, the control animals in group 1 were injected by an equal volume of the buffer by
the same qualified person35. Three days after the STZ injection, animals with stable fasting blood glucose levels
at > 250 mg/dl were considered diabetic.
Euthanasia and biopsy collection. After eight weeks of treatment, all rats were anesthetized with Xyla-
zine (5 mg/kg, ADWIA Co. S.A.E 10 of Ramadan city, Egypt) and Ketamine (40 mg/kg, Segmatec Pharma-
ceutical Industries Co., Egypt) injection into the peritoneum (IP) and euthanized by decapitation (at 8 am to
minimize the circadian effect)36,37 and the SMG tissues were collected. The right halves were processed for the
histological analysis, and the left halves were snap frozen in liquid nitrogen and kept at − 80 °C until used for
oxidative stress estimation, RT-PCR and ELISA techniques.
Histological analysis. The 4 μm sections of paraformaldehyde-fixed and paraffin-embedded SMG tissues
were stained with hematoxylin and eosin (H&E). For the semithin sections, tissue biopsies were dehydrated
through an ascending series of ethanol (to 100%) and then washed in dry acetone and embedded in epoxy resin
then stained with toluidine blue.
Immunohistochemistry (IHC) and image analysis. The protein expression of SIRT1 (Bioss Antibod-
ies, USA, 1:200), ET-1 (Bioss Antibodies, USA, 1:200), AQP1 (Scervicebio Co., USA, 1:1000), AQP4 (Scervice-
bio Co., USA, 1:1500), AQP5 (ABclonal, USA, 1:200) and autophagy biomarkers LC3 (Abcam, USA, 1:1200),
P62 (ABclonal, USA, 1:200) were determined in each group by incubating tissue sections in primary antibodies
overnight followed by incubation with secondary antibodies to perform IHC. The visualization of slides was
detected using 3,3-Diaminobenzidine (DAB, Abcam, USA), and counterstained with hematoxylin. Then, the
sections were analyzed and photographed using an Olympus microscope (Japan) with installed camera. The
positive reaction was thresholded and calculated in relation to the surface area using Image J. The data were then
decoded and statistically analyzed.
Biochemical analysis of oxidative stress markers. The SMG tissue was homogenized with sodium
phosphate buffer, centrifuged, and the supernatant was used for the biochemical analysis. Oxidative stress mark-
ers; reduced glutathione (GSH), superoxide dismutase (SOD) and malondialdehyde (MDA) were measured
spectrophotometrically38,39.
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Figure 5. ET1 immunostained sections of SMG shows positive brown reaction around interlobular ducts and
blood vessels in control group (A), DM group shows mucous changes in the acini with marked expression of
ET1 in vascular and acinar components of the parenchyma (B). GA application reduced ET-1 expression (C).
AQP1 immunoreaction was localized in the parenchymal vasculature of SMG in control group (D) and GA
treated group (F), while this reaction was markedly increased in DM group (E). GA restored AQP4 expression
levels in diabetic SMG tissue (I) where diabetic group showed a marked reduction in AQP4 expression (H)
compared to the control group (G). (ET-1, AQP1, AQP4 IHC, × 20). Quantitative analysis of immunostaining
%area of ET-1 (J), AQP1 (K) and AQP4 (L). Data were expressed in mean ± SE. P < 0.05 is considered significant.
***Significant compared to control group at P < 0.001, ****Significant compared to control group at P < 0.0001,
###
Significant compared to diabetic group at P < 0.001. ####Significant compared to diabetic group at P < 0.0001.
Assessment of SMG levels of Beclin‑1and LC3II. Rat Beclin-1 ELISA Kit (MBS733192) and Rat LC3II
ELISA kit (MBS169564) were used for quantitative measurement of Beclin-1and LC3II protein levels in the SMG
homogenate according to the manufacturers’ instructions.
Quantitative Assay of LC3 and AQP5 gene expression using RT‑PCR. Total RNA was extracted
from SMG samples, and then RNA quality and purity were assured. Then cDNA was synthesized from RNA.
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Figure 6. AQP5 immunostained sections of SMG shows, apicolateral localization (arrow) in control (A), and
GA (C) groups, with apparent degradation and cytoplasmic translocation in DM (curved arrow) and little
apicolateral expression (arrow) (B) (AQP5, IHC, × 40)). (D) Quantitative analysis of AQP5 immunostaining
(%area) and fold change of AQP5 gene expression (E), Data were expressed in mean ± SE. P < 0.05 is considered
significant. ****Significant compared to control group at P < 0.001, ####Significant compared to diabetic group at
P < 0.0001.
The cDNA was amplified and used in SYBR Green Based Quantitative Real-Time PCR. For Relative Quantifica-
tion (RQ) of LC3 gene expression, a primer with Gene Bank Accession No. NM_022867.2, Forward sequence:
5-ACG-GCT-TCC-TGT-ACA-TGG-TC-3 and Reverse sequence: 5-GTG-GGT-GCC-TAC-GTT-CTG-AT was
used. And for AQP5, a primer with Gene Bank Accession No. NM_012779.2 was used. The forward primer
sequence was 5-GGGCCATCTTGTGGGGATCT-3 and the reverse primer sequence was 5-CCAGTGAGA
GGGGCTGAACC-3. The RQ of both genes expression was performed using comparative 2−ΔΔCt method, where
the amount of the target genes mRNA were normalized to an endogenous reference gene glyceraldehyde 3-phos-
phate dehydrogenase (GAPDH) and relative to a control40.
Statistical analysis. Data were tested for normal distribution by Shapiro–Wilk test. Quantitative data were
analyzed using Graph Prism 8 (GraphPad Software, Inc., CA, USA) to test the significance between different
groups using analysis of variance (ANOVA) followed by Tukey’s test. Data were presented as mean ± standard
error (SE). Significance was inferred at P < 0.05.
Received: 10 May 2021; Accepted: 15 December 2021
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Acknowledgements
The authors deeply acknowledge the Researchers Supporting program (TUMA-Project-2021-3), Almaarefa Uni-
versity, Riyadh, Saudi Arabia for supporting steps of this work.
Author contributions
All authors contributed equally to this research. Maysarah was added for her major contribution in revision.
Competing interests
The authors declare no competing interests.
Additional information
Correspondence and requests for materials should be addressed to N.M.E. or A.M.E.
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