Title: Biochemical and histopathological evidence for beneficial effects of empagliflozin
pretreatment on acetic acid-induced colitis in rats
Version: 2 Date: 05 Jan 2023
Reviewer:  Paul Aimé Noubissi
Reviewer's report:
Biochemical and histopathological evidence for beneficial effects of empagliflozin pretreatment
on acetic acid-induced colitis in rats
This study aimed to validate the biochemical and histopathological evidence for beneficial
effects of empagliflozin pretreatment on acetic acid-induced colitis in rats. However, there are
some minor problems that need to be addressed.
Abstract
- Background: Rephrase the first sentence, it is not clear. Also, claiming that “ulcerative
colitis (UC) is a long-term disorder that characterizes by oxidative stress” is not correct. We
have pro-oxidative and anti-oxidative stress. So, which one do the authors mean? Also, write
“ulcerative colitis (UC) is a long-term disorder that is characterized by oxidative stress…” and
not “ulcerative colitis (UC) is a long-term disorder that characterizes by oxidative stress…”
- The authors wrote “This research investigates the preventive effects of EMPA as an anti-stress
oxidative agent in rats” Better “This research investigates the preventive effects of EMPA as an
anti- oxidative stress agent in rats”
Secondly, you evaluated the anticolitis or the antioxidative stress? Be clear
Methods:
Write “Twenty-four hours (24 h) after AA…” not “24 hours after AA…
Results
- The abstract might present the only source of knowledge for many researchers who has no
access to the whole work, therefore it is highly recommended to add some digital results in the
abstract rather than elastic sentences. The authors should include values in the result section of
the abstract. At the moment, they are barely described.
- Empagliflozin is abbreviated EMPA or Empa as it is written elsewhere, be consistent.
Conclusions:
From the abstract conclusion, one can think the only ulcerative colitis symptom is the oxidative
stress. What about the colon weight/length ratio, macroscopic and histological score?
Introduction
- The authors wrote “The pathophysiology of UC is not clearly understood but it had shown
that several factors such as environment, genetic, autoimmunity, and intestinal microbiota are
involved in the pathophysiology of this disease”. Rephrase this statement and write for example
“The pathophysiology of UC is not clearly understood but it had shown that several factors such
as environment, genetic, autoimmunity, and intestinal microbiota are involved.”
- The authors wrote “Various animal models of intestinal inflammation have been established
and acetic acid-induced colitis is one of the widely used animal models of inflammatory bowel
disease”. Rephrase this statement and write for example “Various animal models of intestinal
inflammation have been established and acetic acid-induced colitis is one of the widely used.”
- The authors wrote “Although many types of treatment have been proposed…” Rephrase this
statement and write “Although many treatments have been proposed…”
- “… and demonstrate significant side effects on prolonged use..” What demonstrates? Rephrase
the statement.
- “Although the exact mechanism for these effects is not fully understood (12), investigations
reported that number of potential effects such as anti-inflammatory, anti-oxidative and
antiapoptotic properties through which Empa could exert its beneficial effects (13, 14)”.
Rephrase this statement.
- “It has been reported that in streptozotocin-induced type 1 diabetic rats Empa can reduces ROS
in pancreatic #-cells (15).” Write but “…Empa can reduce” so delete “s” from reduce.
Experimental design
- The animal grouping is not clear, please, rephrase the entire statement.
- What do you mean by “Empa 10 mg/kg per day + AA (AA+ Empa)”? it will be better to write
but Empa “(empagliflozin, 10 mg/kg administered once per day).
Authors wrote “Diluted AA (2 ml, 4% v/v in 0.9% saline) was instilled slowly into the rat colons
by using….” Better write “Diluted AA (2 ml, 4% v/v in 0.9% saline) was slowly instilled into
the rat colons using….”
Diluted AA (2 ml, 4% v/v in 0.9% saline) was instilled slowly into the rat colons by using a soft
pediatric catheter (2.7 mm diameter) inserted into the rat anus for 8 cm. (put a full stop)
Authors wrote “After AA administration, rats were holed horizontally for 2 min to…” Write but
“After AA administration, rats were held horizontally for 2 min to…” so were held not were
holed.
- “…rats were held horizontally for 2 min to…” Why horizontally? Not vertically?
- “In order to pretreatment in AA+ Empa or AA + Dexa groups, EMPA or Dexa respectively
was injected intraperitoneally …” Rephrase this statement. So, what about the AA group?
- Animals were sacrificed twenty-four hours after the colitis induction. At this stage, what were
evidences of colitis being induced? Did colitis succeeded to be induced in all the groups?
- Before sacrifice, why did you not evaluate the quality and quantity of stools emission, the body
weight variation? This may be signs of colitis been induced.
- Authors wrote “The remaining tissues were stored at -80 # till biochemical analysis.” How did
you proceed to biochemical analysis from the stored tissues? Rather, you have to tell us how the
homogenates used for biochemical analysis were prepared.
- Authors wrote “The abdomens of experimental animals were dissected, and colons excised by
tracing backward? Why did you excise the colon before measuring its length?  How did you
measure it?
- Authors wrote “Colon lengths measuring 10 cm were cut out and opened longitudinally and
rinsed in saline and the wet weights were measured.” Do you mean the colon of all rats measured
10 cm? And what about rats which colon measured more or less than 10 cm?
Colon weight-to-length ration = total weight of the colon/the length of the entire colon.
- Under the Section Histological evaluation, authors wrote “One-half of them were transferred
to a -80 centigrade freezer for examining specific inflammatory markers” The results for the
examined specific inflammatory markers are absent in the results.
Authors wrote “The MDA level, which is an indicator of lipid peroxidation, was determined
according to the method of Ohkawa et al. with slight modifications (26, 27). Reduced
glutathione or GSH, commonly known non-enzymatic antioxidants involved in detoxification
of xenobiotics, was measured as described previously by Hu et al. (28).” Please, describe these
methods for MDA and GSH levels determination.
Results
Effect of empagliflozin on colon weight-to-length ratio
- The results under this section were presented without units; for example, the weight-to-length
ratio of the control group was 58.8 ± 5.69. Please authors should add the unit. Same with 108.8
± 5.51 in the AA group, … with 81.8 ± 4.18 in AA + Dexa group, or with 81 ± 5.28 in AA +
Empa group was. For example, 108.8 ± 5.51 mg/cm
Also, since the Standard Error on the Mean (SEM) are expressed with two decimals, also write
the Mean with two decimals, ie for example 58.80 ± 5.69 mg/cm and not 58.8 ± 5.69 mg/cm or
81.00 ± 5.28 mg/cm not 81 ± 5.28 mg/cm.
- On Fig 1., it was indicated that ## stands for p < 0.01 vs. AA group. How can you explain
that the Empa group with the weight-to-length ratio of 81.00 ± 5.28 mg/cm and the AA + Dexa
group with 81.80 ± 4.18 mg/cm were significantly different compared to AA group; and not the
control group with 58.80 ± 5.69 mg/cm?
Effect of empagliflozin on macroscopic changes of colitis
- Authors here “Harvested colons were opened longitudinally and washed in normal saline after
which they were examined.” This statement is good in methodology, not under results.
- Authors here “Acetic acid caused severe ulceration and hemorrhage of colonic mucosa and
extensive necrosis of tissue over a wide surface in this group was observed…” Not clear,
rephrase this statement.
- Be consistent while describing Figure 2. You started with Fig. 2A, then Fig. 2B. Why did you
from here moved directly to Fig. 2D before talking about the Fig. 2C?
- Also, these figures need to be labelled.
- On figure legends, authors wrote “…(B) Macroscopic appearance of colonic mucosa of AA
group showing severe ulceration, erosions and tissue necrosis of the mucosa and submucosa.”
Can you show those severe ulceration, erosions and tissue necrosis of the mucosa and submucosa
on Fig. 2B? Because I can not see them.
- Also, is there any difference between Fig. 2 B and Fig.2C, if yes, the feces content in Fig. 2B.
- … while the macroscopic score in AA +Empa group was of 2.5 ± 0.57, write but 2.50 ± 0.57
- Fig. 2 lacks the scale it should have. Also, the pictures in Fig. 2 are not beautiful enough, so
it is recommended to beautify them. For example, keep the length and width of these pictures
consistent, and align them.
- On Fig. 2 legends, authors wrote “Treatment with Empa or Dexa attenuated the extent and
severity of the morphological alterations associated with AA. (E) Mean scores of the tissue
damage observed in colonic specimens of animals in different experimental groups. The results
were expressed as mean ± SEM (n = 6) and analyzed by ANOVA followed by Tukey test. #p
< 0.05 vs. AA group; ###p < 0.001 vs. AA, &p < 0.05 vs. AA + Empa group. AA: Acetic acid,
Empa: empagliflozine, Dexa: Dexamethasone.” In this statement, bring “Treatment with Empa
or Dexa attenuated the extent and severity of the morphological alterations associated with AA.”
to the result comments before the Fig, not in the Fig legend.
- Also, delete this statement “and analyzed by ANOVA followed by Tukey test.”
- What differences are there between macroscopic and histological scores? How did you evaluate
each?
Effect of empagliflozin on histopathological changes
- “Microscopic evaluation of hematoxylin and eosin-stained (H&E) sections of colonic tissues
of normal mice indicated intact epithelium and mucosal layer (Figure 3A)” . Can you please,
indicate on Fig. 3 the intact epithelium and mucosal layer?
- Also, authors indicated here that the colonic sections were obtained from mice. So, actually
did you work with mice or rats? Be consistent.
- Please indicate on the following on Figure 3B: focal crypt distortions and abscesses necrotic
destruction of surface epithelium, muco-depletion, cellular infiltration, and hyperemia of
submucosal capillaries. So, label the diagrams of Fig. 3
- Significantly, treatment with AA + Empa indicated remarkable recovery of the colonic tissue
compared to the AA-induced colitis damage (Figure 3C). How did you measure the significance
value among diagrams?
- Fig 3 lacks the magnification power. It has to be tired directly under the diagram.
- Observe the same comments I did about the Fig 2 legends on Fig. 3 legend.
Effect of empagliflozin on oxidative stress and antioxidant activity
- It is highly recommended to add some digital results rather than elastic sentences. For example,
when you write “MDA level was significantly increased in AA group versus control group
(P<0.001).” what was the MDA value in AA or in control group?
- Observe the same comments I did about the Fig 2 legends on Fig. 4 legend.
- Why did you not evaluate the levels of some pro or anti-inflammatory cytokines in colonic
tissue homogenate?
Discussion
- “In the current study application of AA disturbed the colonic mucus” How did you evaluate
that?
- “In the present study, pre-treatment with Empa in the AA induced colitis significantly
decreaesed….” Decreased not decreaesed”
- “It has been showed that intrarectal administration of AA…” It has been shown… not “showed”
- “…with DNA and increase stress oxidative and cell death…” write but “…and increase
oxidative stress…” not “stress oxidative”.
- In general, English editing is required.
Title: Biochemical and histopathological evidence for beneficial effects of empagliflozin
pretreatment on acetic acid-induced colitis in rats
Version: 2 Date: 05 Jan 2023
Reviewer:  Paul Aimé Noubissi
Reviewer's report:
Biochemical and histopathological evidence for beneficial effects of empagliflozin pretreatment
on acetic acid-induced colitis in rats
This study aimed to validate the biochemical and histopathological evidence for beneficial
effects of empagliflozin pretreatment on acetic acid-induced colitis in rats. However, there are
some minor problems that need to be addressed.
Abstract
- Background: Rephrase the first sentence, it is not clear. Also, claiming that “ulcerative
colitis (UC) is a long-term disorder that characterizes by oxidative stress” is not correct. We
have pro-oxidative and anti-oxidative stress. So, which one do the authors mean? Also, write
“ulcerative colitis (UC) is a long-term disorder that is characterized by oxidative stress…” and
not “ulcerative colitis (UC) is a long-term disorder that characterizes by oxidative stress…”
- The authors wrote “This research investigates the preventive effects of EMPA as an anti-stress
oxidative agent in rats” Better “This research investigates the preventive effects of EMPA as an
anti- oxidative stress agent in rats”
Secondly, you evaluated the anticolitis or the antioxidative stress? Be clear
Methods:
Write “Twenty-four hours (24 h) after AA…” not “24 hours after AA…
Results
- The abstract might present the only source of knowledge for many researchers who has no
access to the whole work, therefore it is highly recommended to add some digital results in the
abstract rather than elastic sentences. The authors should include values in the result section of
the abstract. At the moment, they are barely described.
- Empagliflozin is abbreviated EMPA or Empa as it is written elsewhere, be consistent.
Conclusions:
From the abstract conclusion, one can think the only ulcerative colitis symptom is the oxidative
stress. What about the colon weight/length ratio, macroscopic and histological score?
Introduction
- The authors wrote “The pathophysiology of UC is not clearly understood but it had shown
that several factors such as environment, genetic, autoimmunity, and intestinal microbiota are
involved in the pathophysiology of this disease”. Rephrase this statement and write for example
“The pathophysiology of UC is not clearly understood but it had shown that several factors such
as environment, genetic, autoimmunity, and intestinal microbiota are involved.”
- The authors wrote “Various animal models of intestinal inflammation have been established
and acetic acid-induced colitis is one of the widely used animal models of inflammatory bowel
disease”. Rephrase this statement and write for example “Various animal models of intestinal
inflammation have been established and acetic acid-induced colitis is one of the widely used.”
- The authors wrote “Although many types of treatment have been proposed…” Rephrase this
statement and write “Although many treatments have been proposed…”
- “… and demonstrate significant side effects on prolonged use..” What demonstrates? Rephrase
the statement.
- “Although the exact mechanism for these effects is not fully understood (12), investigations
reported that number of potential effects such as anti-inflammatory, anti-oxidative and
antiapoptotic properties through which Empa could exert its beneficial effects (13, 14)”.
Rephrase this statement.
- “It has been reported that in streptozotocin-induced type 1 diabetic rats Empa can reduces ROS
in pancreatic #-cells (15).” Write but “…Empa can reduce” so delete “s” from reduce.
Experimental design
- The animal grouping is not clear, please, rephrase the entire statement.
- What do you mean by “Empa 10 mg/kg per day + AA (AA+ Empa)”? it will be better to write
but Empa “(empagliflozin, 10 mg/kg administered once per day).
Authors wrote “Diluted AA (2 ml, 4% v/v in 0.9% saline) was instilled slowly into the rat colons
by using….” Better write “Diluted AA (2 ml, 4% v/v in 0.9% saline) was slowly instilled into
the rat colons using….”
Diluted AA (2 ml, 4% v/v in 0.9% saline) was instilled slowly into the rat colons by using a soft
pediatric catheter (2.7 mm diameter) inserted into the rat anus for 8 cm. (put a full stop)
Authors wrote “After AA administration, rats were holed horizontally for 2 min to…” Write but
“After AA administration, rats were held horizontally for 2 min to…” so were held not were
holed.
- “…rats were held horizontally for 2 min to…” Why horizontally? Not vertically?
- “In order to pretreatment in AA+ Empa or AA + Dexa groups, EMPA or Dexa respectively
was injected intraperitoneally …” Rephrase this statement. So, what about the AA group?
- Animals were sacrificed twenty-four hours after the colitis induction. At this stage, what were
evidences of colitis being induced? Did colitis succeeded to be induced in all the groups?
- Before sacrifice, why did you not evaluate the quality and quantity of stools emission, the body
weight variation? This may be signs of colitis been induced.
- Authors wrote “The remaining tissues were stored at -80 # till biochemical analysis.” How did
you proceed to biochemical analysis from the stored tissues? Rather, you have to tell us how the
homogenates used for biochemical analysis were prepared.
- Authors wrote “The abdomens of experimental animals were dissected, and colons excised by
tracing backward? Why did you excise the colon before measuring its length?  How did you
measure it?
- Authors wrote “Colon lengths measuring 10 cm were cut out and opened longitudinally and
rinsed in saline and the wet weights were measured.” Do you mean the colon of all rats measured
10 cm? And what about rats which colon measured more or less than 10 cm?
Colon weight-to-length ration = total weight of the colon/the length of the entire colon.
- Under the Section Histological evaluation, authors wrote “One-half of them were transferred
to a -80 centigrade freezer for examining specific inflammatory markers” The results for the
examined specific inflammatory markers are absent in the results.
Authors wrote “The MDA level, which is an indicator of lipid peroxidation, was determined
according to the method of Ohkawa et al. with slight modifications (26, 27). Reduced
glutathione or GSH, commonly known non-enzymatic antioxidants involved in detoxification
of xenobiotics, was measured as described previously by Hu et al. (28).” Please, describe these
methods for MDA and GSH levels determination.
Results
Effect of empagliflozin on colon weight-to-length ratio
- The results under this section were presented without units; for example, the weight-to-length
ratio of the control group was 58.8 ± 5.69. Please authors should add the unit. Same with 108.8
± 5.51 in the AA group, … with 81.8 ± 4.18 in AA + Dexa group, or with 81 ± 5.28 in AA +
Empa group was. For example, 108.8 ± 5.51 mg/cm
Also, since the Standard Error on the Mean (SEM) are expressed with two decimals, also write
the Mean with two decimals, ie for example 58.80 ± 5.69 mg/cm and not 58.8 ± 5.69 mg/cm or
81.00 ± 5.28 mg/cm not 81 ± 5.28 mg/cm.
- On Fig 1., it was indicated that ## stands for p < 0.01 vs. AA group. How can you explain
that the Empa group with the weight-to-length ratio of 81.00 ± 5.28 mg/cm and the AA + Dexa
group with 81.80 ± 4.18 mg/cm were significantly different compared to AA group; and not the
control group with 58.80 ± 5.69 mg/cm?
Effect of empagliflozin on macroscopic changes of colitis
- Authors here “Harvested colons were opened longitudinally and washed in normal saline after
which they were examined.” This statement is good in methodology, not under results.
- Authors here “Acetic acid caused severe ulceration and hemorrhage of colonic mucosa and
extensive necrosis of tissue over a wide surface in this group was observed…” Not clear,
rephrase this statement.
- Be consistent while describing Figure 2. You started with Fig. 2A, then Fig. 2B. Why did you
from here moved directly to Fig. 2D before talking about the Fig. 2C?
- Also, these figures need to be labelled.
- On figure legends, authors wrote “…(B) Macroscopic appearance of colonic mucosa of AA
group showing severe ulceration, erosions and tissue necrosis of the mucosa and submucosa.”
Can you show those severe ulceration, erosions and tissue necrosis of the mucosa and submucosa
on Fig. 2B? Because I can not see them.
- Also, is there any difference between Fig. 2 B and Fig.2C, if yes, the feces content in Fig. 2B.
- … while the macroscopic score in AA +Empa group was of 2.5 ± 0.57, write but 2.50 ± 0.57
- Fig. 2 lacks the scale it should have. Also, the pictures in Fig. 2 are not beautiful enough, so
it is recommended to beautify them. For example, keep the length and width of these pictures
consistent, and align them.
- On Fig. 2 legends, authors wrote “Treatment with Empa or Dexa attenuated the extent and
severity of the morphological alterations associated with AA. (E) Mean scores of the tissue
damage observed in colonic specimens of animals in different experimental groups. The results
were expressed as mean ± SEM (n = 6) and analyzed by ANOVA followed by Tukey test. #p
< 0.05 vs. AA group; ###p < 0.001 vs. AA, &p < 0.05 vs. AA + Empa group. AA: Acetic acid,
Empa: empagliflozine, Dexa: Dexamethasone.” In this statement, bring “Treatment with Empa
or Dexa attenuated the extent and severity of the morphological alterations associated with AA.”
to the result comments before the Fig, not in the Fig legend.
- Also, delete this statement “and analyzed by ANOVA followed by Tukey test.”
- What differences are there between macroscopic and histological scores? How did you evaluate
each?
Effect of empagliflozin on histopathological changes
- “Microscopic evaluation of hematoxylin and eosin-stained (H&E) sections of colonic tissues
of normal mice indicated intact epithelium and mucosal layer (Figure 3A)” . Can you please,
indicate on Fig. 3 the intact epithelium and mucosal layer?
- Also, authors indicated here that the colonic sections were obtained from mice. So, actually
did you work with mice or rats? Be consistent.
- Please indicate on the following on Figure 3B: focal crypt distortions and abscesses necrotic
destruction of surface epithelium, muco-depletion, cellular infiltration, and hyperemia of
submucosal capillaries. So, label the diagrams of Fig. 3
- Significantly, treatment with AA + Empa indicated remarkable recovery of the colonic tissue
compared to the AA-induced colitis damage (Figure 3C). How did you measure the significance
value among diagrams?
- Fig 3 lacks the magnification power. It has to be tired directly under the diagram.
- Observe the same comments I did about the Fig 2 legends on Fig. 3 legend.
Effect of empagliflozin on oxidative stress and antioxidant activity
- It is highly recommended to add some digital results rather than elastic sentences. For example,
when you write “MDA level was significantly increased in AA group versus control group
(P<0.001).” what was the MDA value in AA or in control group?
- Observe the same comments I did about the Fig 2 legends on Fig. 4 legend.
- Why did you not evaluate the levels of some pro or anti-inflammatory cytokines in colonic
tissue homogenate?
Discussion
- “In the current study application of AA disturbed the colonic mucus” How did you evaluate
that?
- “In the present study, pre-treatment with Empa in the AA induced colitis significantly
decreaesed….” Decreased not decreaesed”
- “It has been showed that intrarectal administration of AA…” It has been shown… not “showed”
- “…with DNA and increase stress oxidative and cell death…” write but “…and increase
oxidative stress…” not “stress oxidative”.
- In general, English editing is required.
Please review this paper.
user
Title: Biochemical and histopathological evidence for beneficial effects of empagliflozin
pretreatment on acetic acid-induced colitis in rats
Version: 2 Date: 05 Jan 2023
Reviewer:  Paul Aimé Noubissi
Reviewer's report:
Biochemical and histopathological evidence for beneficial effects of empagliflozin pretreatment
on acetic acid-induced colitis in rats
This study aimed to validate the biochemical and histopathological evidence for beneficial
effects of empagliflozin pretreatment on acetic acid-induced colitis in rats. However, there are
some minor problems that need to be addressed.
Abstract
- Background: Rephrase the first sentence, it is not clear. Also, claiming that “ulcerative
colitis (UC) is a long-term disorder that characterizes by oxidative stress” is not correct. We
have pro-oxidative and anti-oxidative stress. So, which one do the authors mean? Also, write
“ulcerative colitis (UC) is a long-term disorder that is characterized by oxidative stress…” and
not “ulcerative colitis (UC) is a long-term disorder that characterizes by oxidative stress…”
- The authors wrote “This research investigates the preventive effects of EMPA as an anti-stress
oxidative agent in rats” Better “This research investigates the preventive effects of EMPA as an
anti- oxidative stress agent in rats”
Secondly, you evaluated the anticolitis or the antioxidative stress? Be clear
Methods:
Write “Twenty-four hours (24 h) after AA…” not “24 hours after AA…
Results
- The abstract might present the only source of knowledge for many researchers who has no
access to the whole work, therefore it is highly recommended to add some digital results in the
abstract rather than elastic sentences. The authors should include values in the result section of
the abstract. At the moment, they are barely described.
- Empagliflozin is abbreviated EMPA or Empa as it is written elsewhere, be consistent.
Conclusions:
From the abstract conclusion, one can think the only ulcerative colitis symptom is the oxidative
stress. What about the colon weight/length ratio, macroscopic and histological score?
Introduction
- The authors wrote “The pathophysiology of UC is not clearly understood but it had shown
that several factors such as environment, genetic, autoimmunity, and intestinal microbiota are
involved in the pathophysiology of this disease”. Rephrase this statement and write for example
“The pathophysiology of UC is not clearly understood but it had shown that several factors such
as environment, genetic, autoimmunity, and intestinal microbiota are involved.”
- The authors wrote “Various animal models of intestinal inflammation have been established
and acetic acid-induced colitis is one of the widely used animal models of inflammatory bowel
disease”. Rephrase this statement and write for example “Various animal models of intestinal
inflammation have been established and acetic acid-induced colitis is one of the widely used.”
- The authors wrote “Although many types of treatment have been proposed…” Rephrase this
statement and write “Although many treatments have been proposed…”
- “… and demonstrate significant side effects on prolonged use..” What demonstrates? Rephrase
the statement.
- “Although the exact mechanism for these effects is not fully understood (12), investigations
reported that number of potential effects such as anti-inflammatory, anti-oxidative and
antiapoptotic properties through which Empa could exert its beneficial effects (13, 14)”.
Rephrase this statement.
- “It has been reported that in streptozotocin-induced type 1 diabetic rats Empa can reduces ROS
in pancreatic #-cells (15).” Write but “…Empa can reduce” so delete “s” from reduce.
Experimental design
- The animal grouping is not clear, please, rephrase the entire statement.
- What do you mean by “Empa 10 mg/kg per day + AA (AA+ Empa)”? it will be better to write
but Empa “(empagliflozin, 10 mg/kg administered once per day).
Authors wrote “Diluted AA (2 ml, 4% v/v in 0.9% saline) was instilled slowly into the rat colons
by using….” Better write “Diluted AA (2 ml, 4% v/v in 0.9% saline) was slowly instilled into
the rat colons using….”
Diluted AA (2 ml, 4% v/v in 0.9% saline) was instilled slowly into the rat colons by using a soft
pediatric catheter (2.7 mm diameter) inserted into the rat anus for 8 cm. (put a full stop)
Authors wrote “After AA administration, rats were holed horizontally for 2 min to…” Write but
“After AA administration, rats were held horizontally for 2 min to…” so were held not were
holed.
- “…rats were held horizontally for 2 min to…” Why horizontally? Not vertically?
- “In order to pretreatment in AA+ Empa or AA + Dexa groups, EMPA or Dexa respectively
was injected intraperitoneally …” Rephrase this statement. So, what about the AA group?
- Animals were sacrificed twenty-four hours after the colitis induction. At this stage, what were
evidences of colitis being induced? Did colitis succeeded to be induced in all the groups?
- Before sacrifice, why did you not evaluate the quality and quantity of stools emission, the body
weight variation? This may be signs of colitis been induced.
- Authors wrote “The remaining tissues were stored at -80 # till biochemical analysis.” How did
you proceed to biochemical analysis from the stored tissues? Rather, you have to tell us how the
homogenates used for biochemical analysis were prepared.
- Authors wrote “The abdomens of experimental animals were dissected, and colons excised by
tracing backward? Why did you excise the colon before measuring its length?  How did you
measure it?
- Authors wrote “Colon lengths measuring 10 cm were cut out and opened longitudinally and
rinsed in saline and the wet weights were measured.” Do you mean the colon of all rats measured
10 cm? And what about rats which colon measured more or less than 10 cm?
Colon weight-to-length ration = total weight of the colon/the length of the entire colon.
- Under the Section Histological evaluation, authors wrote “One-half of them were transferred
to a -80 centigrade freezer for examining specific inflammatory markers” The results for the
examined specific inflammatory markers are absent in the results.
Authors wrote “The MDA level, which is an indicator of lipid peroxidation, was determined
according to the method of Ohkawa et al. with slight modifications (26, 27). Reduced
glutathione or GSH, commonly known non-enzymatic antioxidants involved in detoxification
of xenobiotics, was measured as described previously by Hu et al. (28).” Please, describe these
methods for MDA and GSH levels determination.
Results
Effect of empagliflozin on colon weight-to-length ratio
- The results under this section were presented without units; for example, the weight-to-length
ratio of the control group was 58.8 ± 5.69. Please authors should add the unit. Same with 108.8
± 5.51 in the AA group, … with 81.8 ± 4.18 in AA + Dexa group, or with 81 ± 5.28 in AA +
Empa group was. For example, 108.8 ± 5.51 mg/cm
Also, since the Standard Error on the Mean (SEM) are expressed with two decimals, also write
the Mean with two decimals, ie for example 58.80 ± 5.69 mg/cm and not 58.8 ± 5.69 mg/cm or
81.00 ± 5.28 mg/cm not 81 ± 5.28 mg/cm.
- On Fig 1., it was indicated that ## stands for p < 0.01 vs. AA group. How can you explain
that the Empa group with the weight-to-length ratio of 81.00 ± 5.28 mg/cm and the AA + Dexa
group with 81.80 ± 4.18 mg/cm were significantly different compared to AA group; and not the
control group with 58.80 ± 5.69 mg/cm?
Effect of empagliflozin on macroscopic changes of colitis
- Authors here “Harvested colons were opened longitudinally and washed in normal saline after
which they were examined.” This statement is good in methodology, not under results.
- Authors here “Acetic acid caused severe ulceration and hemorrhage of colonic mucosa and
extensive necrosis of tissue over a wide surface in this group was observed…” Not clear,
rephrase this statement.
- Be consistent while describing Figure 2. You started with Fig. 2A, then Fig. 2B. Why did you
from here moved directly to Fig. 2D before talking about the Fig. 2C?
- Also, these figures need to be labelled.
- On figure legends, authors wrote “…(B) Macroscopic appearance of colonic mucosa of AA
group showing severe ulceration, erosions and tissue necrosis of the mucosa and submucosa.”
Can you show those severe ulceration, erosions and tissue necrosis of the mucosa and submucosa
on Fig. 2B? Because I can not see them.
- Also, is there any difference between Fig. 2 B and Fig.2C, if yes, the feces content in Fig. 2B.
- … while the macroscopic score in AA +Empa group was of 2.5 ± 0.57, write but 2.50 ± 0.57
- Fig. 2 lacks the scale it should have. Also, the pictures in Fig. 2 are not beautiful enough, so
it is recommended to beautify them. For example, keep the length and width of these pictures
consistent, and align them.
- On Fig. 2 legends, authors wrote “Treatment with Empa or Dexa attenuated the extent and
severity of the morphological alterations associated with AA. (E) Mean scores of the tissue
damage observed in colonic specimens of animals in different experimental groups. The results
were expressed as mean ± SEM (n = 6) and analyzed by ANOVA followed by Tukey test. #p
< 0.05 vs. AA group; ###p < 0.001 vs. AA, &p < 0.05 vs. AA + Empa group. AA: Acetic acid,
Empa: empagliflozine, Dexa: Dexamethasone.” In this statement, bring “Treatment with Empa
or Dexa attenuated the extent and severity of the morphological alterations associated with AA.”
to the result comments before the Fig, not in the Fig legend.
- Also, delete this statement “and analyzed by ANOVA followed by Tukey test.”
- What differences are there between macroscopic and histological scores? How did you evaluate
each?
Effect of empagliflozin on histopathological changes
- “Microscopic evaluation of hematoxylin and eosin-stained (H&E) sections of colonic tissues
of normal mice indicated intact epithelium and mucosal layer (Figure 3A)” . Can you please,
indicate on Fig. 3 the intact epithelium and mucosal layer?
- Also, authors indicated here that the colonic sections were obtained from mice. So, actually
did you work with mice or rats? Be consistent.
- Please indicate on the following on Figure 3B: focal crypt distortions and abscesses necrotic
destruction of surface epithelium, muco-depletion, cellular infiltration, and hyperemia of
submucosal capillaries. So, label the diagrams of Fig. 3
- Significantly, treatment with AA + Empa indicated remarkable recovery of the colonic tissue
compared to the AA-induced colitis damage (Figure 3C). How did you measure the significance
value among diagrams?
- Fig 3 lacks the magnification power. It has to be tired directly under the diagram.
- Observe the same comments I did about the Fig 2 legends on Fig. 3 legend.
Effect of empagliflozin on oxidative stress and antioxidant activity
- It is highly recommended to add some digital results rather than elastic sentences. For example,
when you write “MDA level was significantly increased in AA group versus control group
(P<0.001).” what was the MDA value in AA or in control group?
- Observe the same comments I did about the Fig 2 legends on Fig. 4 legend.
- Why did you not evaluate the levels of some pro or anti-inflammatory cytokines in colonic
tissue homogenate?
Discussion
- “In the current study application of AA disturbed the colonic mucus” How did you evaluate
that?
- “In the present study, pre-treatment with Empa in the AA induced colitis significantly
decreaesed….” Decreased not decreaesed”
- “It has been showed that intrarectal administration of AA…” It has been shown… not “showed”
- “…with DNA and increase stress oxidative and cell death…” write but “…and increase
oxidative stress…” not “stress oxidative”.
- In general, English editing is required.
assistant