Exosomal circEZH2_005, an intestinal injury biomarker, alleviates intestinal ischemia/reperfusion injury by mediating Gprc5a signaling

Intestinal ischemia/reperfusion (I/R) injury is a severe clinical condition without optimal diagnostic markers nor clear molecular etiological insights. Plasma exosomal circular RNAs (circRNAs) are valuable biomarkers and therapeutic targets for various diseases, but their role in intestinal I/R injury remains unknown. Here we screen the expression profile of circRNAs in intestinal tissue exosomes collected from intestinal I/R mice and identify circEZH2_005 as a significantly downregulated exosomal circRNA. In parallel, circEZH2_005 is also reduced in the plasma of clinical cardiac surgery patients who developed postoperative intestinal I/R injury. Exosomal circEZH2_005 displays a significant diagnostic value for intestinal injury induced by I/R. Mechanistically, circEZH2_005 is highly expressed in intestinal crypt cells. CircEZH2_005 upregulation promotes the proliferation of Lgr5+ stem cells by direct interaction with hnRNPA1, and enhanced Gprc5a stability, thereby alleviating I/R-induced intestinal mucosal damage. Hence, exosomal circEZH2_005 may serve as a biomarker for intestinal I/R injury and targeting the circEZH2_005/hnRNPA1/Gprc5a axis may be a potential therapeutic strategy for intestinal I/R injury.

other areas of the intestine and is the large intestine also involved in the production of this circular RNA?
3) Authors do not specify which kit is used to purify the human exosomes from patients and how rapidly this procedure is, which is of significance in clinical diagnosis and evaluation of suitability of this marker for I/R diagnosis.How long would a purification take if it was to be used to diagnose a patient?4) In figure 5-7 where biochemical experiments are performed by overexpression of circEZH2_005, the authors should use a mutated version of the circular RNA with the mutation in the hnRNPA1 which they identify as a direct binder to the circEZH2_005.In the crypt rescue experiments involving overexpression of circEZH2_005, using a mutated version of circEZH2expressed from a virus to assess the ability increase the number of stem cells would be necessary.
5) The authors should test whether the decrease /induction of circEZH2_005 in the intestine is regulated by hypoxia inducible factors (HIFs).With a large number of reports showing induction of circular RNA by HIFs in cancer and other disease, it begs a question if these factors are involved in regulating stem cell activity during I/R, and it has clinical significance with a number of clinically relevant drugs that regulate HIF activity.Mice with conditional deletion of HIFs in the intestinal stem cells need to be used to address this question.

Minor comments:
1) In figure 7 the micrographs of the intestine are provided but no histological quantitation is included.
2) The description of how Lrg5+ stem cells were purified is missing in the methods.
3) The explanation for Chiu's score and its significance needs to be explained.Similarly, the significance of findings in figure 3H needs to be explained.4) In lines 190-193 of the manuscript references and a context need to be provided for circRNAs involvement in disease regulation.5) In the methods section when researchers are blinded to the samples, it needs to be stated clearly and grammatically.

Reviewer #2 (Remarks to the Author):
major concers 1.The authors first measured the levels of CircEZH2_005 (EZH2 for short) in the plasma of patients undergoing cardiopulmonary bypass at various postoperative time points.It is true that postoperative cardiopulmonary bypass can cause inadequate blood supply to the small intestine, but the severity of ischemia is much lower than in the animal model applied in this paper.How to explain this mismatch.
2. The mouse IIR model uses the ischemia 60 min and reperfusion 120 min timepoint.
Although a minority of articles also use this time-point, reading through most articles on intestinal ischemia-reperfusion injury in mice, it is rarely used by investigators because very few mice can successfully survive to 120 minutes of reperfusion (even if the ischemic time is 30-45 minutes).Furthermore, 60 minutes of SMA ischemia in mice will cause widespread intestinal necrosis (not applicable to the study of various programmed cell deaths), such that mice will have difficulty surviving even through the 120-minute reperfusion period.This differs too much from the intestinal ischemia in patients undergoing clinical cardiopulmonary bypass.I wish the authors could provide a more comprehensive experimental procedure, an atlas, and operation specification for 60 minutes of intestinal ischemia and 120 minutes of reperfusion in mice, including whether/how to use anesthesia plan and ventilator, detailed anesthesia protocol, surgical procedures，the model of vascular clamps, perioperative monitoring methods, and fluid rehydration methods.
3. IFABP is a classical biomarker of intestinal ischemia-reperfusion injury.If EZH2 and IFABP are "similar", what is its advantage?Does it have advantages in terms of sensitivity, specificity, etc.? 4. Statistical problems: What is the reason for using standard errors instead of standard deviations?Have all data been tested for normal distribution and variance homogeneity?How are some data that apparently fail to be normally distributed statistically different?minor concerns 1.In Figure 5a, HR should be IR.
2. Note the font of some of the Greek letters.

Reviewer #3 (Remarks to the Author):
The study is well-structured with novel findings.It is worth publication; however, I suggest several amendments prior to it can be accepted.Methods are explained in detail, and the figures possess good quality.
The quality of the writing is not satisfying and requires vigorous editing and improvement.
The introduction is weak, unnecessarily long, and lacks a dynamic flow.circRNAs are not necessarily carried by exosomes.The authors are suggested to briefly discuss the structure, function, and clinical significance of exosomal circRNAs in a dynamic flow.A review of recent studies highlighting the importance of exosomal circRNAs in I/R injury and the novelty of the current study should be added appropriately to the introduction.
2-50 patients to evaluate exosomal circRNA expression seems a small population 3-Investigation of circEZH2_005 distribution in intestinal tissues seems unnecessary for discussing in a title 4-All claims should be treated cautiously using some terms like suggesting, may, can, etc. 5-The discussion is too long and requires shortening with a focus on explaining the study findings and their significance in therapy and diagnosis