Application of Piezo1 inhibitor in preparation of medicine for treating necrotizing enterocolitis

Abstract

The invention discloses an application of a Piezo1 inhibitor in preparation of a medicine for treating necrotizing enterocolitis. The Piezo1 inhibitor can be applied in preparation of the medicine for treating the necrotizing enterocolitis. The Piezo1 inhibitor can also be applied to preparation of drugs for relieving NEC intestinal epithelial barrier injury. The Piezo1 inhibitor can also be applied to preparation of drugs for reducing calcium influx in intestinal epithelial cells and calcium / calmodulin dependent protein kinase activity. The application has the beneficial effects that the effect and mechanism of the Piezo1 for regulating and controlling the calcium ion influx and promoting the NEC severity degree by the calcium / calmodulin dependent protein kinase activity level are discussed for the first time from multiple aspects of animals, tissues, cells, molecules and the like, and the effect and mechanism of the activated intestinal epithelial cell Piezo1 in NEC inflammatory injury and barrier repair are clarified for the first time; the knockout of the gene can improve the NEC severity of mice, and a new intervention target is provided for clinical treatment.

Description

Technical Field

[0001] The present invention relates to application of a Piezo1 inhibitor in preparing medicines, and in particular to application of a Piezo1 inhibitor in preparing medicines for treating necrotizing enterocolitis. Background Art

[0002] Currently, necrotizing enterocolitis (NEC) is a serious neonatal gastrointestinal emergency, common in extremely low birth weight infants. It is characterized by intestinal inflammation, cell death and tissue necrosis, often leading to high morbidity and mortality. Existing treatments include fasting, gastrointestinal decompression, antibiotic treatment, surgical intervention and supportive care, but these methods often have limited effects, and postoperative complications such as short bowel syndrome seriously affect the quality of life of patients.

[0003] The exact etiology of NEC remains elusive, but it is generally believed that it involves a complex interaction of multiple factors, including intestinal immaturity, microbial dysbiosis, and inappropriate inflammatory responses. Intestinal epithelial cells play a key role in maintaining intestinal barrier integrity and immune homeostasis. Recent studies have shown that Piezo1, a mechanosensitive ion channel, is a key regulator of the response of various tissue cells to mechanical stimulation. Piezo1 is widely expressed in intestinal epithelial cells and is associated with the regulation of epithelial integrity and inflammatory signaling pathways, but the specific role of Piezo1 in the pathological mechanism of NEC and its potential as a therapeutic target have not been reported so far. Piezo1 has the effect of aggravating the inflammatory response of necrotizing enterocolitis in mice. Summary of the invention

[0004] The purpose of the present invention is to solve the problem that there is no specific drug for treating necrotizing enterocolitis in the clinic, and to provide an application of a Piezo1 inhibitor in the preparation of a drug for treating necrotizing enterocolitis.

[0005] The Piezo1 inhibitor provided by the present invention can be used in the preparation of medicines for treating necrotizing enterocolitis.

[0006] The Piezo1 inhibitor provided by the present invention can also be used in the preparation of a drug for alleviating intestinal epithelial barrier damage in NEC.

[0007] The Piezo1 inhibitor provided by the present invention can also be used in the preparation of drugs for reducing calcium influx and calcium / calmodulin-dependent protein kinase activity in intestinal epithelial cells.

[0008] The Piezo1 inhibitor is a substance that inhibits the activity of Piezo1 or a substance that reduces the level of Piezo1. The substance that inhibits the activity of Piezo1 is GsMTx4 or a calcium / calmodulin-dependent protein kinase inhibitor or a calcium chelator; the substance that reduces the level of Piezo1 is an interfering RNA or a microRNA or a gene knockout material.

[0009] Beneficial effects of the present invention:

[0010] The Piezo1 inhibitor provided by the present invention can be used in the preparation of a drug for treating necrotizing enterocolitis, and its active ingredient is a substance that inhibits the activity of Piezo1 or a substance that reduces the level of Piezo1; the drug or pharmaceutical composition provided by the present invention has the following effects:

[0011] (a) prevention and / or treatment of necrotizing enterocolitis;

[0012] (b) Relieve and / or improve intestinal pathological damage caused by necrotizing enterocolitis;

[0013] (c) improve the survival rate of necrotizing enterocolitis;

[0014] (d) Alleviate intestinal permeability;

[0015] (e) reduce intestinal epithelial barrier damage;

[0016] (f) inhibiting the expression levels of intestinal inflammatory factors TNF-α and / or IL-6;

[0017] This invention is the first to explore the role and mechanism of Piezo1 in regulating calcium ion influx and calcium / calmodulin-dependent protein kinase activity levels in promoting the severity of NEC from multiple levels such as animals, tissues, cells, and molecules. This invention is the first to clarify the role and mechanism of Piezo1 activation in intestinal epithelial cells in NEC inflammatory damage and barrier repair. Its knockout can improve the severity of NEC in mice, providing a new intervention target for clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the expression level of Piezo1 in NEC intestinal tissue in Example 1. Figure 1 A: Real-time fluorescence quantitative PCR technique was used to detect the expression level of Piezo1 in intestinal tissue cDNA; Figure 1 B: The expression level of Piezo1 was detected by immunohistochemistry.

[0019] Figure 2 This is a schematic diagram of the intestinal pathological damage of each treatment group in Example 1. Figure 2 A is the gross picture and HE staining picture of the intestinal tissue of suckling mice. Figure 2B is the statistical graph of HE pathological disease scores in the intestinal tissues of suckling mice in each treatment group.

[0020] Figure 3 Schematic diagram of the survival curve of suckling mice in each treatment group.

[0021] Figure 4 Schematic diagram of the expression levels of tight junction proteins in the intestine of suckling mice in each treatment group.

[0022] Figure 5 Schematic diagram of the expression level of Tunel in the intestine of suckling mice in each treatment group.

[0023] Figure 6 Schematic diagram of mouse gene identification results.

[0024] Figure 7 This is the effect of intestinal epithelial cell-specific Piezo1 knockout on NEC intestinal pathological damage in mice in Example 2. Figure 7 A is the gross image and HE staining image of intestinal tissue in each treatment group. Figure 7 B is the statistical graph of HE pathological disease scores of intestinal tissues in each treatment group.

[0025] Figure 8 Schematic diagram of the effect of intestinal epithelial cell-specific Piezo1 knockout on the survival rate of NEC in mice.

[0026] Fig. 9 Schematic diagram of the effect of intestinal epithelial cell-specific Piezo1 knockout on intestinal permeability and intestinal Tunel expression levels in mice.

[0027] Fig.10 The effect of the Piezo1 channel state on the in vitro NEC intestinal epithelial barrier in Example 3. Fig.10 A is the immunofluorescence image of tight junction proteins in each treatment group. Fig.10 B is the statistical graph of tight junction protein mRNA expression levels in each treatment group.

[0028] Fig.11 Schematic diagram of the Piezo1 channel status and the effect of Piezo1 knockout in intestinal epithelial cells on intracellular calcium ion concentration. Fig.11 A is the effect of Piezo1 channel status on intracellular calcium ion concentration. Fig.11 B shows the effect of Piezo1 knockout in intestinal epithelial cells on intracellular calcium ion concentration.

[0029] Fig.12 Schematic diagram of detecting the effect of Piezo1 on pCaMKII / CaMKII by Western blotting experiment.

[0030] Comparison between the experimental group and the control group: *P<0.05, **P<0.01, ***P<0.001, ns means no statistical difference. DETAILED DESCRIPTION

[0031] See also Figures 1 to 12 As shown:

[0032] The Piezo1 inhibitor provided by the present invention can be used in the preparation of medicines for treating necrotizing enterocolitis.

[0033] The Piezo1 inhibitor provided by the present invention can also be used in the preparation of a drug for alleviating intestinal epithelial barrier damage in NEC.

[0034] The Piezo1 inhibitor provided by the present invention can also be used in the preparation of drugs for reducing calcium influx and calcium / calmodulin-dependent protein kinase activity in intestinal epithelial cells.

[0035] The Piezo1 inhibitor is a substance that inhibits the activity of Piezo1 or a substance that reduces the level of Piezo1. The substance that inhibits the activity of Piezo1 is GsMTx4 or a calcium / calmodulin-dependent protein kinase inhibitor or a calcium chelator; the substance that reduces the level of Piezo1 is an interfering RNA or a microRNA or a gene knockout material.

[0036] The specific test process is as follows:

[0037] Experimental Example 1: Effects of the wild-type mouse NEC model on Piezo1 and intestinal epithelial cells

[0038] 1. Test methods

[0039] 1.1 Test subjects

[0040] 7-day-old C57BL / 6J suckling mice, either sex, were randomly divided into groups, with 6 to 8 mice in each group;

[0041] 1.2 Trial Grouping

[0042] The mice were divided into 4 groups: 1. breast milk control group (Ctrl); 2. NEC model group (NEC); 3. breast milk feeding group and intraperitoneal administration of Piezo1 activator group (Ctrl+Yoda1); 4. NEC model group and administration of Piezo1 activator group (NEC+Yoda1)

[0043] 1.3 Test steps:

[0044] The mouse NEC model was established by the "hypoxia-formula-oral gavage of feces from surgical NEC children" method [1-2], which was induced for 4 days. At the beginning of the modeling, the newborn mice were separated from the mother mice and gavaged with formula milk at 40μl / g body weight 5 times a day. The formula milk was prepared by Abbott Similac infant formula and PetAg Esbilac puppy formula at a ratio of 2:1, and each 1ml of formula milk contained 12.5μl of fecal homogenate from surgical NEC children. During the modeling period, the newborn mice were subjected to hypoxia twice a day, and the newborn mice were placed in a 5% O2-95% N2 hypoxia box for 10 minutes. The above operation was performed for a total of 4 days. In the NEC+Yoda1 group, Yoda1 was injected intraperitoneally into the suckling mice before modeling every day, 2.5umol / kg / day, once a day, for 4 consecutive days, and the body weight and survival status were monitored every day; on the 5th day, the suckling mice were killed by the method, and the intestinal tissues of the suckling mice were obtained. A part of the intestine was used for HE staining, immunohistochemistry and immunofluorescence detection; a part of the ileum was used to extract tissue mRNA and protein for subsequent RT-PCR and Western blot detection.

[0045] Experimental results:

[0046] Piezo1 expression

[0047] Real-time PCR and immunohistochemistry were used to detect the expression level of Piezo1 in NEC intestinal tissues. Figure 1 As shown, the expression level of Piezo1 in NEC samples was higher than that in the control group.

[0048] 2.2 HE pathological disease score[3]

[0049] Scoring criteria:

[0050] 0 points, no damage;

[0051] 1 point, slight separation of the submucosal layer and / or lamina propria;

[0052] 2 points, moderate separation of the submucosal layer and / or lamina propria;

[0053] 3 points: severe separation of the submucosal layer and / or lamina propria; severe edema of the submucosal layer and muscle layer and regional villous loss;

[0054] 4 points: villous loss and necrosis.

[0055] The gross observation and HE staining images of the intestinal tissues of the suckling mice in each treatment group are shown in the figure. Figure 2 As shown in A, the statistical results of intestinal tissue HE pathological disease scores are Figure 2 As shown in B, Figure 2 It can be seen that intraperitoneal injection of Piezo1 activators increased NEC intestinal pathological damage.

[0056] 2.3 Survival of suckling mice

[0057] The results are as follows Figure 3 shown.

[0058] Breast milk control group and Ctrl+Yoda1 group: survival rate 100%;

[0059] NEC model group: survival rate 75%;

[0060] NEC+Yoda1 group: survival rate 60%.

[0061] 2.4. Expression of intestinal tight junction proteins and intestinal permeability detection

[0062] Immunofluorescence staining and Western blot were used to observe the expression of intestinal tight junction proteins ZO-1, Claudin-1, Occludin, and cell adhesion protein E-cadherin, and intestinal permeability was detected by measuring serum FITC-dextran content. Figure 4 As shown. Figure 4 It can be seen that intraperitoneal injection of Yoda1 destroyed the intestinal epithelial barrier and increased the intestinal permeability of NEC mice.

[0063] 2.5. Detection of intestinal epithelial cell apoptosis

[0064] Immunofluorescence staining was used to observe the expression of protein Tunel. The results are as follows Figure 5 Intraperitoneal injection of Yoda1 aggravated intestinal epithelial cell death in NEC mice.

[0065] Experimental Example 2: Effect of intestinal epithelial cell-specific Piezo1 knockout mice on intestinal epithelial cell Piezo1 regulation of NEC inflammatory response

[0066] 1. Test methods

[0067] 1.1. Test subjects

[0068] Conditional Piezo1 knockout mice (Piezo1fl / fl) were purchased from the laboratory of Saiye Biotechnology Co., Ltd., and Villin-Cre transgenic mice were purchased from Jackson Laboratory in the United States. Piezo1fl / fl mice and Villin-Cre mice were hybridized to obtain intestinal epithelial cell-specific Piezo1 knockout (Piezo1vill KO) mice, and Villin-Cre gene-negative Piezo1fl / fl mice in the same litter were used as experimental control mice.

[0069] 1.2 Experimental Grouping

[0070] The mice were divided into 4 groups, namely Piezo1fl / fl+breast-feeding control group, Piezo1fl / fl+NEC model group, Piezo1vill KO+breast-feeding control group and Piezo1vill KO+NEC model group, with 6-10 mice in each group.

[0071] 2. Experimental results:

[0072] 2.1 Mouse gene identification

[0073] The band position of Piezo1fl / fl mice detected by PCR was located at 210bp ( Figure 6 A), the band position of Villin-Cre mice, located at 304 bp ( Figure 6 B).

[0074] 2.2 Effects of intestinal epithelial cell-specific Piezo1 knockout on NEC pathological damage in mice

[0075] After establishing the NEC model with Piezo1fl / fl and Piezo1vill KO mice, intestinal damage and mouse survival were detected. Intestinal macroscopic and H&E staining showed that the degree of villus destruction in Piezo1vill KO mice was significantly reduced compared with that in Piezo1fl / fl mice ( Figure 7 A), Statistical results of HE pathological disease scores in Piezo1vill KO mice Figure 7 As shown in B, Figure 7 It can be seen that intestinal epithelial cell-specific Piezo1 knockout attenuated NEC intestinal pathological damage.

[0076] 2.3 Survival of suckling mice

[0077] The results are as follows Figure 8 As shown in the figure, the survival rate of the two groups of mice was 100% in the breast milk control group, 75% in the Piezo1vill KO+NEC model group, and 66.7% in the Piezo1fl / fl+NEC model group.

[0078] 2.4. Intestinal permeability test and intestinal epithelial cell apoptosis test

[0079] Intestinal permeability was measured by serum FITC-dextran content. Fig. 9 As shown in A, intestinal epithelial cell-specific Piezo1 knockout alleviated NEC-related intestinal barrier damage; immunohistochemical experiments observed the expression of intestinal tissue protein Tunel, and the results were as follows Fig. 9 (B) Intestinal epithelial cell-specific knockout of Piezo1 reduced the extent of intestinal epithelial cell death in NEC mice.

[0080] Experimental Example 3: Effects and Mechanisms of Piezo1 in Cultured Intestinal Epithelial Cells on the Regulation of NEC in Vitro 1. Experimental Methods

[0081] 1.1. Test subjects

[0082] Caco-2 cells were used to construct shRNA-Piezo1 interference plasmids using shRNA interference technology to silence Piezo1 gene expression (the shPiezo1 control group was treated with blank plasmids), Yoda1 (Piezo1-specific agonist) was used to activate Piezo1 channels, KN-93 (CaMKII inhibitor) and BAPTA (calcium chelator) were used to treat, and the effects of intestinal epithelial cell Piezo1 on intracellular calcium ion concentration and CaMKII pathway were observed.

[0083] 1.2 Experimental Grouping

[0084] An in vitro NEC model was constructed by stimulating Caco-2 cells with LPS. Yoda1 (Piezo1-specific agonist) was used to activate the Piezo1 channel, and GsMTx4 (Piezo1-specific inhibitor) was used to inhibit the Piezo1 channel. Finally, the cells were divided into 4 groups (N=3), namely, Ctrl group, LPS group, LPS+Yoda1 group and LPS+GsMTx4 group.

[0085] 1.3 Experimental Grouping

[0086] There were 6 groups (N=3) in total, namely shCtrl group, shCtrl+LPS group, shCtrl+LPS+Yoda1 group, shPiezo1 group, shPiezo1+LPS group, and shPiezo1+LPS+Yoda1 group.

[0087] 1.4 Experimental Grouping

[0088] There were 8 groups (N=3) in total. Under LPS stimulation, they were shCtrl group, shCtrl+Yoda1 group, shCtrl+KN-93 / BAPTA+Yoda1 group, shCtrl+KN-93 / BAPTA group, shPiezo1 group, shPiezo1+Yoda1 group, shPiezo1++KN-93 / BAPTA+Yoda1 group, and shPiezo1++KN-93 / BAPTA group.

[0089] 2. Experimental results:

[0090] 2.1 Effects of Piezo1 channel status on the intestinal epithelial barrier of NEC in vitro

[0091] Immunofluorescence staining and Real-time PCR were used to observe the expression of intestinal epithelial cell tight junction proteins ZO-1, Claudin-1, and cell adhesion protein E-cadherin. Fig.10 As shown, Piezo1 activation aggravated LPS-induced intestinal epithelial barrier disruption, while inhibition of Piezo1 channels attenuated the extent of barrier disruption.

[0092] 2.2. Effects of Piezo1 channel status and Piezo1 knockout in intestinal epithelial cells on intracellular calcium concentration

[0093] After the Caco-2 cells with different stimulations were washed twice with HANKs solution, Fluo-8AM (final concentration 5 μmol / L) was dissolved in D-HANKs solution in a dark environment and added to the cells for incubation at 37°C for 30 minutes to 1 hour. The intracellular calcium ion fluorescence value was then measured with an ELISA reader. The results are as follows: Fig.11 As shown, the intracellular calcium ion concentration increased significantly after Piezo1 channel activation, while the cellular calcium ion concentration decreased significantly after Piezo1 knockout, indicating that Piezo1 plays a vital role in regulating Ca2+ influx.

[0094] 2.3 Effect of Piezo1 on the CaMKII pathway

[0095] Western blot revealed that p-CaMKII / CaMKII was significantly downregulated after Piezo1 knockout in intestinal epithelial cells ( Fig.12 ), while the CaMKII pathway was significantly activated after Yoda1 stimulation ( Fig.12 A), indicating that Piezo1 acts through the CaMKII pathway. After the use of CaMKII inhibitors or calcium chelators, p-CaMKII / CaMKII was significantly downregulated ( Fig.12 B), and Yoda1 stimulation also failed to reactivate CaMKII activity.

Claims

1. Use of a Piezo1 inhibitor in the preparation of a drug for treating necrotizing enterocolitis.

2. Application of a Piezo1 inhibitor in the preparation of a drug for alleviating intestinal epithelial barrier damage in NEC.

3. Use of a Piezo1 inhibitor in the preparation of a drug for reducing calcium influx and calcium / calmodulin-dependent protein kinase activity in intestinal epithelial cells.

4. Use of a Piezo1 inhibitor according to claim 1 in the preparation of a drug for treating necrotizing enterocolitis, characterized in that: The Piezo1 inhibitor is a substance that inhibits Piezo1 activity or reduces Piezo1 levels. The substance that inhibits Piezo1 activity is GsMTx4 or a calcium / calmodulin-dependent protein kinase inhibitor or a calcium chelator; the substance that reduces Piezo1 levels is an interfering RNA or a microRNA or a gene knockout material.

5. The use of a Piezo1 inhibitor according to claim 2 in the preparation of a drug for alleviating intestinal epithelial barrier damage in NEC is characterized by: The Piezo1 inhibitor is a substance that inhibits Piezo1 activity or reduces Piezo1 levels. The substance that inhibits Piezo1 activity is GsMTx4 or a calcium / calmodulin-dependent protein kinase inhibitor or a calcium chelator; the substance that reduces Piezo1 levels is an interfering RNA or a microRNA or a gene knockout material.

6. Use of a Piezo1 inhibitor according to claim 3 in the preparation of a drug for reducing calcium influx and calcium / calmodulin-dependent protein kinase activity in intestinal epithelial cells, characterized in that: The Piezo1 inhibitor is a substance that inhibits Piezo1 activity or reduces Piezo1 levels. The substance that inhibits Piezo1 activity is GsMTx4 or a calcium / calmodulin-dependent protein kinase inhibitor or a calcium chelator; the substance that reduces Piezo1 levels is an interfering RNA or a microRNA or a gene knockout material.

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