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Dietary nitrate maintains homeostasis of oxidative stress and gut microbiota to promote flap survival in type 2 diabetes mellitus rats

BMC Endocrine Disorders volume 24, Article number: 184 (2024) Cite this article

Abstract

Background

Random-pattern skin flaps are commonly used to repair skin tissue defects in surgical tissue reconstruction. However, flap necrosis in the distal area due to ischemia injury is still challenging for its applications in plastic surgery. The complications of diabetes will further increase the risk of infection and necrosis.

Methods

This study induced type 2 diabetes mellitus (T2DM) rats with a high-fat diet and STZ. The survival rate of the skin flap was observed by adding inorganic sodium nitrate to drinking water. Histology and immunohistochemistry were used to detect the damage to the skin flap. The nitrate content was measured by total nitric oxide and nitrate/nitrite parameter assay. Dihydroethidium and malondialdehyde (MDA) assays were used to value oxidative stress. Rat colon feces were collected for 16s rRNA gene sequence.

Results

Our studies showed that nitrate administration leads to anti-obesity and anti-diabetic effects. Nitrate directly increased the survival area of skin flaps in diabetic rats and mean blood vessel density by enhancing angiogenesis, inhibiting apoptosis, and reducing oxidative stress. The 16s rRNA sequence revealed that nitrate may regulate the homeostasis of the gut microbiota and re-store energy metabolism.

Conclusion

Dietary nitrate has been shown to maintain the homeostasis of oxidative stress and gut microbiota to promote flap survival in rats with T2DM.

Peer Review reports

Background

Random-pattern skin flap transplantation is the main method for plastic repair of tissue defects caused by trauma, tumors, or malformations [1, 2]. It’s widely used with the advantage that the position and direction of the flap are not limited [3, 4]. Flap survival is closely associated with patient prognosis and the quality of life [5, 6]. Distal ischemic necrosis, a common clinical complication due to insufficient blood supply is the most crucial threat to flap survival, especially when the length-to-width ratio of the flap exceeds 2:1, which limits the use of flaps [7, 8]. This damage is primarily caused by oxidative stress and apoptosis.

Type 2 diabetes mellitus (T2DM) is a common metabolic disease mainly caused by insulin resistance. Significant hyperglycemia may lead to pathological conditions such as oxidative stress, chronic inflammation, and imbalanced energy metabolism [9]. The risk of tissue flap transplantation failure in patients with T2DM is 1.83 times of the general population [10], and the incidence of postoperative infection and other surgical complications is also significantly increased [10,11,12]. Currently, there are no effective methods for preventing or treating tissue flap necrosis associated with diabetes mellitus.

Inorganic nitrate is the most important resource for supplementing nitric oxide (NO) through the nitrate-nitrite-NO pathway [13]. Dietary nitrate may improve insulin sensitivity, metabolic abnormalities, and organ blood perfusion and regulate inflammatory reactions by restoring NO homeostasis in diabetic patients [14,15,16,17]. It has also been shown to improve vascular function after acute or chronic ischemic injury [18]. In ischemia-reperfusion (IR) injury of the heart, liver, and other vital organs, nitrate can reduce the early generation of intracellular reactive oxygen species (ROS) to relieve oxidative stress and reduce cell damage [18,19,20]. Previous studies have found that nitrite supplementation promotes healing of skin wounds in T2DM rats [21]. As mentioned above, we aimed to investigate the therapeutic effect of dietary nitrate and its mechanism of action on random-pattern skin flaps with T2DM.

Materials and methods

Animals

Animal experiments were performed with the approval of the Animal Welfare Management Association (approval number: KQYY-202010-001). Male Sprague-Dawley rats aged 4–6 weeks (100–150 g) were purchased from SPF Biotechnology Co. (Beijing, China) and housed in a standard condition, and free fed with food and water at least 7 days of local adaptation before experimental use (rats weighing 190–210 g).

Diabetes induction

Rats were fed with a high-fat diet (HFD, 60 kcal% fat, 20 kcal% carbohydrates, 20 kcal% proteins, D12492, SPF Biotechnology) for 4 weeks. Then diabetes was induced by 40 mg/kg intraperitoneal injection of Streptozotocin (STZ, Phygene® Scientific, Fuzhou, China) dissolved in sodium citrate buffer (0.1 M, pH = 4.5) [22]. After 72 h to one week, random and fasting blood glucose was detected with an automatic blood glucose analyzer (HGM-121, Omron Corporation, Kyoto, Japan). The random blood glucose test levels ≥ 250 mg/dL and fasting serum glucose levels ≥ 150 mg/dL were considered diabetes in this study [23,24,25].

Study design

Rats were then randomly divided into three groups(n = 6); Control group (Ctrl): rats consuming distilled water and a standard rodent chow (4% fat, SPF-F02-002, SPF Biotechnology), diabetes group (DM): diabetic rats drinking distilled water, diabetes + nitrate group (Nit): diabetic rats drinking sodium nitrate water (2mM) [26,27,28] for 3 weeks. Nitrate supplementation was started one week after confirmation of diabetes.

Blood glucose levels and body weight were recorded weekly.

Skin flap model

Three weeks after STZ injections, rats were anesthetized by intraperitoneal injection of pentobarbital sodium (40 mg/kg). The random-pattern skin flap was based on the modified McFarlane flap model [29,30,31]. After shaving hair and disinfecting, a caudal skin flap (3 cm×9 cm) in the central dorsum was marked on the rat’s back and separated from the subcutaneous deep fascia. The rats were anesthetized and euthanized by pentobarbital sodium overdose through intraperitoneal injection on postoperative days 3 or 7 to collect blood samples, kidneys, and flap tissues. The area of flap tissue collection is shown in Supplement Fig. S1.

Flap necrosis evaluation

On postoperative days 3 and 7, the dorsal flap was removed along the original incision after the rats were sacrificed. The skin surface and internal tissue surface are photographed. The survival and necrosis area were determined by appearance, color, and necrosis. The percentage of flap necrosis was calculated by the ImageJ software (National Institutes of Health, Bethesda, USA) with the formula: necrosis area/total area×100%.

Hematoxylin & eosin (HE) and Masson staining

The flap tissues were fixed with 4% paraformaldehyde embedded in paraffin and cut into 5-µm-thick slices. HE staining was performed to observe histological changes and a Masson’s trichrome staining kit (G1346, Solarbio, Beijing, China) was used to check the collagen fiber area. A light optical microscope (Olympus, Corp, Tokyo, Japan) was used to visualize the slices. Histological score and collagen fiber area were valued as described [32, 33].

Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL)

The apoptosis assay was performed using the TUNEL Assay Kit (C1086, Beyotime, Suzhou, China). Following the TUNEL reaction, slides (5 μm thickness) were sealed by fluoroshield with DAPI (F6057, Sigma, St. Louise, USA). Images were captured in six random fields per slide under a confocal microscope and the number of TUNEL-positive cells was counted by Image J software.

Immunohistochemistry (IHC) staining

Slides for IHC after dewaxing were antigen-repaired through microwave heating as previously described [34]. Sections were incubated with primary antibodies (CD31, ab182981, Abcam, Boston, USA; CD68, 28058-1-AP, Proteintech, Rosemont, USA) were incubated overnight at 4 °C. The following day, a secondary antibody was used for IHC (SP-9000, ZSGB-Bio, Beijing, China). Five different views in each section were captured. IHC of CD31 was used to calculate the microvessel density (MVD). The positive areas of CD31 and CD68 were calculated using Image J software.

Western blot analysis

The western blot was performed as previously described [19]. Flap tissues were collected, and protein was extracted and quantified by the BCA assay kit (P0011, Beyotime, Shanghai, China). The primary antibodies (BAX, 60267-1-Ig, Proteintech; BCL-2, 68103-1-Ig, Proteintech; CASPASE 3, A2156, Abclonal, Wuhan, China; iNOS, 18985-1-AP, Proteintech; eNOS, 27120-1-AP, Proteintech; KEAP1, 10503-2-AP, Proteintech; NRF2, 16396-1-AP, Proteintech; HO1, 10701-1-AP, Proteintech; NOX2, 19013-1-AP, Proteintech; NOX4, 14347-1-AP, Proteintech; β-actin, AC026, Abclonal) were incubated overnight at 4 °C after proteins were transferred to polyvinylidene fluoride membranes (ISEQ00010, Millipore, Massachusetts, USA). Next, the membranes were incubated with secondary antibodies (AS014 and AS003, Abclonal) the next day. Results were repeated three times and the data was analyzed by Image J and GraphPad Prism 8.0 (GraphPad Software, San Diego, USA).

Real-time polymerase chain reaction (RT-PCR)

The total RNA in the flaps was extracted by a Total RNA Extraction Kit (CW0581M, CWbio, Suzhou, China) with the manufacturer’s protocol. Then, the total RNA was reverse transcribed to cDNA using a PrimeScript RT Reagent Kit (RR047A, TaKaRa, Otsu, Japan), and quantitative RT-PCR was performed using the Ultra SYBR Mixture (CW2601M, CWbio). Quantitative RT-PCR analysis was performed further in Bio-Rad IQ5 thermocycler (Bio-Rad, CA, USA) following the procedures below: 10 min at 95 °C; 10 s at 95 °C; 30 s at 65 °C; 32 s at 72 °C; 40 cycles for total. Relative mRNA levels were normalized to values for β-actin mRNA expression and calculated by the 2−ΔΔCt method. Set the gene expression of the Ctrl group to 1(fold). The final gene expression levels were expressed as multiples of the Ctrl group. The primers used in RT-PCR are listed in Supplementary Table S1.

Enzyme-linked immunosorbent assay (ELISA)

Protein extraction and concentration measurement method as described above. ELISA was performed using commercial kits (specific to rats) to measure the content of TNF-α (438204, BioLegend, San Diego, CA, USA) and IL-1β (1310122, Dakewe, Shenzhen, China). The TNF-α and IL-1β content were assessed using a microplate reader at 450 nm (corrected at 570 nm). The sensitivity of TNF-α and IL-1β were 2 and 30 pg/mL, respectively. All inter-assay coefficient of variations (CVs) were below 10%, and all intra-assay CVs were below 15%.

Nitrate level in serum, kidney, and flap tissue

Flap and kidney tissues were obtained and homogenized to collect the supernatant and the protein content was measured using a BCA kit. Next, samples of flap, kidney, and serum were filtered using 10,000 MW filters and diluted. The total nitric oxide and nitrate/nitrite parameter assay kit (KGE001, R&D, MN, USA) were used to measure the concentration of nitrate. The sensitivity was 0.78 µmol/L. All inter- and intra-assay CVs were below 5%.

Measurement of superoxide dismutase (SOD) activity and Malondialdehyde(MDA) content

SOD activity and MDA content assessments were used to evaluate the oxidative stress status in flaps. The SOD activity and MDA content of the flap were measured using a total superoxide dismutase assay kit (S0101, Beyotime) and an MDA assay kit (S0131, Beyotime). The sensitivity of the SOD kit was 1 U/mL. Inter- and intra-assay CVs were 6.2% and 11.4%. The sensitivity of the MDA kit was 1 µmol/L. Inter- and intra-assay CVs were 8.4% and 13.9%.

Reactive oxygen species (ROS) assessments

The ROS generation in the flap was assessed by a dihydroethidium (DHE)-ROS assay kit (BB-47051, BestBio, Suzhou, China). Briefly, flap tissues were rapidly frozen and cut into 10-µm-thick slices and mounted on glass slides. Then slices were incubated with DHE at 37 °C for 30 min in darkness. Cell nuclei were stained with DAPI. Red fluorescence was observed using an Olympus fluorescence microscope at 610 nm wavelength and further analyzed by Image J software.

16 S rRNA gene sequencing technique to detect gut microbiota

The large intestine contents of the three groups were collected for 16s amplicon sequencing when the rats were sacrificed after the operation. The DNA extraction and library construction were performed by OE Biotech (Shanghai, China). Sequencing was performed on an Illumina NovaSeq6000 with two paired-end read cycles of 250 bases each (Illumina Inc., San Diego, CA). The representative read of each ASV was selected using QIIME 2. The microbial diversity in large intestine contents was estimated using the alpha diversity that includes the Chao1 index and Shannon index. The 16 S rRNA gene amplicon sequencing and analysis were conducted by OE Biotech.

Statistical analysis

Data are presented as mean ± standard error of the mean (SEM). All graphs were generated using GraphPad Prism 8.0 software. The data differences among groups were compared using Student’s t-tests or two-way analysis of variance (ANOVA). P-values < 0.05 were considered statistically significant.

Results

Dietary nitrate regulates body weight and blood glucose in T2DM rats

The experimental procedure is illustrated in Fig. 1A. After four weeks on a high-fat diet (HFD), T2DM was induced in rats by intraperitoneal injection of streptozotocin (STZ). Compared to the control group, T2DM rats in the DM and Nit groups had increased blood glucose levels and decreased body weight during the entire experimental period (Fig. 1B-C). Dietary nitrate significantly affected the body weight and random blood glucose in the third and fourth weeks (Fig. 1D-E).

Fig. 1
figure 1

Dietary nitrate regulates body weight and blood glucose in T2DM rats: (A) The experimental procedure: After 4 weeks of high-fat diet (HFD), STZ was injected intraperitoneally, nitrate was fed 1 week later, skin flap surgery was performed 3 weeks later, and anesthesia photos were taken on 3d and 7d after surgery; (B) Rat body weight per week; (C) Rat random blood glucose after STZ injection per week; (D, E) The rat body weight and random blood glucose after STZ injection at the 3rd and 4th week. * P < 0.05, ** P < 0.01

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Dietary nitrate alleviates necrosis of random flap in T2DM rats

The flaps were photographed and harvested on postoperative days 3 and 7 (Fig. 2A). The necrosis rates of the skin flaps differed considerably among the three groups (Fig. 2B, C). From day 3 onward, the circumscriptions between the surviving and necrotic areas became visible distal to the flaps. On the tissue side of the flap, the color and texture of the flap in the DM and control groups differed significantly. In the DM group, the skin flap was bleak in color, the capillaries were reduced, the distal area was significantly swollen, and the necrotic area was grayish-yellow with a necrotic ulcer. The distal portion (area III) was nearly necrotic in three groups due to the length-to-width ratio of the flap exceeded 2:1. The middle area showed the most significant difference (area II). The flaps of the control group were normal in area II, whereas the DM group had a larger region of necrosis than the Nit group (P < 0.01).

Fig. 2
figure 2

Dietary nitrate alleviates necrosis of random flap in T2DM rats. (A) Digital photograph of the skin side and tissue side of flaps on day 3 and day 7 after surgery(scale bar, 1 cm); (B, C) The percentage of damage area on day 3&7 of each group; (D, F) H&E and Masson staining of tissue sections of each group on day 3&7 (scale bar, 200 μm); (E, G) Histological score and Collagen volume fraction (collagen area/total area) of each group on day 3&7. * P < 0.05, ** P < 0.01, *** P < 0.001

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Based on these results, the tissues selected for subsequent experiments were all located in the middle region (Supplementary Figure S1). The middle region of the flap tissue was fixed and dehydrated, and histological hematoxylin and eosin staining was performed (Fig. 2D). Histological scores were performed according to the aspects of tissue inflammation, vacuolar degeneration, and hair follicle injury (Fig. 2E). Compared to the DM group, the Nit group had significantly lower histological scores (P < 0.001). Masson staining showed that the collagen fiber area in the Nit group recovered from the decrease in collagen fibers compared to the DM group (P < 0.01) (Fig. 2E-G).

Dietary nitrate maintained vascular density and reduced apoptosis of random flap

Immunohistochemistry of CD31 was used to detect the distribution of blood vessels and calculate microvessel density (Fig. 3A-B). At 3 and 7 days after the operation, the positivity rate of CD31 significantly decreased in the DM group, whereas the Nit group was able to maintain partial microvessel density. Tissue apoptosis was detected using the terminal deoxynucleotidyl transferase dUTP nick end labeling assay and western blotting. The number of apoptotic cells in the Nit group was remarkably lower than that in the DM group (P < 0.01) (Fig. 3C-D). Western blotting for BAX, BCL-2, and CASPASE 3 was performed to verify tissue apoptosis (Fig. 3E). The relative expression levels of the apoptotic protein BAX and CASPASE 3 significantly increased in the DM group (P < 0.05), while that of the anti-apoptotic protein BCL-2 was reduced (P < 0.05) (Fig. 3F-H). Collectively, these results suggest that inorganic sodium nitrate significantly reduced apoptosis in flaps that experienced ischemic injury due to T2DM.

Fig. 3
figure 3

Dietary nitrate maintained vascular density and reduced apoptosis. (A) The expression of CD31 of the flaps in three groups on day 3&7 detected in immunohistochemistry (scale bar, 50 μm); (B) The microvessel density calculated on day 3&7; (C, D) TUNEL staining (green) and the number of TUNEL positive cells of flap tissue in the three groups on day 3&7 (scale bar, 50 μm); (E) The expression of BAX, BCL-2, CASPASE 3 and β-actin in three groups on day 3&7 in western blotting; (F-G) Quantitative analysis of the expression of BAX, BCL-2 and CASPASE 3. Full-length blots are presented in Supplementary Figure S3. * P < 0.05, *** P < 0.001

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Dietary nitrate relieved inflammatory responses of the flap with T2DM

IL-1β, TNF-α, IL-6 and IL-17 are all considered as pro-inflammatory factors. The relative mRNA levels (Fig. 4A-B) and ELISA (Fig. 4E-F) results showed that IL-1β and TNF-α increased in the DM group. While the expression and content of IL-1β and TNF-α in the Nit group decreased compared to the DM group (P < 0.05). In addition, the mRNA levels of IL-6 and IL-17 in the DM group were also higher than the other two groups(Figs. 4C-D). Immunohistochemistry for CD68 showed that in the DM group, macrophages in flap tissues increased compared to the control group. However, the Nit group presented a significant decrease in CD68 expression compared to that in the DM group (p < 0.01) (Fig. 4G-H). The increased expression of pro-inflammatory factors and the macrophage aggregation indicate a more severe inflammatory response in the DM group and it might be relieved by inorganic sodium nitrate administration. .

Fig. 4
figure 4

Dietary nitrate relieved inflammatory responses. (A-D) The relative mRNA expression of IL-1β, TNF-α, IL-6 and IL-17 of the flaps in the three groups on day 3&7 in qRT-PCR assay; (E, F) The serum concentration of IL-1β and TNF-α in Elisa assay; (G, H) The expression and quantitative analysis of CD68 of the flaps in three groups on day 3&7 detected in immunohistochemistry (scale bar, 50 μm). * P < 0.05, ** P < 0.01, *** P < 0.001

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Dietary nitrate could maintain NO homeostasis in T2DM rats

Nitrate levels in the serum, kidney, and flap were measured using a total nitrate/nitrite/NO assay kit (Fig. 5A-C). It was found that the nitrate levels in the serum, kidney, and flap tissues were significantly higher in the Nit group than other groups, while it had no significant difference between the Control and DM groups, which suggested that dietary nitrates may increase nitrate levels in blood and tissues effectively. Protein and mRNA expression of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) was also detected (Fig. 5D-H). In the DM group, the expression of iNOS increased, whereas eNOS decreased. In the Nit group, the increased iNOS expression was alleviated, and the expression of eNOS was restored.

Fig. 5
figure 5

The effects of dietary nitrate on NO homeostasis maintaining in T2DM rats. (A-C) Nitrate levels in the serum, kidney, and flap of the rats in the three groups on day 3&7; (D-F) The expression and quantitative analysis of iNOS and eNOS of the flaps in all groups on day 3&7 in western blot analysis; (G, H) The relative mRNA expression of iNOS and eNOS of the flaps in the three groups on day 3&7 in qRT-PCR assay. Full-length blots are presented in Supplementary Figure S4. * P < 0.05, ** P < 0.01, *** P < 0.001

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Dietary nitrate attenuated oxidative stress in T2DM rats

Oxidative stress in the flap tissues was assessed by dihydroethidium (DHE) staining, superoxide dismutase (SOD), and malondialdehyde (MDA) determination. Specifically, intracellular ROS was positively stained with DHE. SOD measures intracellular antioxidant content, and MDA is considered an indicator of oxidative stress. As demonstrated in our results, nitrate application significantly reduced the fluorescence intensity of DHE staining in the three days after the operation (Fig. 6A-B). The SOD activity was significantly lower in the DM group than in the control group (Fig. 6C). The MDA concentration showed a reverse trend (Fig. 6D). Nitrate administration increased SOD activity and decreased MDA content in the Nit group compared to those in the DM group.

Fig. 6
figure 6

Dietary nitrate attenuated oxidative stress in T2DM rats. (A) The ROS content (Red) in the three groups on day 3 in DHE staining (scale bar, 50 μm); (B) The quantitative analysis of ROS content; (C, D) The measurement of SOD activity and MDA content of the flaps in all groups on day 3 & day 7; (E, F) The expression and quantitative analysis of KEAP1, NRF2, HO1, NOX2 and NOX4 of the flaps in all groups on day 3 & day 7 in western blot analysis. Full-length blots are presented in Supplementary Figure S5. * P < 0.05, ** P < 0.01, *** P < 0.001

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Considering the antioxidant capacity of the NRF2 pathway, we analyzed KEAP1, NRF2, and HO1 proteins in the three groups. The NADPH oxidase family of enzymes is one of the main sources of ROS. The expression levels of NOX2 and NOX4 were also examined (Fig. 6E-F). Western blotting results showed a significant upregulation of NRF2 and HO1 protein levels (P < 0.05) in nitrate-treated diabetic rats compared to the DM group. In contrast to NRF2, the protein expression of KEAP1 was increased in the DM group and decreased in the Nit group. In the DM groups, NOX2 and NOX4 levels increased significantly (P < 0.05) compared to those in the control group, but were lower in the Nit group, suggesting a protective effect of dietary nitrate.

Nitrate regulated the gut microbiota composition in T2DM rats

The overall structure of the gut microbiota was analyzed using the alpha diversity metric, which was measured using ACE richness, Chao1, Simpson index, and Shannon diversity index (See Supplementary Figure S2). Principal coordinate analysis (PCoA) was used in the present study to indicate community changes in different samples (Fig. 7A). Specifically, the abundance of Firmicutes in the DM and Nit groups decreased, whereas that of Proteobacteria was higher than that in the control group (Fig. 7B). Nitrate reduced the proportion of Bacteroides in T2DM rats. Notably, there was a characteristic Nitrospirota in the intestinal flora of rats in the Nit group; however, this bacterium was not detected in the DM group (Fig. 7C). Additionally, the three groups of animal flora showed significant differences at the genus level (Fig. 7D). The relative abundance of Pseudomonas in the DM group was significantly higher than in the other two groups (Fig. 7E). Linear discriminant analysis Effect Size (LEfSe) analysis showed the effects of nitrate on the intestinal flora in rats. In the DM group, T2DM rats showed increased harmful flora, such as Desulfovibrionaceae, Neisseria, and Pseudomonasceae, while nitrate levels decreased. In the Nit group, it was found that the intestinal Nitrospirota and Pelomonas were increased (Fig. 7F-G). Functional prediction analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) indicated that the function of nitrate-treated gut microbiota was linked to the enrichment of genes for carbohydrate metabolism, membrane transport, energy metabolism, nucleotide metabolism, signal transduction, and cellular community (Fig. 7H).

Fig. 7
figure 7

Nitrate regulated the gut microbiota composition in T2DM rats. (A) Principal coordinates analysis (PCoA) of three groups; (B) The top 15 microbiota community composition of three groups at the phylum level; (C) The top 10 boxplot of three groups at phylum level; (D) The different heatmap of three groups at genus level; (E) The relative abundance of Pseudomonas at genus level; (F, G) LEFSe analysis results of score plot and cladogram; (H) The KEGG predicted pathways at level 2

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Discussion

T2DM, the most common disease of the endocrine system, is mainly characterized by hyperglycemia and is usually accompanied by microcirculation disorders and multisystem complications [35, 36]. Studies have shown that disturbances in microvascular function are worse in patients with T2DM than in healthy ones [37, 38]. The disturbed microvascular function may be caused by endothelial dysfunction, vessel structural changes, and reduced NO bioavailability [39, 40].

NO is a multifunctional signal molecule that plays key roles in regulating vascular homeostasis by relaxing vascular tension, inhibiting platelet aggregation [41] and modulating inflammatory responses [40]. NO signaling plays an essential role in IR. It may counteract the increase in ROS caused by hypoxia and reduce oxidative stress and inflammation [42]. Under normal conditions, NO homeostasis is maintained mainly by endothelial NO synthase (eNOS) via L-arginine oxidation [43].

Although NO is essential for the body, it is quickly consumed and metabolized into nitrites and nitrates [13, 21]. NOS activity can be assessed by measuring the nitrate concentration under normal physiological conditions. However, the concentration of nitrate in the blood of patients with T2DM is decreased, and the activity of eNOS is also decreased [44]. Meanwhile, the activity of iNOS, which is also the source of NO but is mostly found in pathological conditions, is increased in these patients [45]. It was proved that excessive iNOS activity can aggravate disease progression [46, 47]. Thus, T2DM may aggravate ischemia injury and increase the possibility of distal necrosis of flaps [37, 38]. It was confirmed in our study that random flaps of T2DM rats had more severe ischemia injury and tissue apoptosis. Correspondingly, the lower eNOS expression, and higher iNOS expression in these tissues, indicate that NO homeostasis was seriously damaged. Dietary nitrate significantly increased the nitrate content in T2DM rats and may maintain NO homeostasis in the flap region by restoring eNOS expression and reducing iNOS expression. It indicates the therapeutic potential of dietary nitrate on flap necrosis in diabetic ones.

During tissue ischemia and reperfusion, ROS are usually produced explosively [48], which may increase oxidative stress and damage of various tissues. The damage caused by ROS in endothelial cells may enhance their adhesion to neutrophils and monocytes, change the visible components of the blood, and promote thrombosis formation [49]. Patients with T2DM are in a chronic state of oxidative stress generally, which may also affect endothelial function [50, 51]. Free tissue flap transplantation in these patients may have higher ROS production and more severe oxidative stress after surgery. Because of the excellent therapeutic effect of nitrate on both T2DM and IR injuries, we attempted to apply dietary nitrate in treating random skin flap injuries in T2DM rats. Dietary nitrate effectively increased the survival area of the flap in T2DM rats and reduced tissue apoptosis and inflammation. In addition, dietary nitrate enhanced SOD activity and reduced ROS production and MDA content in rats with T2DM, thereby relieving oxidative stress. The NADPH oxidase (NOX) family, which generates superoxide anions, was considered the primary source of ROS [52]. The decreased activity of NOX2 and NOX4, the key members of the NOX family, was found in diabetic rats feeding dietary nitrate and may lead to the reduction of ROS content.

Nuclear factor erythroid 2-related factor 2 (NRF2), one of the members of the cap ‘n’ collar family of basic region leucine zipper transcription factors, was proved to play an indispensable role in the induction of endogenous antioxidant enzymes against oxidative stress, such as SOD and Hemeoxygenase1 (HO1), which may reduce ROS content [53]. Kelch-like ECH-associated protein 1 (KEAP1) is the main repressor protein of NRF2, by binding to it to produce NRF2-Keap1 complex [54]. The downregulation of KEAP1 and the upregulation of NRF2 and HO1 were found in the Nit group, which indicates that the NRF2 pathway can be activated by dietary nitrate. It may be the key to the reduction of ROS content and oxidative stress damage.

Patients with T2DM usually showed intestinal microbial disturbances, and the numbers of some common butyrate-producing bacteria and mucinous Ackermannia were reduced [55]. The therapeutic effect of the regulation of gut microbiota on T2DM was proved in previous studys [56, 57]. Some drugs commonly used to treat T2DM, such as metformin and acarbose, also showed significant regulatory effects on the intestinal flora. In this study, we analyzed the colonic flora of rats and found distinctive Nitrospirota in the Nit group. Nitrospirota can metabolize nitrates and nitrites [58], allowing rats to consume more nitrides and maintain high nitrate levels. In addition, compared to the DM group, the nitrate diet reduced the number of harmful bacteria, such as Neisseria, Pseudomonadaceae, and Desulfovibrionaceae. KEGG analysis showed that the nitrate diet regulated the intestinal functions of rats related to energy metabolism and signal transduction. Above all, it indicates that dietary nitrate may improve the intestinal flora structure and maintain gut microbiota homeostasis, thus restoring the energy metabolism of T2DM rats. And it might also be one of the mechanisms for preventing weight loss and ameliorating blood glucose levels.

Conclusions

In summary, our results indicate that dietary nitrate ameliorates flap survival in T2DM rats. Supplementation with inorganic nitrate can effectively increase the nitrate level of T2DM ones, improve NO metabolism, and maintain the homeostasis of oxidative stress and gut microbiota. The regulation of oxidative stress may be dependent on the activation of the NRF2 pathway and the inhibition of the NOX family. Our study also supports that dietary nitrate may be one of the effective strategies for restoring the metabolism of one with T2DM by modifying gut microbiota.

Data availability

The data supporting the present study will be made available from the corresponding author on request.

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Acknowledgements

The authors acknowledge the technical support of OE Biotech (Shanghai, China).

Funding

This research was funded by the Beijing Stomatological Hospital, Capital Medical University Young Scientist Program, grant number YSP202008; the National Natural Science Foundation of China, grant number 81974144; Beijing Municipal Administration of Hospitals Incubating Program, code: PX2024056; Innovation Foundation of Beijing Stomatological Hospital, Capital Medical University, code: 23-09-19.

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Author notes

  1. Qifang Niu and Delong Li contributed equally to this research.

Authors and Affiliations

  1. Department of Stomatology, Beijing Tiantan Hospital, Capital Medical University, Beijing, China

    Qifang Niu

  2. Department of Oral and Maxillofacial-Head and Neck Oncology, Beijing Stomatological Hospital, Capital Medical University, Beijing, China

    Delong Li, Zhien Feng, Zhengxue Han & Yang Yang

  3. Department of Stomatology, First Medical Center, Chinese PLA General Hospital, Beijing, China

    Wenwen Guo

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  1. Qifang Niu
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  4. Zhien Feng
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  6. Yang Yang
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Contributions

Conceptualization, Y.Y. and Z.H.; methodology, Q.N. and Y.Y.; validation, Q.N., D.L. and W.G.; formal analysis, D.L.; investigation, Z.F.; resources, Y.Y. and Z.H.; data curation, Q.N. and D.L.; writing—original draft preparation, Q.N. and D.L.; writing—review and editing, Y.Y. and Z.H.; visualization, D.L. and Z.F.; supervision, Y.Y. and Z.H.; project administration, Y.Y. and Z.H.; funding acquisition, Y.Y. and Z.H. All authors have read and agreed to the published version of the manuscript.

Corresponding author

Correspondence to Yang Yang.

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Ethics approval and consent to participate

All experiments were performed according to the ARRIVE guidelines (https://arriveguidelines.org) and the guidelines and regulations of the Animal Ethics Regulation Committee of Beijing Stomatological Hospital (Capital Medical University, Beijing, China). The animal experiments were approved by the Animal Welfare Management Association of Beijing Stomatological Hospital (approval number: KQYY-202010-001). All animal procedures are carried out in accordance with relevant guidelines and regulations. The consent to participate is not applicable in this study.

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The authors declare no competing interests.

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Niu, Q., Li, D., Guo, W. et al. Dietary nitrate maintains homeostasis of oxidative stress and gut microbiota to promote flap survival in type 2 diabetes mellitus rats. BMC Endocr Disord 24, 184 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12902-024-01691-5

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BMC Endocrine Disorders

ISSN: 1472-6823

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