Electrophysiology, molecular modelling, and functional analysis of the effects of dietary quercetin and flavonoid analogues on Kir6.1 channels in rat stomach fundus smooth muscle.

Flavonoids, ubiquitously distributed in the plant world, are regularly ingested with diets rich in fruit, vegetables, wine, and tea. During digestion, they are partially absorbed in the stomach. The present work aimed to assess the in vitro effects of quercetin and ten structurally related flavonoids on the rat gastric fundus smooth muscle, focussing on ATP-dependent K+ (Kir6.1) channels, which play a central role in the regulation of resting membrane potential, membrane excitability and, consequently, of gastric motility. Whole-cell currents through Kir6.1 channels (IKir6.1) were recorded with the patch-clamp technique and the mechanical activity of gastric fundus smooth muscle strips was studied under isometric conditions. Galangin ≈ tamarixetin > quercetin > kaempferol > isorhamnetin ≈ luteolin ≈ fisetin > (±)-taxifolin inhibited pinacidil-evoked, glibenclamide-sensitive IKir6.1 in a concentration-dependent manner. Morin, rutin, and myricetin were ineffective. The steric hindrance of the molecule and the number and position of hydroxyl groups on the B ring played an important role in the activity of the molecule. Molecular docking simulations revealed a possible binding site for flavonoids in the C-terminal domain of the Kir6.1 channel subunit SUR2B, in a flexible loop formed by residues 251 to 254 of chains C and D. Galangin and tamarixetin, but not rutin relaxed both high K+- and carbachol-induced contraction of fundus strips in a concentration-dependent manner. Furthermore, both flavonoids shifted to the right the concentration-relaxation curves to either pinacidil or L-cysteine constructed in strips pre-contracted by high K+, rutin being ineffective. In conclusion, IKir6.1 inhibition exerted by dietary flavonoids might counterbalance their myorelaxant activity, affect gastric accommodation or, at least, some stages of digestion.

Near-infrared fluorophore IR-61 delays postovulatory aging of mouse oocytes through suppressing oxidative stress mediated by mitochondrial protection.

Postovulatory aging can trigger deterioration of oocyte quality and subsequent embryonic development, and thus reduce the success rates of assisted reproductive technology (ART). The molecular mechanisms underlying postovulatory aging, and preventative strategies, remain to be explored. The near-infrared fluorophore IR-61, a novel heptamethine cyanine dye, has the potential for mitochondrial targeting and cell protection. In this study, we found that IR-61 accumulated in oocyte mitochondria and reduced the postovulatory aging-induced decline in mitochondrial function, including mitochondrial distribution, membrane potential, mtDNA number, ATP levels, and mitochondrial ultrastructure. In addition, IR-61 rescued postovulatory aging-caused oocyte fragmentation, defects in spindle structure, and embryonic developmental potential. RNA sequencing analysis indicated that the postovulatory aging-induced oxidative stress pathway might be inhibited by IR-61. We then confirmed that IR-61 decreased the levels of reactive oxygen species and MitoSOX, and increased GSH content in aged oocytes. Collectively, the results indicate that IR-61 may prevent postovulatory aging by rescuing oocyte quality, promoting successful rate in ART procedure.

Near-infrared fluorophore IR-61 improves the quality of oocytes in aged mice via mitochondrial protection.

Maternal aging is associated with a decline in oocyte quality, which leads to the decreased fertility. Therefore, developing approaches to reduce aging-induced deterioration of oocyte quality in older women is important. Near-infrared cell protector-61 (IR-61), a novel heptamethine cyanine dye, has the potential for antioxidant effects. In this study, we found that IR-61 can accumulate in the ovaries and improved ovarian function of naturally aged mice; it also increased the oocyte maturation rate and quality by maintaining the integrity of the spindle/chromosomal structure and reducing the aneuploidy rate. In addition, the embryonic developmental competence of aged oocytes was improved. Finally, RNA-sequencing analysis indicated that IR-61 might perform the beneficial effects on aged oocytes by regulating mitochondrial function, this was confirmed by immunofluorescence analysis of mitochondrial distribution and reactive oxygen species. Taken together, our findings demonstrate that IR-61 supplementation in vivo can increase oocyte quality and protect oocytes from aging-induced mitochondrial dysfunction, and thus could improve the fertility of older women and efficiency of assisted reproductive technology.

Near-infrared Nrf2 activator IR-61 dye alleviates radiation-induced lung injury.

Oxidative stress injury and subsequent inflammatory response are considered to play critical roles in radiation-induced lung injury (RILI). Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor that regulates oxidative stress response and represses inflammation, but its therapeutic value in RILI remains elusive. Our previous studies have shown that the near-infrared (NIR) IR-61 dye evokes intracellular antioxidant defense by enhancing Nrf2 signaling and promoting anti-inflammatory effects. We established a model of RILI in mice exposed to whole-thoracic irradiation. The results showed that IR-61 treatment notably improved pulmonary functions by decreasing lung density and diminishing airway resistance. In addition, IR-61 significantly ameliorated radiation-induced inflammatory cell infiltration and proinflammatory cytokine (IL-1ß, IL-6, and TNF-α) release, thereby mitigating inflammatory response. Furthermore, IR-61 mitigated radiation-induced lung fibrosis by decreasing the collagen deposition and the levels of fibrogenesis-related factors (collagen I, collagen III, α-SMA, and fibronectin). More importantly, IR-61 was found to accumulate in the mitochondria of macrophages in irradiated lung tissues. Therefore, the functions of IR-61 in macrophages were further studied in irradiated macrophage cell lines, MH-s and RAW 264.7 in vitro. The results indicated that IR-61 upregulated the expression of Nrf2 and heme oxygenase-1 (HO-1) and decreased the levels of reactive oxygen species (ROS) and pro-inflammatory cytokines (IL-1ß and IL-6) in macrophages after radiation. In summary, our study suggests that IR-61 effectively mitigates RILI by activating Nrf2 signaling in irradiated lung tissues. In particular, Nrf2-mediated anti-inflammatory and antioxidant effects in irradiated lung tissue macrophages play critical roles in protecting against RILI.

[Experimental study of mitochondrion-targeted small molecule IR-61 ameliorated exhaustive exercise-induced cardiac injury in rats].

OBJECTIVE: To investigate the effects of mitochondrion-targeted cyanine fluorescent small molecule IR-61 on cardiac injury induced by exhaustive exercise in rats. METHODS: Thirty-six adult male SD rats were randomly divided into 3 groups(n=12),control group (Ctrl), exhaustive exercise group (EE) and IR-61+ exhaustive exercise group (IR-61+EE). IR-61+EE group were intraperitoneally injected with 2 mg/kg IR-61 at the same time on day 1, 4 and 7. One hour after the end of the last drug administration, the two exhaustive exercise groups were subjected to exhaustive exercise modeling. The rats were placed on an animal treadmill with a slope of 0° at a speed of 10~15 m/min to coordinate their limbs running posture, and then ran at a speed of 25~30 m/min until exhaustion about 15 minutes later. After the animal models established, ECG was recorded by physiological recorder, myocardial injury was observed by light microscope, mitochondrial injury was observed by transmission electron microscope, myocardial cell apoptosis was detected by TUNEL method, markers of myocardial injury were detected by ELISA, and myocardial mitochondrial respiration rate was measured by high-resolution Oxygraph-2K mitochondrial instrument. RESULTS: ① Compared with Ctrl group, heart rate was increased, PR interval was shortened, QRS interval was prolonged, QTc was prolonged and ST segment was depressed significantly in EE group (P＜0.05). In EE group, myocardial fiber fracture and mitochondrial inner chamber swelling were obvious, mitochondrial crest was fuzzy, mitochondrial outer membrane was incomplete, and a large number of mitochondrial rupture and fusion were visible. In EE group, TUNEL staining cells were abundant, chromatin concentration and marginalization, nuclear membrane lysis, chromatin fragmentation into massive apoptotic bodies, apoptosis score increased (P＜0.05). The levels of creatine kinase isoenzyme-MB (CK-MB), cardiac troponin I(cTn-I) and N-terminal B-type natriuretic peptide (NT-proBNP) were increased in EE group (P＜0.05). Basal respiration rate, oxidative respiration rate of fatty acids and respiration rate of complex Ⅰ, Ⅱ and Ⅳ were all decreased (P＜ 0.05). ② Compared with EE group, the heart rate in IR-61+EE group was increased, PR interval was prolonged, QRS interval was shortened, QTc was shortened, ST segment was not significantly depressed (P＜0.05). In IR-61+EE group, myocardial fiber arrangement was loose, no obvious fracture was observed, mitochondrial inner ventricle was swelling, mitochondrial outer membrane was intact, TUNEL stained cells and unstained cells were observed, the overall morphology was more similar to Ctrl group. Apoptosis index was decreased (P＜0.05), the levels of CK-MB and cTn-I were decreased in IR-61+EE group (P＜0.05). The oxidative respiration rate of fatty acids and the respiration rate of complex Ⅱ and Ⅳ were increased (P＜0.05). CONCLUSION: Mitochondrion-targeted cyanine fluorescent small molecule IR-61 can improve cardiac electrical activity, reduce myocardial cell injury and mitochondrial injury, reduce myocardial cell apoptosis, and improve the myocardial mitochondrial energy metabolism condition in exhausted rats.

IR-61 Improves Voiding Function via Mitochondrial Protection in Diabetic Rats.

Diabetic bladder dysfunction (DBD) afflicts nearly half of diabetic patients, but effective treatment is lacking. In this study, IR-61, a novel heptamethine cyanine dye with potential antioxidant effects, was investigated to determine whether it can alleviate DBD. Rats were intraperitoneally injected with IR-61 or vehicle after diabetes was induced with streptozotocin. Before evaluating the effects of IR-61 in improving DBD by filling cystometry, we detected its distribution in tissues and subcellular organelles by confocal fluorescence imaging. Near infrared (NIR) imaging showed that IR-61 could accumulate at high levels in the bladders of diabetic rats, and confocal images demonstrated that it was mainly taken up by bladder smooth muscle cells (BSMCs) and localized in mitochondria. Then, filling cystometry illustrated that IR-61 significantly improved the bladder function of diabetic rats. The histomorphometry results showed that IR-61 effectively mitigated the pathological changes in bladder smooth muscle (BSM) in diabetic rats. Furthermore, IR-61 remarkably reduced the number of apoptotic BSMCs and the unfavorable expression of proteins related to the mitochondrial apoptotic pathway (Bcl-2, BAX, Cytochrome C, and cleaved Caspase-9) in diabetic rats. Moreover, the frozen section staining and transmission electron microscopy results proved that IR-61 significantly reduced the reactive oxygen species (ROS) levels and prevented the mitochondrial mass and morphology damage in the BSM of diabetic rats. In addition, IR-61 upregulated the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its associated antioxidant proteins in the BSM of diabetic rats. Together, these results indicate that IR-61 can improve the voiding function of rats with DBD by protecting the mitochondria of BSMCs from oxidative stress, which is possibly mediated through the activation of the Nrf2 pathway.

The near-infrared dye IR-61 restores erectile function in a streptozotocin-induced diabetes model via mitochondrial protection.

This study aimed to evaluate the therapeutic effect of IR-61, a novel mitochondrial heptamethine cyanine dye with antioxidant effects, on diabetes mellitus-induced erectile dysfunction (DMED). Eight-week-old male Sprague-Dawley rats were intraperitoneally injected with streptozotocin (STZ) to induce type 1 diabetes. Eight weeks after STZ injection, all rats were divided into three groups: the control group, DM group, and DM + IR-61 group. In the DM + IR-61 group, the rats were administered IR-61 (1.6 mg kg-1) twice a week by intravenous injection. At week 13, erectile function was evaluated by determining the ratio of the maximal intracavernous pressure to mean arterial pressure, and the penises were then harvested for fluorescent imaging, transmission electron microscopy, histological examinations, and Western blot analysis. Whole-body imaging suggested that IR-61 was highly accumulated in the penis after intravenous injection. IR-61 treatment significantly improved the maximal ICP of diabetic rats. Additionally, IR-61 ameliorated diabetes-induced inflammation, apoptosis, and phenotypic transition of corpus cavernosum smooth muscle cells (CCSMCs) in penile tissue. IR-61 also attenuated mitochondrial damage, reduced reactive oxygen species production in the corpus cavernosum and upregulated sirtuin1 (SIRT1), sirtuin3 (SIRT3), nuclear factor (erythroid-derived 2)-like 2 (Nrf2), and heme oxygenase expression in penile tissue. In conclusion, IR-61 represents a potential therapeutic option for DMED by protecting the mitochondria of CCSMCs, which may be mediated by activation of the SIRT1, SIRT3, and Nrf2 pathways.

The near-infrared dye IR-61 restores erectile function in a streptozotocin-induced diabetes model via mitochondrial protection / 亚洲男科学杂志(英文版)

This study aimed to evaluate the therapeutic effect of IR-61, a novel mitochondrial heptamethine cyanine dye with antioxidant effects, on diabetes mellitus-induced erectile dysfunction (DMED). Eight-week-old male Sprague-Dawley rats were intraperitoneally injected with streptozotocin (STZ) to induce type 1 diabetes. Eight weeks after STZ injection, all rats were divided into three groups: the control group, DM group, and DM + IR-61 group. In the DM + IR-61 group, the rats were administered IR-61 (1.6 mg kg

Molecular analysis of ATP-sensitive K(+) channel subunits expressed in mouse portal vein.

BACKGROUND: Several combinations of inwardly rectifying K(+) channel 6.x family pore-forming (KIR6.x) subunits associated with sulphonylurea receptor (SUR.x) subunits have been detected among ATP-sensitive K(+) (KATP) channels. It remains to be established which of these is expressed in native vascular smooth muscle. METHODS: Pharmacological and electrophysiological properties of KATP channels in mouse portal vein were investigated using tension measurements and patch-clamp techniques. Molecular biological analyses were also performed to investigate the structural properties of these channels. RESULTS: Spontaneous contractions in mouse portal vein were reversibly reduced by pinacidil and MCC-134, and the pinacidil-induced relaxation was antagonized by glibenclamide and U-37883A. In cell-attached mode, pinacidil activated glibenclamide-sensitive K(+) channels with a conductance (35 pS) similar to that of KIR6.1. RT-PCR analysis revealed the expression of KIR6.1, KIR6.2 and SUR2B transcripts. Using real-time PCR methods, the quantitative expression of KIR6.1 was much greater than that of KIR6.2. Immunohistochemical studies indicated the presence of KIR6.1 and SUR2B proteins in the smooth muscle layers of mouse portal vein and in single smooth muscle cells dispersed from mouse portal vein. CONCLUSIONS: The results indicate that native KATP channels in mouse portal vein are likely to be composed of a heterocomplex of KIR6.1 and SUR2B subunits.