Effect of visfatin on K_ATP channel upregulation in colonic smooth muscle cells in diabetic colon dysmotility

Animals’ models

Male Sprague-Dawley (SD) rats (initial weight = 90–110 g; 4 weeks old), were obtained from the Animal Core Facility of Nanjing Medical University. They were allowed to acclimatize to their surroundings for 1 w and then randomly divided into two experimental groups: Control and T2DM (n = 10 per group). T2DM was induced by a single intraperitoneal (i.p.) injection of 0.1 mol/L streptozotocin (STZ, 30 mg/kg body weight) (Sigma, St. Louis, MO, USA) dissolved in citrate buffer after 8 w of high fat-high sugar chow feeding consisted of 67% standard laboratory chow, 2.5% cholesterol, 0.5% sodium cholate, 10% lard and 15% carbohydrate. Control rats fed with standard chow and received equal volumes of buffer by i.p. injection [17]. Ethics approval for all animal experiments was obtained from the Institutional Animal and Use Committee of Nanjing Medical University (Approval ID: NJMU20110587).

Intraperitoneal glucose tolerance tests

An intraperitoneal glucose tolerance test (IPGTT) was performed at 18 weeks of age. After 6 h of fasting (8:00 to 14:00), rats were weighed, fasting glucose and insulin level was determined from tail vein blood, and then 50% glucose (2 g/kg) was injected into the intraperitoneal cavity, followed by measurement of glucose levels after 30, 60, 90, and 120 min. The IPGTT assay was performed twice in two days. Glucose levels were determined using an AccuChek Compact Plus glucometer (Roche, Basel, Switzerland).

Serum visfatin assay

Serum visfatin was determined using an enzyme-linked immunosorbent assay (ELISA) kit (E-EL-R1067c; Elabscience Biotechnology Co., Ltd, Wuhan, China) according to the manufacturer's protocol. Briefly, standards and samples were added to wells that were pre-coated with a rat VF-specific antibody; all samples were assayed in duplicate. After 90 minutes of incubation at 37°C, the plate was washed with 1× wash buffer and incubated with biotinylated detection antibody specific for rat VF for 1 h at 37°C. After washing again, an avidin-horseradish peroxidase (HRP)-conjugate was added to each well and incubated for 30 minutes at 37°C. Then, the substrate reagent and stop solution were added to the wells in turn. Finally, absorbance was measured at 450 nm.

Distal colon ejection response

Rats were anesthetized with diethyl ether, and then a glass bead, about 5 mm in diameter, was inserted into the anus of each rat and pushed into the distal colon to a depth of 3 cm using a plastic rod. The rod was withdrawn slowly so that the glass bead remained in the colon. The time of the bead ejection was defined as the time interval between placing the bead and its discharge.

Muscle strip contractility assays

The distal colonic tissues (above the pelvic brim) [18] were isolated from control and diabetic rats, and the smooth muscle layer were carefully separated out, washed with Krebs solution (118 mmol/L sodium chloride, 4.7 mmol/L potassium chloride, 1.2 mmol/L magnesium sulphate, 1.2 mmol/L monopotassium phosphate, and 11.1 mmol/L glucose, and the pH was adjusted to 7.2~7.4 with 1M NaOH), and dissected into strips measuring approximately 0.3 × 0.8 cm. The strips were mounted in 15 mL organ baths filled with Krebs solution (37°C, 5% CO₂/95% O₂). One end of the strip was fixed to a hook on the bottom of the chamber, while the other end was fixed to an isometric force transducer. All the muscle strips were placed under 1 g of resting tension and allowed to equilibrate for at least 30 min. Then, 10⁻⁴ mol/L acetylcholine (ACh) (Sigma) was added to induce maximal contractility in each strip. Changes in tension at the transducer were processed through an amplifier and recorded using a dedicated data acquisition system (Alcott-Biotech, Shanghai, China). The mean of the contractile response was determined for muscle strip of each rat treated with or without the K_ATP channel blocker, glibenclamide (10 μM). In another experiment, drugs, including the vehicle (phosphate-buffered saline (PBS)) and VF (200 ng/mL), were added sequentially and directly to the Krebs solution when the contractions of the strips from normal SD rats became steady after ACh stimulation; the change of muscle activity was then recorded.

Immunohistochemical staining

Immunohistochemical staining (IHC) was performed to detect VF, SUR2B, and Kir6.1 expression in the colonic smooth muscle layer of control and diabetic rats. The 4% paraformaldehyde solution-fixed, paraffin-embedded colonic tissue sections were sectioned into 5-μm slices, and imaged using IHC staining using rabbit anti-visfatin polyclonal antibodies (1:500, Abcam, Cambridge, MA, USA), rabbit anti-Kir6.1 polyclonal antibodies (1:200, NOVUS, Vancouver, Canada), and mouse anti-SUR2B monoclonal antibodies (1:500, Sigma). Positive cells appeared brown-yellow, and negative cells were blue-purple. Finally, the number of positive cells was counted using the ‘count small-cells’ function using Image-Pro Plus (version 5.0, Media Cybernetics, Rockville, MD, USA), while area percent and integrated optical density (IOD) percent were measured with the ‘measure stain’ function. Five fields were analyzed on each slide, and the average was taken as the measured value. The scoring was done blind to the tissue source.

Culture and treatment of colonic smooth muscle cells

Primary colonic smooth muscle cells (CSMCs) were isolated from colon tissues as described previously [19]. Briefly, the serosal and mucosal layers were removed and the remaining muscularis was cut into pieces. After type II collagenase digestion (Sigma-Aldrich, St. Louis, MO, USA), CSMCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (Gibco), 100 U/ml penicillin, and 100 μg/ml streptomycin (Beyotime, Jiangsu, China). Only cells that were more than 95% positive for smooth muscle-specific α-actin were used. CSMCs were incubated with 0, 50, 100, 200, or 300 ng/mL VF for various periods after 24 h of serum starvation. To investigate the potential effect of K_ATP channels, CSMCs were transfected with Kir6.1 expression plasmids (Shanghai Genechem Co., Ltd., Shanghai, China) using Lipofectamine 2000 (Invitrogen, Waltham, MA, USA). At 6 h post-transfection and after 12 h of serum starvation, the cells were treated with VF. To further investigate the potential effect of nuclear factor kappa B (NF-κB) and reactive oxygen species (ROS), the NF-κB inhibitor BAY 11-7082 (10 mg/mL) (Beyotime) and the ROS inhibitor N-acetyl-L-cysteine (NAC, 1 mmol/L) (Beyotime) were added separately to the medium 1 h before VF treatment.

Measurement of intracellular Ca²⁺ concentrations

CSMCs were incubated with 5 μmol/L Fluo-3/AM (Beyotime) for 30 mins at 37°C, washed twice with PBS to remove non-specifically bound dye on the cell surface, and then incubated for a 30 minutes to allow complete deesterification of intracellular AM esters. The cells were resuspended in calcium-free Hank's Balanced Salt Solution (HBSS) base buffer or calcium-containing HBSS buffer (Gibco). Then, the fluorescence intensity (indicating changes in the Ca²⁺ concentration) was measured for 200 seconds with excitation at 488 nm and emission at 515 nm using a confocal laser scanning microscope (LSM510, Zeiss, Wetzlar, Germany). Variations in Ca²⁺ fluorescence intensity were expressed as ratios (F/F₀) of fluorescence counts (F) relative to baseline values before stimulation (F₀) [20]. All procedures were carried out in the dark. This experiment was performed three times with different CSMC preparations.

Measurement of contraction by scanning micrometry

An aliquot of cell suspension containing 10⁴ muscle cells/ml was added to HEPES medium containing the test agents. The reaction was terminated by the addition of Ach (10⁻⁵ mol/L final concentration). Every 50 isolated cells were counted for use as a control. The length of isolated cells treated with a contractile agent was measured at random. The average length of treated cell group was then compared with the average length of untreated cells group. The contraction was expressed as the percentage decrease in the mean cell length from the control. Percentage decrease was calculated as:

$$\text{Cell contraction (\%)}=\text{100}-\frac{\text{mean cell length of experimental group}}{\text{mean cell length of control groupa}}\times 100$$

Western blotting

The frozen colonic smooth muscle tissues or cultured cells were homogenized in lysis buffer (Beyotime) to extract protein. The protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Beyotime). The protein samples were separated by sodium dodecylsulfate/polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% (w/v) skim milk for 1 h at room temperature, and probed with rabbit anti-myosin light chain (MLC) monoclonal antibodies (1:1000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phosphorylated (Ser19) MLC monoclonal antibodies (1:1000, Cell Signaling), mouse anti-SUR2B monoclonal antibodies (1:1000, Sigma), rabbit anti-Kir6.1 polyclonal antibodies (1:500, NOVUS), rabbit anti-phosphorylated IκB-α, and rabbit anti-phosphorylated IκB-α (1:1000, Cell Signaling) at 4°C overnight. Rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibodies (1:1000, Beyotime) were used as an internal reference. After washing three times with Tris-buffered saline and Tween 20 (TBST) for 5 minutes, the membranes were probed with corresponding HRP-conjugated secondary antibodies (Beyotime) for 1 h at room temperature. Immunoreactive Protein bands were detected using enhanced chemiluminescence western blotting reagents (Thermo Fisher Scientific, Waltham, MA, USA) and analyzed using Image J (NIH, Bethesda, MD, USA).

Quantitative real-time reverse transcription polymerase chain reaction

Total RNA was isolated from the CSMCs or liquid nitrogen-frozen colon tissue using the TRIzol reagent (Invitrogen) and quantified using a Nanodrop 2000 Spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). After reverse transcription, quantitative real-time polymerase chain reaction (qPCR) was performed using SYBR Premix Ex Taq (Takara, Shiga, Japan) according to the manufacturer’s instructions. The primer sequences were as follows: Kir6.1 forward, 5′-TGCTCTTCGCTATCATGT-3′, reverse, 5′-GTTTTCTTGACCACCTGGAT-3′; SUR2B forward, 5′-ATGAAGCCACTGCTTCCATC-3′, reverse, 5′-ATCCGTCAAAGTTGGCAAAG-3′; GAPDH forward, 5′-GGCCTTCCGTGTTCCTACC-3′, reverse, 5′-CGCCTGCTTCACCACCTTC-3′. GAPDH was used as an internal reference. The relative level of mRNA was quantified using the 2^−ΔΔCt method. Each experiment was performed in triplicate.

NF-κB activity assay

NF-κB activity was evaluated using an NF-κB Activation-Nuclear Translocation Assay Kit (Beyotime) according to the manufacturer’s instructions. Briefly, CSMCs cultured in cover glass bottom dishes were fixed using staining blocking buffer, incubated with anti-NF-κB/P65 antibodies at 4°C overnight, incubated with Cy3-labeled goat anti-rabbit fluorescent antibodies at room temperature for 1 h, and then stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 mins. The cells were visualized using a confocal laser scanning microscope (LSM510, Zeiss). Besides, phosphorylation of IκB-α by western blot analysis was also used to evaluate the activity of NF-κB.

Flow cytometric measurement of ROS

ROS were measured using a ROS assay kit (Beyotime). Treated CSMCs were loaded with 10 μM 2,7-Dichlorodi-hydrofluorescein diacetate (DCFH-DA) for 20 minutes at 37°C, and then washed with serum-free DMEM three times. The cells were resuspended in PBS. All the operations were carried out in the dark until flow cytometry analysis. Measurement of ROS formation was performed using flow cytometry with a 488 nm excitation beam and a 515 nm emission beam.

Statistical analysis

SPSS v14.0 (IBM Corp., Armonk, NY, USA) was used for statistical analysis. All results are expressed as the mean ± standard deviation (SD). The differences between the treatment groups were analyzed using Student’s t test or one-way analysis of variance (ANOVA). Statistical significance was accepted at P < 0.05.

Evaluation of the animal model

One week after modeling, the eight rats in the T2DM group had blood glucose levels >16.7 mmol/L, and were considered diabetic and suitable for further research. The body weights of the STZ-induced rats (T2DM group n = 8) were lower than those of the controls (n = 10) (Figure 1A). The fasting blood glucose values of the T2DM group were significantly higher than those of the controls. After stimulation with 50% glucose, the peak blood glucose exceeded normal, and the peak was delayed (Figure 1B), indicating that the T2DM rats were established successfully. The fasting serum insulin level was lower in the diabetic T2DM group (P < 0.01, Figure 1C). Compared with that of the controls, the serum VF concentrations were higher in the T2DM rats (P < 0.001, Figure 1D). IHC staining showed a statistically significant increase in the VF levels in the colonic smooth muscle layers obtained from T2DM rats (P < 0.01, Figure 1E).

Figure 1. Evaluation of the animal model. (A) Body weight following an injection of STZ in the control and treatment groups. (B) Glucose tolerance test results in the two groups at 18 w. (C) Fasting serum insulin level in the two groups at 18 w. (D) Serum VF levels in the two groups at 18 w. (E) Immunohistochemical staining of VF in the colon muscle layers in the two groups at 8 w. Left panel: Representative image; right panel: Quantitative analysis. Low magnification, 200×, scale bar = 100 μm; high magnification, 400×, scale bar = 50 μm. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, control group n = 10, T2DM group n = 8.

Colonic motility dysfunction of T2DM rats

Compared with the controls, the distal colon ejection response time was increased in the T2DM rats. (P < 0.01, Figure 2A). Muscle strip contractility assays showed that ACh (10⁻⁴ mol/L) increased the tension of distal colonic strips in both the normal and T2DM rats. The maximum contractile tension was increased about twice under the treatment of glibenclamide in the normal group, while it increased to about 5 fold in the T2DM group (P < 0.01, Figure 2B). Western blotting demonstrated that MLC phosphorylation, which is a prerequisite for smooth muscle contraction, was decreased significantly in colonic muscle tissues of T2DM rats (P < 0.05, Figure 2C). All these results indicated colonic motility dysfunction in T2DM rats.

Figure 2. Colonic motility dysfunction of T2DM rats. (A) Distal colonic transit time at 8 w following an injection of STZ in the diabetic and control groups. (B) Distal colonic strips of diabetic and control rats were treated with vehicle (DMSO) or glibenclamide (10 μM). Quantification of maximum contractile force of a circular muscle strip stimulated by 10⁻⁴ mol/L ACh. (C) Western blotting analysis of the phosphorylation of MLC in the colon muscle layers. Relative expression was normalized to that of GAPDH. Left panel: Representative image; right panel: Quantitative analysis from three independent experiments. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, control group n = 10, T2DM group n = 8.

VF significantly inhibited the contractility of colonic smooth muscle strips from normal SD rats

An in vitro study of distal colonic strips recorded the contractility of the colon (Figure 3). There was no change when the vehicle was added to the Krebs solution; however, the contractile tension decreased significantly after VF (200 ng/ml) stimulation. (P < 0.01, n = 6), suggesting that VF impaired colonic contractility.

Figure 3. VF significantly inhibited the contractility of colonic smooth muscle strips from normal SD rats. Distal colonic strips of normal rats were treated with vehicle (PBS solution) or VF (200 ng/mL). Circular muscle strip contractility was recorded. (A) Representative image. (B) Quantitative analysis of contractile force. (C) Quantitative analysis of contractile frequency. ^*P < 0.05, n = 6.

The expression of K_ATP channels were altered in T2DM rats

Immunofluorescence staining of distal colonic sections showed that levels of Kir6.1 and SUR2B subunits of K_ATP channels were increased in the muscle layer in T2DM rats compared with that in the controls (both P < 0.01, Figure 4A). Western blotting revealed that the protein levels of Kir6.1 and SUR2B in the colonic smooth muscle of T2DM rats were increased considerably (both P < 0.01, Figure 4B). Thus, colonic K_ATP expression was increased in T2DM rats.

Figure 4. The expression of K_ATP channels were altered in T2DM rats. (A) Immunohistochemical staining of Kir6.1 and SUR2B subunits of K_ATP channels in the colon muscle layers in the two groups of rats at 18 w. Left panel: Representative image; right panel: Quantitative analysis. Low magnification, 200×, scale bar = 100 μm; high magnification, 400×, scale bar = 50 μm. (B) Western blotting analysis of Kir6.1 and SUR2B subunits of K_ATP channels in the colon muscle layers in the two groups of rats at 18 w. Relative expression was normalized to that of GAPDH. Left panel: Representative image; right panel: Quantitative analysis. ^*P < 0.05, ^**P < 0.01, control group n = 10, T2DM group n = 8.

VF reduced MLC phosphorylation and intracellular Ca²⁺ concentration in isolated colonic smooth muscle cells

Western blotting demonstrated significantly reduced levels of phosphorylated MLC in the 200 ng/mL VF group (P < 0.01, Figure 5A); 200 ng/mL was considered the most effective VF concentration and was used in subsequent experiments. The cells were treated with 200 ng/mL VF for 6–48 h, and the maximal effect was observed at 24 and 48 h (both P < 0.001; Figure 5B. The difference between the two groups was not statistically significant (P > 0.05), so 24 hours was chosen as the optimal intervention time.

Figure 5. VF reduced MLC phosphorylation and intracellular Ca²⁺ concentration in isolated colonic smooth muscle cells. (A) CSMCs were treated with vehicle (PBS solution) or 0, 50, 100, 200, and 300 ng/mL VF for 24 h. Western blotting analysis of the phosphorylation of MLC in CSMCs stimulated by 0.1 μM ACh for 30 s. Relative expression was normalized to that of GAPDH. Left panel: Representative image; right panel: Quantitative analysis from three independent experiments. (B) CSMCs were treated with vehicle (PBS solution) or 200 ng/mL VF for 0–48 h. Western blotting analysis of the phosphorylation of MLC in SMCs stimulated by 0.1 μM Ach for 30 s. Relative expression was normalized to that of GAPDH. Left panel: Representative image; right panel: Quantitative analysis from three independent experiments. (C) Changes in fluorescence intensity caused by [Ca²⁺]_i relative to baseline (F/F0) in CSMCs treated with vehicle (PBS solution) or 200 ng/mL VF for 24 h. F0 was derived from the averaged intensity of the first 0–30 seconds. CSMCs were incubated in regular HBSS base buffer. Left panel: Fluorescence intensity of [Ca²⁺]_i; right panel: Quantitative analysis of peak F/F0 and a representative image of fluorescence at peak F/F0 from three independent experiments (three cells in each experiment). (D) Changes in fluorescence intensity caused by [Ca²⁺]_i relative to baseline (F/F0) in CSMCs treated with vehicle (PBS solution) or 200 ng/mL VF for 24 h. F0 was derived from the averaged intensity of the first 0–30 s. CMSCs were incubated in calcium-free HBSS base buffer. Left panel: Fluorescence intensity of [Ca²⁺]_i; right panel: Quantitative analysis of peak F/F0 and a representative image of fluorescence at peak F/F0 from three independent experiments. ^**P < 0.01, ^***P < 0.001.

The effect of VF on Ca²⁺ mobilization in vitro was then investigated. Laser confocal microscopy showed no significant difference in the increase in intracellular Ca²⁺ concentration between the normal control group and the vehicle control group after ACh stimulation (P > 0.05, Figure 5C); in comparison, the increase in intracellular Ca²⁺ concentration in the VF group was significantly lower (P < 0.01, Figure 5C), suggesting that VF could inhibit the increase of intracellular Ca²⁺ concentration in CSMCs. The effect of VF on the different components of Ca²⁺ mobilization was investigated further by resuspending the CSMCs in calcium-free HBSS base buffer. In the absence of extracellular Ca²⁺, all increases in intracellular Ca²⁺ would represent the release of Ca²⁺ from intracellular stores only. Under these conditions, there was no difference in the increase in intracellular Ca²⁺ concentration between the VF and control groups (P > 0.05, Figure 5D), suggesting that VF has no significant effect on the release of Ca²⁺ from intracellular calcium pools; thus, VF may mainly suppress the ACh-induced increase of Ca²⁺ concentration in CSMCs by reducing Ca²⁺ influx.

Altered VF expression induced Kir6.1 upregulation

We next examined whether VF increases K_ATP levels in CSMCs. The expression of the Kir6.1 subunit increased significantly when treated with 200 ng/mL VF for 24 h (P < 0.001, Figure 6A). qRT-PCR also showed that the expression of the Kir6.1 subunit mRNA in the VF group was significantly higher than that in the vehicle group (P < 0.01, Figure 6B). However, VF did not influence the expression of the SUR2B subunit. Therefore, we focused on the Kir6.1 subunit.

Figure 6. Altered VF expression induced Kir6.1 upregulation. (A) CSMCs were treated with vehicle (PBS solution) or 0, 50, 100, 200, and 300 ng/mL VF for 24 h. Western blotting analysis of K_ATP subunit protein levels in all the groups. Relative expression was normalized to that of GAPDH. Left panel: Representative image; right panel: Quantitative analysis from three independent experiments. (B) qRT-PCR analysis of K_ATP subunit mRNA levels in CSMCs treated with vehicle (PBS solution) or 200 ng/mL VF for 24 h. Relative expression was normalized to that of GAPDH. Left panel: Kir6.1 subunit; right panel: SUR 2B subunit. ^**P < 0.01, ^***P < 0.001.

Overexpression of Kir6.1 decreases intracellular Ca²⁺ concentration ([Ca²⁺]_i) and MLC phosphorylation

The Kir6.1 subunit was overexpressed a using plasmid to identify the relationship between Kir6.1 and intracellular Ca²⁺ mobilization and MLC phosphorylation in CSMCs.

Compared with that in the control group, the level of Kir6.1 mRNA of the Kir6.1 overexpression group increased significantly, indicating successful transfection (P < 0.001, Figure 7A). The intracellular p-MLC level and Ca²⁺ concentration of the Kir6.1 overexpression group were significantly reduced compared with those in the control group (both P < 0.01, Figure 7B–7C). This suggested that Kir6.1 exerts negative feedback on Ca²⁺ signals and MLC Phosphorylation in CSMCs. VF intervention increased the expression of Kir6.1 in CSMCs, indicating that VF might be involved in the contractile dysfunction of CSMCs by upregulating Kir6.1 levels.

Figure 7. Overexpression of the Kir6.1 subunit decreases intracellular Ca²⁺ concentration ([Ca²⁺]_i) and MLC phosphorylation. (A) qRT-PCR analysis of Kir6.1 subunit mRNA levels in CSMCs transfected with blank or Kir6.1 expression plasmids. Relative expression was normalized to that of GAPDH. (B) Western blotting analysis of Kir6.1 subunit protein levels and phosphorylation of MLC in CSMCs transfected with blank or Kir6.1 expression plasmids. Relative expression was normalized to that of GAPDH. Left panel: Representative image; right panel: Quantitative analysis from three independent experiments. (C) Changes in fluorescence intensity caused by [Ca²⁺]_i relative to baseline (F/F0) in CSMCs transfected with blank or Kir6.1 expression plasmids. F0 was derived from the averaged intensity of the first 0–30 s. CMSCs were incubated in calcium-free HBSS base buffer. Left panel: Fluorescence intensity of [Ca²⁺]_i; right panel: Quantitative analysis of peak F/F0 and a representative image of fluorescence at peak F/F0 from three independent experiments. ^**P < 0.01, ^***P < 0.001.

Kir6.1 upregulation was induced via the ROS/NF-κB-mediated pathway

Flow cytometry showed that ROS levels were increased significantly in VF-treated CSMCs (Figure 8A). The results showed that the levels of phosphorylated IκB-α and translocation of NF-κB/P65 from cytoplasm to nuclei were increased in CSMCs exposed to exogenous VF for different times (15, 30, 45, and 60 min), and the maximal effect was observed at 45 minutes (Figure 8B). Pretreatment with the ROS inhibitor NAC for 1 h reversed the VF-induced increase in NF-κB/P65 activity significantly (Figure 8C), suggesting that ROS act upstream of NF-κB/P65. In addition, Kir6.1 upregulation was reduced significantly by NAC pretreatment and NF-κB inhibitor BAY 11-7082 treatment, separately (both P < 0.01, Figure 8D). This suggested strongly that VF induces Kir6.1 upregulation in CSMCs through the ROS/NF-κB pathway. In addition, we found that the contraction of CSMCs in the present study refers to the initial peak contraction occurred at 60 upon the addition of Ach. As shown in Figure 8E, the initial contraction of CSMCs were decreased significantly under the treatment of VF. However, this can be reversed by NAC and BAY 11-7082 pretreatment, suggesting that VF decreased the contractility of smooth muscle cells through the ROS/NF-κB pathway.

Figure 8. Kir6.1 upregulation was induced via the ROS/NF-κB-mediated pathway. (A) Flow cytometry analysis of the levels of ROS in CSMCs treated with vehicle (PBS solution) or VF (200 ng/mL), with or without NAC (1 mmol/L) for 24 h. Left panel: Representative image; right panel: Quantitative analysis from three independent experiments. Positive control: Rosup, 50mg/ml. Control: No treatment. (B) Upper panel: Representative double immunofluorescence staining of NF-κB/P65 activity in CSMCs treated with vehicle (PBS solution) or VF (200 ng/L) for 0–60 min. Nuclei were identified by DAPI staining. Lower panel: Western blotting analysis of phosphorylated IκB-αin CSMCs treated with vehicle (PBS solution) or VF (200 ng/L) for 0–60 min. (C) Representative double immunofluorescence staining of NF-κB/P65 activity in CSMCs treated with vehicle (PBS solution) or VF (200 ng/L), with or without NAC (N-acetyl-L-cysteine, 1 mmol/L), for 1 hour. Nuclei were identified by DAPI staining. (D) Western blotting analysis of Kir6.1 subunit protein in CSMCs treated with vehicle (PBS solution) or VF (200 ng/L), with or without NAC (1 mmol/L) or BAY 11-7082 (10 mg/mL), for 1 hour. Upper panel: Representative image; Lower panel: Quantitative analysis from three independent experiments. (E) Measurement of contraction of CSMCs treated with vehicle (PBS solution) or VF (200 ng/L), with or without NAC (1 mmol/L) or BAY 11-7082 (10 mg/mL), for 45 min by scanning micrometry. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.