Determination of total reactive sulfide species in biological matrix after administration of hydrogen sulfide donors or cellular oxidative stress

2.1. Chemicals and reagents

NBDCl standard substance (Batch NO. RH560817, purity 98%), dithiothreitol (DTT, AR, purity≧98%), and ammonium acetate (purity≧99.5% for HPLC) were sourced from Shanghai ANPEL laboratory Technologies Inc. (Shanghai, China). Ammonium hydroxide (AR, 25%-28%) was sourced from Chinasun Specialty Products Co., Ltd. (Jiangsu, China). Methanol (HPLC grade) and tris(2-carboxyethyl) phosphine (TCEP, AR, purity≥98%) were purchased from MERCK (KgaA, Germany). Ibuprofen (ID 690F-1E05, purity 99.9%) was sourced from the National Institutes for Food and Drug Control, China. Sodium sulfide (Na₂S∙9H₂O, purity 99.9%, Adamas reagent Co., Ltd, Cat NO.19978A) was used as the source of H₂S. Na₂S∙9H₂O injection (150 mg/ ampoule, Batch NO. 20170801) used in the pharmacokinetic study was provided by West China School of Pharmacy Sichuan University. EDTA (AR, 99%) was obtained from Shanghai Yuanye Bio-technology Co., Ltd (Shanghai, China). NBDSH (1 g, purity ≧95% by Nuclear Magnetic Resonance Spectroscopy) was synthesized by Shanghai Nafu biotechnology Co., Ltd. (Shanghai, China). NBDOEt and 4, 4’ -Thiobis (7-nitro-2,1,3-benzoxadiazole) (TBNBD) were synthesized as in our previous work [45]. RPMI-1640 cell culture media (Item NO. 11875, Lot NO. 2572144) was from Gibco (Invitrogen; Darmstadt, Germany). Cell culture fetal bovine serum (FBS, Cat NO. Z7181FBS, Batch NO. 42302202S) was from Zeta Life Inc. (California, USA). Penicillin-Streptomycin liquid (100×, Cat NO. P1400), trypsin digestion solutions (0.25%, without phenol red, Cat NO. T1350) and thioglycolate broth medium (Cat NO. LA8740) were from Solarbio Science & technology co., Ltd. (Beijing, China). Escherichia coli Lipopolysaccharides (LPS, Cat NO. L5293-2ML, Lot NO. 0000219279) and Adenosine 5’-triphosphate Sodium salt hydrate (ATP, Cat NO. A2383-1G, PCode 1001150755) were from Merck KGaA (Darmstadt, Germany). RNA fast 200 total RNA rapid extraction kit (Cat NO. 220011) was obtained from Shanghai Fastagen Bio-technology Co., Ltd (Shanghai, China). ReverTra AceTM qPCR RT Kit (Lot NO. 256200) was obtained from TOYOBO life science (Shanghai, China). TB Green Premix Ex Taq Ⅱ (Til RNaseH Plus, Cat NO. RR420A, Lot NO. AM81832A) was obtained from Takara Bio Inc. (Beijing, China). Deionizer water (18.2 MΩ/cm) was prepared from the Milli-Q purification system (Millipore, Bedford, MA, USA).

2.2. Animals

Sprague-Dawley (SD) rats (18/sex) were procured from Sipple-Bikai Laboratory Animal Co., LTD (Shanghai, China) at 2 months of age. C57BL/6 mice were procured from Slack Jingda Laboratory Animal Co., LTD (Hunan, China) at 2 months of age and kept in a specific pathogen free (SPF grade) system. Animals were kept in standard cages under 12 h light/dark cycle and provided with ad libitum access to water and food throughout the experimental period. Animal studies were conducted in accordance with the recommendations in the Guidelines for the Care and Use of Laboratory Animals and relevant Chinese laws and regulations. The experimental procedures and protocols for SD rats were approved by the Institutional Animal Care and the Use Committee of Shanghai Jiao Tong University (IACUC No. A2018075). The experimental procedures and protocols for C57BL/6 mice were approved by the Animal Ethics Committee of Guangxi Institute for Drug Control (IACUC No. LLSC20240301-1).

2.3. Preparation of solutions

NBDOEt, NBDCl, NBDSH and TBNBD stock solutions were prepared in methanol to a concentration of 5 mM. Ibuprofen stock solution was prepared in methanol to 1 mg∙mL^-1.

Hypoxic deionizer water was prepared by ultrasonic degassing (15 min) in combination with nitrogen gas saturation (1 h). Na₂S stock solution was prepared by dissolving Na₂S∙9H₂O crystal with hypoxic deionizer water in a plastic volumetric flask under nitrogen gas flow to make a concentration of 20 mM, the oxidized Na₂S at the surface of the crystal was discarded before weighing by rinsing with the solvent and wiping dry; nitrogen gas was filled into the headspace of the volumetric flask and the stock solution was used within 30 min. Na₂S calibration and quality control (QC) series working solutions were prepared by gradual dilution of the stock solutions with hypoxic deionizer water and kept on ice with no head space in Eppendorf (EP) plastic tubes.Considering compatibility with he mass spectrometer, all the reaction solvents at pH 6.5, 9.5, and 10.5 were prepared by adjusting the pH of ammonium acetate (NH₄Ac) solution using ammonium hydroxide (NH₄OH).

The reaction solvent for derivatization of TRSS in plasma was prepared by accurately weighing 38.54 mg of ammonium acetate into a 100 mL volumetric flask, dissolving and diluting to volume under nitrogen gas flow with hypoxic methanol that was saturated by nitrogen gas for 30 min, and then adjusting pH to 9.5 using ammonium hydroxide. Then, the derivatization solution was prepared by the addition of NBDCl and the internal standard ibuprofen into the reaction solvent under nitrogen gas flow to make a concentration of 150 μM and 50 ng∙mL^-1, respectively.

2.4. Pretreatment of plasma samples

150 μL of the reaction solution was transferred into a 0.5 mL EP plastic tube under nitrogen gas flow, filled with nitrogen gas and capped. The blood sample was centrifuged at 12000 rpm for 3 min immediately after collection, and 30 μL of plasma was quickly added into the test tube containing 150 μL of reaction solution under nitrogen gas flow. It was filled with nitrogen gas, capped and reacted for 2 h on a decolorization shaker at 25 rpm (Room Temperature). Then, 5 uL of 40 mM Ethylene diamine tetra acetic acid (EDTA) was added into the reaction solution, followed by vortexing for 3 min and centrifugation at 12000 rpm (4˚C) for 10 min. The supernatant was finally analyzed by LC-MS/MS. The reaction solution should be protected from light at all times.The injection volume was 10 µl.

2.5. Method validation

2.6. Pharmacokinetic study

Twelve rats were fasted for 12 h and randomly divided into two groups (n = 6) to receive Na₂S at 5 and 10 mg∙kg^-1, respectively. 19.5 and 9.7 mL sterile injection solvents were added through disposable medical syringes into 150 mg Na₂S·9H₂O to make 2.5 and 5 mg∙mL^-1 Na₂S injection, which were administered by tail vein injection right after dissolution. The injection of Na₂S into rats was finished within 2 min but should not be fast in case the rats die of toxicity. Approximately 0.2 mL blood samples were collected from the posterior orbital venous plexus of the rats into 0.5 mL heparinized plastic EP tubes under nitrogen gas flow at 10, 5 and 0 min before dosing and 2, 4, 6, 8, 10, 15, 20, 30, 45, 60 and 120 min after injection. The test tubes were filled with nitrogen gas and centrifuged at 12000 rpm for 3 min right after blood sample collection, and 30 μl of plasma was immediately added into a test tube containing 150 μl derivatization reaction solution and treated as described in section 2.4.

2.7. Cell culture and activation of NLRP3 inflammasome

LPS was reconstituted in cell culture media and ATP in sterile water. The stock solutions were diluted in corresponding media to prepare intermediate concentration solutions and stored at −20˚C. 3% sterile thioglycolate broth medium was prepared by dissolving 3 g of solid thioglycolate broth medium in 100 mL deionized water and autoclave at 121° for 15 min.

Mice peritoneal macrophages were harvested from C57BL/6 mice (aged 10 weeks, mixed sex). Mice were given intraperitoneal injections of 3% sterile thioglycolate broth medium to induce peritoneal inflammation and euthanized by cervical dislocation after 3 days; then macrophages were collected by peritoneal lavage using 15﹢10 mL of serum-free RPMI 1640 medium. The collected cells were resuspended in RPMI-1640 medium supplemented with 10% FBS, 100 U/mL Penicillin and 100 µg∙mL^-1 Streptomycin and plated in 6-well plates to a density of 2 × 10⁶ cells/well. Cells were cultured overnight at 37˚C under an atmosphere of 5% CO₂ before use.

To activate the NLRP3 inflammasome, cells were primed with LPS (100 ng∙mL^-1 for 4 h) followed by stimulation with ATP (5 mM, 30 min)[48-50]. Activation of NLRP3 inflammasome by priming with LPS was confirmed by the detection of the mRNA expression levels of Tnfa, Il1b and Il6 gens by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Mouse macrophage RNA was extracted using Fastagen RNA fast200 total RNA rapid extraction kit, and mRNA was converted to cDNA using TOYOBO ReverTra AceTM qPCR RT Kit according to the manufacturer’s instructions. qPCR reactions were performed on the BIO-RAD CFX96 Real-Time System based on the optimal concentrations of primers and probes recommended by the manufacturer. The thermal profile consisted of pre-denaturation at 95˚C for 1 min, then 40 cycles of denaturation at 95˚C for 10 s, annealing at 60˚C for 20 s, and extension at 72˚C for 30 s. This was plotted as a melting curve. The difference between samples was determined using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal standards. Data were analyzed using Bio-Rad CFX Manager Version 3.1.1517.0823. Oligonucleotide primers were shown in Table 1.

For detection of TRSS, the cell culture medium was discarded, and cells were digested by adding 500 µl 0.25% trypsin solution into each well. Then 1 mL of cell culture medium was added to stop digestion, and the cell suspension was transferred to a 2 mL EP plastic tube and centrifuged at 1500 rpm for 5 min. The supernatant was discarded and 400 μL of derivatization solution (Section 2.3) containing 150 µM NBDCl and 150 ng∙mL^-1 ibuprofen was added into the test tube under nitrogen gas flow. The test tube was filled with nitrogen gas, capped and put into a thermomixer for derivatization reaction (800 rpm, 25˚C). After the reaction, the reaction solution was centrifuged at 12000 rpm for 10 min and the supernatant was subjected to LC-MS/MS analysis. The injection volume was 5 µl.

2.8. LC–MS/MS analysis

LC-MS/MS detection of TRSS in rat plasma was performed on a triple quadrupole tandem mass Spectrometer (Micromass Quattro Ultima, Waters Corporation, Milford, MA, USA) coupled to the HPLC system (Waters 2695) through an electrospray ionization (ESI) source; MassLynx 4.1 software was used for system control, data acquisition and statistical Processing. Chromatographic separation was dependent on an Ultimate XB C8 column (50 mm × 4.6 mm, 5 μm). The mobile phase was methanol (A) and 5 mM ammonium acetate solution (B)the gradient program are shown in Table 2, only three-tenth of the post-column flow (1 mL∙min^-1) was split into the EIS source by a three-way cock. Ibuprofen was used as the internal standard. The analytes were detected under the negative ion mode using multiple reaction monitoring (MRM). The mass spectrometric parameters were optimized as in Table 3.

LC-MS/MS detection of TRSS in macrophages was performed on Shimadzu LCMS-8050 triple quadrupole tandem mass Spectrometer equipped with the LabSolutions software Shimadzu Corporation, Kyoto, Japan. Chromatographic separation was dependent on an Ultimate XB C8 column (2.1 × 100 mm, 1.8 μm), the mobile phase and gradient program was the same as that in detection of rat plasma sample except that the flow rate was 0.3 mL∙min^-1. ESI was performed in the negative ionization mode with a spray capillary voltage of -3 kV. The detector voltage was 1.68 kV. The temperatures of the interface, drying gas, desolvation line and heat block were 300, 526, 250, and 400˚C, respectively. The nebulizing gas flow, heating gas flow, and drying gas flow were 3, 10, and 10 L/min, respectively. Collision-induced dissociation gas pressure was set to 270 kPa. Collision energies for MRM of NBDSH, TBNBD, NBDCl and ibuprofen were 14, 12, 9 and 9 eV, respectively.

3.1. Assay development

In our previous work, we established an LC-MS/MS method for quantification of H₂S in Na₂S·9H₂O bulk drug and injection based on derivatization with NBDOEt [45]. The method was subsequently applied to the detection of H₂S in rat plasma. Considering the extra consumption of the probe by the sulfides in plasma, probe concentration was optimized for the new matrix. Since the optimal reaction concentration of NBDOEt with H₂S at pH 10.5 was 30 μM in our previous work, we observed the response of NBDSH in blank plasma, 2 μM Na₂S aqueous solution and 2 μM Na₂S spiked plasma sample by reaction with 30, 100, 200, 500, 1000, 2000 and 5000 μM NBDOEt. Consistent with the results of our previous work, the response of aqueous samples was suppressed when the concentration of NBDOEt exceeded 100 μM. The peak area of NBDSH in H₂S spiked plasma samples was even lower than that in the blank plasma when the concentration of NBDOEt was lower than 100 μM, and the NBDSH response was suppressed when the concentration of NBDOEt was higher than 2 mM. The NBDSH response of blank plasma and 2 µM H₂S spiked plasma samples by reaction with 200-5000µM NBDOEt are shown in Figure S1. Then, plasma samples were treated using 2 mM NBDOEt. However, when using this optimized concentration, the recovery of H₂S from plasma was 66.4%, 9.6% and 5.6% (n=3) at 0.1, 1 and 10 μM, respectively, which could not be improved by the addition of ascorbic acid, DTT or TCEP. And the reproducibility of the NBDSH peak area was very poor. The recovery of different concentrations of H₂S from rat plasma by reaction with 2 mM NBDOEt are shown in Table S1. According to our previous work, the reaction of NBDOEt with H₂S was less interfered by sulfur compounds, meaning NBDOEt showed stronger specificity to H₂S than other NBD probes [45]. Therefore, we could speculate that once added to the biomatrix or produced in vivo, H₂S would soon be transformed into other forms that could not react with H₂S specific probe and could not be reduced back to H₂S by addition of dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP).

We have also found in our previous work that the ratios of the NBDSH response of blank plasma to that of 2 µM H₂S aqueous solution for NBDCl was 123.5% [45], indicating that NBDCl could react with bound sulfide species in rat plasma to form the same reaction product as with H₂S. Therefore, we try to detect TRSS in vivo using NBDCl as the derivatization reagent after administration of exogenous H₂S. The optimal reaction pH for H₂S and NBDCl in our previous work was 10.5 (0.1% NH₄OH methanol solution) [45], however, the recovery of TRSS was improved in company with better precision when 5 mM ammonium acetate was added into the reaction solvent, the results are shown in Table S2. Even though in 0.1% NH₄OH 5 mM NH₄Ac methanol solution, H₂S aqueous standard solutions could not react with NBDCl efficiently, the reaction efficiency of plasma sulfane sulfur with NBDCl was improved. We speculate that NBDCl was hydrolyzed in the alkaline methanol solution and the intermediate product NBDOMe could react faster with H₂S, which would explain the faster reaction rate of H₂S with NBDCl at higher pH in aqueous solution [51]. However, in the plasma, sulfane sulfur makes up the major part of TRSS and it may react with NBDCl more efficiently than with NBDOMe or the final hydrolysate NBDOH to produce NBDSH, and 0.1% NH₄OH 5 mM NH₄Ac methanol solution could provide the alkaline environment for reaction of sulfane sulfur with NBDCl but also inhibit dramatic hydrolysis of NBDCl. As mentioned in the “Introduction” section, Schilling et al. [44] have shown that when some alkylating agents were used to trap persulfides, the resulting persulfide adducts (R-S-S-Alk) would soon be converted into thioethers (R-S-Alk) due to sulfur deprivation by ambient nucleophiles, the nature of the alkylating agent determines the ability of nucleophiles to deprive the thiosulfoxide of the sulfane sulfur atom [44]. Since we could not detect appropriate amounts of R-S-S-NBD or R-S-S-S-NBD by mass scan when fresh rat plasma sample was extracted with NBDCl reaction solution, we could speculate that NBDCl could not form stable alkylated intermediates with sulfane sulfur, the sulfur atom of the intermediates was deprived by NBDCl itself to form NBDSH given that NBDCl is a better nucleophilic acceptor than other ambient nucleophiles. The proper reaction mechanism for NBDCl and sulfane sulfur is shown in Figure 1.

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Figure 1.

Proper reaction mechanism for 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBDCl) and sulfane sulfur (n=1).

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Then the reaction concentration of NBDCl was optimized for detection of TRSS in plasma. We observed the response of NBDSH by reaction of 2 μM Na₂S spiked plasma sample with 30, 50, 100, 150 and 500 μM NBDCl and found that the signal of NBDSH plateaued at 100 μM and suppressed at 500 μM, the results are shown in Figure S2. Considering the application of the method to detect samples with TRSS concentration higher than 2 μM, the concentration of NBDCl used in our method was set as 150 μM.

In our previous work, 1 mM EDTA was added into the samples to improve the precision of NBDSH, which contains a thiol group that would cause absorption of NBDSH onto the metal surface of the instrument pipeline. Therefore, in this work, 1 mm EDTA was also added into plasma samples after reaction with NBDCl to improve recovery and precision of NBDSH. Moreover, we found in our experiment that excess oxygen presented in the reaction solvent would impair the precision of NBDSH peak area probably through perturbing the initial redox equilibrium of the sulfane sulfur species. Since the simple application of nitrogen-saturated reaction solution was enough to eliminate the reduction in precision, there’s no need to perform all the derivatization steps in a glove box filled with nitrogen gas. Therefore, to eliminate the influence of oxygen on the detection of TRSS, the solvents for NBDCl and Na₂S were degassed by ultrasonic for 15 min and saturated with nitrogen gas for more than 1 h, 0.5 mL EP plastic tube was used to reduce the volume up the reaction solution, and the reaction solution was added into tubes under nitrogen gas flow and the lid was capped before use. The matrix samples were also added into the reaction solvent under nitrogen gas flow to avoid the sharp rise of oxygen content in the reaction solution. Even though oxygen gas would continuously slip into the capped test tube, or the opened test tube protected under nitrogen gas flow, protection of the reaction solvent with nitrogen gas still met the requirement of our sample processing. The precision of NBDSH peak area in 0.5 and 2 µM H₂S spiked plasma samples by derivatization with NBDCl in a normal and hypoxic reaction solvent are shown in Table S3.

According to US Food and Drug Administration (FDA) guidance for industry “Bioanalytical Method Validation and Study Sample Analysis”, the calibration curve should be prepared by fresh matrix spiked calibration standards. For analysis of endogenous molecules (e.g., replacement therapies), the accuracy of the assay constitutes a difficulty when the method cannot discriminate between the therapeutic agent and the endogenous molecule [47]. Considering that surrogate matrix and surrogate analyte were unattainable for sulfane sulfur and H₂S, in this study, the concentration of TRSS in the pharmacokinetic study of an H₂S donor was calibrated using NBDSH spiked plasma standard curve and the background subtraction approach was used to determine the concentration of the analyte in the test samples [46,47]. Typical chromatograms of blank rat plasma, rat plasma sample obtained after injection of Na₂S and Na₂S standard solution after derivatization with NBDCl are shown in Figure 2. The retention time of NBDSH, NBDCl, TBNBD and ibuprofen were 2.34, 2.8, 3.0 and 2.98 min respectively. Even though there was cross-talk between NBDCl/TBNBD and NBDSH due to their common structural fragment, the detection of NBDSH was not interfered with by NBDCl and TBNBD since they could be separated by chromatography. And TBNBD was undetected in biological samples.

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Figure 2.

Representative multiple reaction monitoring (MRM) chromatograms of (a) blank rat plasma, (b) rat plasma sample obtained 4 min after tail intravenous injection of Na₂S 5 mg∙kg_-1, (c) 5 μM Na2S standard solution after derivatization with NBDCl. (1: TBNBD channel; 2: IS channel; 3: NBDSH channel; 4: NBDCl channel.) NBDCl: 4-chloro-7-nitro-2,1,3- benzoxadiazole, NBDSH: 7-nitrobenzofurazan-4-thiol, TBNBD: 4, 4’ -Thiobis (7-nitro-2,1,3-benzoxadiazole), IS: the internal Standard, ibuprofen.

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3.2. Method validation

3.2.2. Stability of TRSS in rat whole blood and plasma

Stability assessment of TRSS in rat whole blood and plasma are shown in Figure 3 and Figure 4, respectively. RSD for the NBDSH peak area of all sample groups were within 15%. Despite minor fluctuation at 10 min after spiked, NBDSH response in 100 nM and 4 µM H₂S spiked blood and plasma samples were considered stable for at least 30 min, the detected NBDSH peak area could represent the actual level of TRSS in vivo. Better precision would be obtained by preparation of sample by centrifugation for only 3 min, immediately after blood collection, protection by filling the test tube with nitrogen gas, and detection immediately right after centrifugation.

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Figure 3.

Stability of termed total reactive sulfide species (TRSS) in 0.1 and 4μM H2S spiked blood samples (n=6).

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Figure 4.

Stability of termed total reactive sulfide species (TRSS) in 0.1 and 4 μM H₂S spiked blood samples (n=6). NBDSH: 7-nitrobenzofurazan-4-thiol.

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3.3. Application of the assay in pharmacokinetic study of H₂S in rats

The development of H₂S donors for the treatment of diseases will benefit from a reliable method for the determination of TRSS in blood, tissues and cells. Despite the controversy in the baseline level of reactive sulfide concentration in blood, cells and tissues in most studies focused on H₂S detection. A short half-life of available blood sulfide and a dose-dependent relationship between H₂S administration and subsequent elevation of sulfide availability were consistently observed in most pharmacokinetic studies [15,17,20]. In this study, after a single dose injection of 5 mg∙kg^-1 and 10 mg∙kg^-1 Na₂S, TRSS in rat plasma was trapped by NBDCl and calculated by NBDSH plasma calibration curve. Baseline levels of TRSS were determined to be approximately 20 µM, and intravenous bolus dosing of Na₂S also resulted in a dose-dependent increase in plasma TRSS levels. The P-value of the Student’s t-test for the two groups of mean plasma concentration of TRSS at different time points was 0.001. The mean plasma concentration-time curve of TRSS after bolus injection of Na₂S at different doses is shown in Figure 5. The results show that the concentration of TRSS reached the peak value (27 µM) at 4 min after administration of 5 mg∙kg^-1 Na₂S and fell back to the base level at 10 min; the peak TRSS concentration in the 10 mg∙kg^-1 Na₂S dose group (36 µM) occurred at 10 min and fell back to a new base level at 30 min. We could see that in the 5 mg∙kg^-1 dose group, the mean concentration of TRSS at 45 min (15 µM) was lower than the initial base level (20 µM) before Na₂S administration, however, in the 10 mg∙kg^-1 dose group, the mean concentration of TRSS at 60 min (approximately 24 µM) was much higher than the initial base level. The former phenomenon could be attributed to the overwhelming detoxification enzyme system, while the latter attributed to the saturation of detoxification capability. The difference in the pharmacokinetic property of Na₂S at different dosages indicates that the natural defense mechanism may respond to the toxic H₂S by increasing production of enzymes involved in its metabolism, which is consistent with the results by Osipov et al. [16] Therefore, caution should be given to the release kinetics when designing H₂S donors. Our method would be a powerful tool to assess the druggability of newly designed controlled release preparation of H₂S.

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Figure 5.

Mean plasma concentration-time curve of termed total reactive sulfide species (TRSS) in rats after tail vein injection of Na₂S at dose of 5 and 10 mg·kg_-1. P=0.001 by Student’s t-test for the mean plasma concentration of TRSS across time points between two dose groups.

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3.4. The level of TRSS in mice macrophages decreased after activation of NLRP3 inflammasome

The method was also applied for the detection of TRSS in mice peritoneal macrophages. Typical chromatograms of NBD derivatives and the internal standard obtained by extraction of LPS and ATP-stimulated macrophages with the derivatization solution containing NBDCl and ibuprofen are shown in Figure 6. The retention time of NBDSH, NBDCl, TBNBD and ibuprofen were 2.33, 2.7, 2.9 and 2.75 min, respectively. We could see that the retention time of NBDSH was stably reproduced on different instruments using different columns when the solid phase material, the mobile phase and the chromatographic elution gradient were kept unchanged, and there was no interference from the matrix appearing at the retention time of NBDSH.

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Figure 6.

Representative multiple reaction monitoring (MRM) chromatograms of NBDSH(1), TBNBD(2), NBDCl(3) and ibuprofen(4) by analyzing (a) the aqueous standard solution containing 500 nM of NBDSH and 100 ng∙mL_-1 of ibuprofen; (b) the extraction solution of LPS (100 ng∙mL_-1, 4h) and ATP (5 mM, 30min) stimulated macrophages with the derivatization solution containing 150 μM NBDCl and 150 ng∙mL_-1 ibuprofen. NBDCL: 4-chloro-7-nitro-2,1,3- benzoxadiazole, NBDSH: 7-nitrobenzofurazan-4-thiol, TBNBD: 4’ -Thiobis (7-nitro-2,1,3- benzoxadiazole).

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Activation of NLRP3 inflammasome in mice peritoneal macrophages by priming with LPS was confirmed by detection of the mRNA expression level of Tnfa, Il1b and Il6 genes by RT-qPCR. As shown in Figure 7, the mRNA level of Tnfa and Il1b were significantly elevated 2 hrs after priming; after priming for 4 hrs, the mRNA level of Tnfa reached a maximum, and the mRNA level of Il1b and Il6 were also approximate to the maximum level. Therefore, we activated the NLRP3 inflammasome using ATP at 4 hrs after priming with LPS.

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Figure 7.

Relative normalized expression of Tnfa, Il1b and Il6 in mice peritoneal macrophages after primed with 100ng∙mL_-1 LPS. n=3, P value was calculated by Student’s t-test. NBDSH: 7-nitrobenzofurazan-4-thiol, LPS: Escherichia coli Lipopolysaccharides, ATP: Adenosin 5’-triphosphate.

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The method was applied to the detection of the change in TRSS in mice peritoneal macrophages. As shown in Figure 8, the level of TRSS (NBDSH peak area normalized by the area of the internal standard) decreased after activation of the NLRP3 inflammasome (P=0.03 by Student’s t-test), which is consistent with the result in the study by Bronowicka-Adamska et al. that say, stimulation of murine J774A.1 cell line (macrophage) with LPS or interferon-gamma (IFNγ) after 24 h resulted in a decrease in H₂S levels [52]. There are very few reports focused on H₂S levels in LPS-activated macrophages. Even though it has been reported that activation of macrophages by LPS contributed to higher mRNA levels of CSE, H₂S concentration in those studies was not detected, or only free H₂S was detected using amperometric microsensor [53-55]. Moreover, a high CSE mRNA level does not necessarily imply a high H₂S level. Mikami et al. have reported that H₂S production in cells is catalyzed by CSE at the steady state and is suppressed given increased intracellular Ca²⁺ concentrations [56]. Fortuitously, the flux of Ca²⁺ into the cytosol is a critical upstream event in NLRP3 activation.

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Figure 8.

The level of termed total reactive sulfide species (TRSS) in mice peritoneal macrophages was decreased after priming with 100 ng∙mL_-1 LPS for 4 h following stimulation with 5 mM ATP for 30 min. *p=0.03 by Student’s t-test.

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The unifying NLRP3 activators, including viral, bacterial and fungal infections, as well as endogenous damage-associated molecular patterns (DAMPs) and environmental irritants which mediated sterile inflammation, all induce cellular stress, which is then sensed by NLRP3. Exactly how the NLRP3 receptor senses cellular stress and which pathways work in to terminate NLRP3 activation and inflammasome assembly remains unclear. Since most NLRP3 activators also induce ion fluxes and organelle damage, it is hard to know whether mitochondrial dysfunction and mitochondrial reactive oxygen species (mtROS) are crucially involved or just associated with NLRP3 activation [57]. It has been supported by increasing evidence that redox-sensitive protein cysteine residues may act as oxidant sensors [58]. At physiological pH, protein cysteine residues with low pKa exist mainly as thiolate anions (S^-) which behave as strong nucleophiles and can generate the sulfenic acid (SOH) form (termed protein S-sulfenylation) by reaction with ROS. The sulfenic form can be irreversibly oxidized into the sulfinic acid (SO₂H) or sulfonic acid (SO₃H) forms or react with HS^- or H₂S to form a per sulfide bond, the latter has been termed protein S-sulfhydration or per sulfidation [59]. Theoretically, the balance between H₂S and ROS determines the redox state of protein cysteine residues, which then affect the function of the redox-regulated protein. It has been reported that in superoxide dismutase 1 (SOD1) -deficient macrophages, the activity of caspase1, a proteinase-activated in response to microbial and ‘danger’ signals by assembling inflammasomes, was specifically inhibited due to reversible oxidation and glutathionylation of the cysteine residues Cys397 and Cys362, which could be contributed to decreased cellular redox potential caused by higher superoxide production [60]. In addition, Mishra et al. also reported that IFN-γ stimulated NO-suppressed IL-1β maturation by suppression of inflammasome activity via S-nitrosylation [61]. Moreover, Carroll and coworkers have established proteomics workflows to reveal the dynamic response of S-sulfenylation and S-sulfinylation to oxidative stress and moderate overlapped proteins were observed with disulfide, S-glutathionylation, and S-nitrosylation proteomes [62-64]. Since soluble macromolecules constitute a considerable proportion of the total cell volume, protein modification could explain the plethora of effects H₂S exhibits. The response of TRSS by derivatization with NBDCl, indicates the relative reactivity of protein polysulfides with the alkylating agent, could reflect the amount of total redox-sensitive cysteine residues. Therefore, monitoring the fluctuation of total TRSS in macrophages after activation of NLRP3 inflammasome would be helpful to uncover the role of H₂S and ROS in the signaling transduction pathways involved in NLRP3 activation.