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Back to Journal »Journal of Inflammation Research» Volume 14

The co-delivery of H2S donor S-propargyl-cysteine ​​can enhance the anti-inflammatory and anti-injury effects of ketoprofen in rats

Authors: Yu Y, Yang Q, Wang Z, Ding Q, Li M, Fang Y, He Q, Zhu YZ 

Published on November 9, 2021, the 2021 volume: 14 pages 5863-5875

DOI https://doi.org/10.2147/JIR.S333326

Single anonymous peer review

Editor who approved for publication: Dr. Monika Sharma

Yue Yu,1,* Qin Yanyang,1,* Zhou Wang,1 Ding Ding,1 Meng Li,1 Yu Dongfang,1 Qi Dahe,1 Yi Zhunzhu1,2 1 State Key Laboratory of Traditional Chinese Medicine Quality Research & Faculty of Pharmacy, Macau University of Science and Technology, Taipa, Macao Special Administrative Region of the People’s Republic of China; 2Shanghai Fudan University Key Laboratory of Bioactive Small Molecules & School of Pharmacy, Shanghai, People’s Republic of China *These authors contributed equally to this work, Macao Special Administrative Region of the People’s Republic of China, 999078 Tel 853 8897 2880 Fax 853 2882 3575 E-mail [Email protection] Purpose: Ketoprofen (KETO) is a traditional non-steroidal anti-inflammatory drug (NSAIDs) with good analgesic and antipyretic effects. However, as NASID, the toxicity of KETO to the gastrointestinal (GI) system may limit its clinical application. S-propargyl-cysteine ​​(SPRC) is an excellent endogenous hydrogen sulfide donor. It has a wide range of anti-inflammatory, anti-oxidative stress and even protection of the cardiovascular system by increasing the concentration of endogenous hydrogen sulfide. Applications. As reported in recent studies, the co-administration of H2S donors may reduce the gastrointestinal toxicity and related side effects caused by a series of NSAIDs. Method: In this study, we established SPRC and KETO co-encapsulated poly(lactic-co-glycolic acid) microspheres ([email protection]), and first studied its particle size, morphology, storage stability and In vitro release characteristics. Then the increase in [email protected] endogenous H2S levels was calculated, and the pharmacodynamic studies (anti-inflammatory and analgesic effects of [email protected], SPRC and KETO on adjuvant arthritis (AIA) in rats were also studied ). Finally, in order to test for potential side effects, the heart, liver, spleen, lung, kidney, stomach, small intestine, and large intestine were removed from the rat and examined by H&E staining. Result: The monodisperse [email protection] can be observed under SEM, and the calculated particle size is about 25.12 μm. The load efficiency (LE) of SPRC and KETO are 6.67% and 2.64%, respectively, while the package efficiency (EE) of SPRC and KETO are 37.20% and 68.28%, respectively. [email protected] Shows the sustained release of SPRC and KETO in vitro for up to 15 days. [email protected] Long-term increase in H2S concentration in the body can be achieved, and SPRC shows that H2S increases immediately and is metabolized within 6 hours. Interestingly, KETO has no effect on the concentration of H2S in the body. After the establishment of the AIA model, SPRC and KETO have no obvious anti-inflammatory and anti-nociceptive effects. On the contrary, [email protected] has a significant relieving effect on foot edema and pain in AIA rats, indicating that its anti-inflammatory and anti-pain effects are somewhat improve. -Nociceptive effects when SRC and KETO are co-administered. In addition, low irritation to the main organs of rats was observed in any experimental group. Conclusion: This study successfully prepared a monodispersion, [email protected] showed sustained in vitro SPRC and KETO release and in vivo H2S release. In pharmacodynamic research, [email protected] not only showed excellent anti-inflammatory and analgesic effects in AIA rats, but also had low stimulation to rats. Keywords: hydrogen sulfide donor, ketoprofen, adjuvant arthritis, anti-inflammatory, analgesic effect

Ketoprofen (KETO) is a non-steroidal anti-inflammatory drug (NSAIDs) with good antipyretic, analgesic and anti-inflammatory effects. 1-6 Compared with other NSAIDs, KETO has obvious analgesic effects on inflammatory pain and postoperative pain, and has fewer side effects. 7,8 In recent years, there have also been related researches in clinical medicine. 2,9,10 Ketoprofen and its related preparations are widely used to treat inflammation (arthritis, scissors synovitis, ankylosing spondylitis, etc.), 11,12 trauma 13,14 and postoperative pain. 7,15 However, as a non-steroidal anti-inflammatory drug, its gastrointestinal toxicity16,17 and other side effects are inevitable, which may limit the clinical application of non-steroidal anti-inflammatory drugs.

Hydrogen sulfide (H2S), which smells like rotten eggs, has long been considered a toxic gas. 18 However, recent research has gradually revealed its potential use as an endogenous gas carrier. 19-22 S-propargyl-cysteine ​​(SPRC) is an analog of garlic extract, which can produce endogenous H2S in the body by up-regulating the expression of cystathionine-γ-lyase (CSE). 23 In recent years, studies have found that SPRC plays an important role in cardiovascular protection, anti-oxidative stress24,25 and anti-inflammatory effects. 23 According to reports, all the therapeutic effects of SPRC are related to the promotion of SPRC to produce endogenous hydrogen sulfide.

Recently, studies have shown that the covalent attachment of H2S release donors and NSAIDs can significantly reduce gastrointestinal toxicity and increase therapeutic efficacy. 26 Among them, ATB-352, a combination of KETO and H2S releasing part, has been extensively studied. 27 A study led by Costa et al. showed that the efficacy and effectiveness of ATB-352 on pain can be observed after surgery. It is enhanced compared with KETO. 26 In addition, the anti-inflammatory effect of ATB-352 on rat periodontitis induced by lipopolysaccharide was also reported. 28 However, as far as we know, it is still unclear whether the co-administration of NSAIDs and H2S donors can achieve better analgesia and reduce gastrointestinal toxicity.

Generally, when one ingredient is used alone, the combination of two drugs is considered a promising strategy to overcome toxicity and side effects. 29-33 According to reports, many efforts have been made to design a single carrier for two or more therapeutic agents. 34-37 Among them, polylactic acid-glycolic acid copolymer (PLGA) has high biocompatibility. And it is widely studied. 38-42 Due to its internal structure separation, the water-in-oil-in-water (W1/O /W2) double emulsion method is usually used to encapsulate two different types of goods. 43,44

In this study, SPRC and KETO were co-encapsulated in PLGA microspheres ([email protection]) through the W1/O/W2 double emulsion method. [Email protection] shows a spherical-like structure and monodisperse particle size. In addition, it can be observed that [email protected] continues to release SPRC and KETO for up to 15 days. In terms of pharmacodynamics, [email protected] showed better anti-inflammatory and anti-nociceptive effects in AIA rats compared with SPRC or KETO alone.

The synthesis of S-propargyl-cysteine ​​(SPRC) is as described above. 45,46 Polylactic acid-glycolic acid copolymer (PLGA, 75:25, acid terminated, 15k) was purchased from Evonik Industries AG (Essen, Germany). Polyvinyl alcohol (PVA) (87-89% hydrolysis, 13k) was purchased from Sigma-Aldrich (St. Louis, USA). Ketoprofen was purchased from Energy Chemical (Shanghai, China). Elisa kits for TNF-α, IL-1β, IL-6 and IL-10 were purchased from MultiSciences (Hangzhou, China). Phosphate buffered saline (PBS), Freund's complete adjuvant (CFA), methanol, monobromodiphenylamine (MBB), hydrocortisone, diethylenetriaminepentaacetic acid (DTPA), dichloromethane (DCM) And acetonitrile were purchased from Macklin Industrial Corporation (Shanghai, China).

SPRC and KETO co-loaded PLGA microspheres ([email protection]) are prepared by the double emulsion solvent evaporation method with almost no modification. 47 In short, first dissolve 200 mg SPRC in 2000 μL distilled water to form the internal water phase (W1). Prepare the oil phase (O) by dissolving 1000 mg PLGA and 45 mg KETO in 20 mL DCM, and then add W1 to O dropwise. Next, place the mixture in a homogenizer (T18 digital ULTRA-TURRAX ®, IKA, Germany) in an ice bath at a speed of 7000 rpm for 2 minutes. Then the obtained primary emulsion (W1/O) was added to 2500 mL 0.5% (w/v) PVA solution, and further emulsified by a homogenizer (T18 digital ULTRA-TURRAX®, IKA, Germany) at a speed of 7000 rpm 4 Minutes to prepare W1/O/W2 emulsion, and harden the W1/O/W2 emulsion under magnetic stirring (400 rpm) for 5 hours. The obtained particles were collected by centrifugation, washed 3 times with distilled water and lyophilized. Finally, freeze-dried particles in the range of 20-30 microns are selected by sieving and named [email protected]

Microtrac S3500 (Montgomeryville, USA) was selected for particle size measurement, and the morphological study was performed by Phenom Pro Desktop SEMS-3400 scanning electron microscope (Thermo Fisher Scientific Inc., Waltham, USA). The particle size is calculated by Mastersizer 3000 from Malvern Panalytical (Worcestershire, United Kingdom). The calculation formula for percentage of yield (PY) is as follows: (1)

Where W: the weight of the corresponding component.

The measurement of loading efficiency and encapsulation efficiency is calculated by HPLC. In short, to measure SPRC, 48,49 first dissolve 50 mg [email protected] in 2 mL DCM, and further extract SPRC with 10 mL distilled water, and analyze by HPLC. For the detection of KETO, 50 mg microspheres were dissolved in 2 mL DCM and further diluted to 10 mL with methanol. The solution was collected and analyzed by HPLC for the KETO concentration, with little modification. 50-52 Finally, LE and EE are calculated as equations (2) and (3) as shown below: (2) (3)

Among them, LE: loading efficiency, EE: encapsulation efficiency, WE: weight of SPRC or KETO encapsulated in [email protected], W[email protected]: weight of [email protected], WT: theoretically loaded in [email protected] SPRC or KETO weighted email protection]

Put the prepared [email protected] in a vacuum desiccator at a temperature of 4 or 25°C and a humidity of 50% to study the impact of the storage environment. [Email protection] Sampling is performed at predetermined time intervals (0, 1, 2, 3, 4, 5, 6 months) to determine any changes in particle size and loading efficiency. [E-mail protection] sampled in "0 months" is regarded as a control.

An in vitro release experiment was carried out, and the method used was almost unchanged. 53 First, 50 mg of differently prepared [Email Protection] was suspended in a vial containing 5 mL of PBS buffer (37°C, pH = 7.4), which contained a constant shaking speed of 100 rpm (Clifton Shaking Bath NE5, Nikel Electro Ltd., Weston-super-Mare, UK). At predetermined time intervals, remove 0.5 mL samples (refilled with an equal amount of fresh PBS) and centrifuge at 12,000 rpm for 5 minutes. The samples were analyzed using a 1290 Infinity II liquid chromatography system (Agilent Technologies Inc., Santa Clara, CA, USA).

The Animal Care and Use Committee of the Municipal Council of Macau approved all the studies described in this article (approval number AL010/DICV/SIS/2018) and conducted experiments under the guidance of the NIH Laboratory Animal Care and Use Guide (8th edition)). All experimental rats were kept in a 12-hour light-dark cycle facility, with constant temperature and humidity, and equipped with a ventilation system. A blood sample was drawn through the tail vein of the rat, each with a volume of about 100 μL. After the experiment, the experimental rats were euthanized with carbon dioxide under the guidance of the Macau University of Science and Technology (MUST) Laboratory Animal Care and Use Guide.

First, the blood samples were dispersed in normal saline for subcutaneous injection. Each sample contained the same amount of SPRC or KETO, which was calculated from the weight of the rat. SPRC was 50 mg kg-1 and KETO was 10 mg kg-1. Collect blood samples from rats to heparin sodium tubes at predetermined times (0, 1, 2, 6, 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, 144, 156 and 168 h) And analyze. Each group contains 5 rats.

The measurement of H2S concentration adopts the reported method with slight modifications54,55. The measurement scheme for H2S detection in vivo is shown in Figure 1. In short, 15 μL of serum sample, 25 μL of MBB acetonitrile solution and 35 μL of 0.3% DTPA containing Tris-HCl buffer (pH 9.5) were mixed and incubated in an anoxic incubator for 30 minutes. Then add 25 μL of sulfosalicylic acid to stop the reaction, and centrifuge at 12,000 rpm for 10 min. Finally, 30 μL of supernatant, 267 μL of acetonitrile and 3 μL of internal standard (hydrocortisone methanol solution) were mixed and analyzed by LC-MS. Figure 1. Endogenous H2S detection mechanism.

Figure 1. Endogenous H2S detection mechanism.

The samples were analyzed using Agilent 1200 series HPLC system (Agilent Technologies Inc., Santa Clara, CA, USA) and Agilent 6460 triple quadrupole (Agilent Technologies Inc., Santa Clara, CA, USA). ZORBAX Eclipse Plus 95 C18, 2.1*50 mm, 1.8 μm column (Agilent Technologies Inc., Santa Clara, CA, USA) was used, and the temperature was set to 35°C. The mobile phase is composed of water (A) and acetonitrile (B), and the gradient delivery is as follows: 0-0.5 minutes, 5% B; 0.5-0.6 minutes, 5-20% B; 0.6-5.0 minutes, 20-47.5% B; 5.0 –5.1 minutes, 47.5-95% B; 5.1-6.0 minutes, 95% B, flow rate 0.3 mL min-1. The mass spectrometer operates in positive ion mode. The selected scan type is MRM, the gas temperature is 325°C, and the gas flow rate is 10 L min-1. The scanning time is 500 ms, and the start-stop quality is 100~1000. The injection volume is 5 μL.

The rat adjuvant arthritis (AIA) model was established by subcutaneous injection of CFA at the base of the tail according to the manufacturer's instructions. 25 rats weighing 180-200 g were randomly divided into the following 5 groups: control group (n=5), no intervention; AIA model group (n=5), SPRC group (n=5), KETO group (n= 5) In the [email protected] group (n=5), the control group was injected with 0.1 mL of saline.

According to our previous research, 45 this study finally selected a SPRC dose of 50 mg kg-1, and then a KETO dose of 10 mg kg-1 for further research. After CFA injection, SPRC received subcutaneous injections of 50 mg kg-1 SPRC, 10 mg kg-1 KETO and [email protected] (equivalent to 50 mg kg-1 SPRC and 10 mg kg-1 KETO) according to the weight of the rat. The dosing interval was selected as q72h once, and the AIA model group was injected with 3 mL of saline subcutaneously.

The paw volume was measured by Ugo Basile 7140 volume measuring instrument (Ugo Basile, Gemonio VA, Italy) on 0, 5, 10, 15, 20, 25 and 30 days after CFA injection. The arthritis index ranges from 0 to 4 points for each limb, 0=no signs of inflammation, 1 to 4=increased inflammation, and the highest score for each rat is 16 points. Table 1 shows the arthritis index scoring system. 56 Table 1 Arthritis Scoring System

Table 1 Arthritis scoring system

Place the rat in a transparent squirrel cage with a wire mesh at the bottom. Allow the rat to enter the cage freely for at least 30 minutes to ensure that the rat can adapt to the new environment. Von Frey filaments with different forces of 0.6, 1, 2, 4, 6, 8, 10, and 15 g were applied, and the pain threshold was detected by the up and down method. 57 With a slow increase in intensity, the mechanical withdrawal threshold is defined as the rat's sudden contraction. The mechanical withdrawal threshold was measured and recorded when the positive reaction was observed on the 0th, 5th, 10th, 15th, 20th, 25th and 30th days after CFA injection.

As mentioned earlier, the hind limbs of different groups of rats were scanned and reconstructed by Siemens Inveon PET/CT (Siemens, Munich, Germany). The exposure time is set to 800 ms, and the voltage and current are set to 70 kV and 400 mA, respectively.

On the 30th day, blood was collected from rats in each group, and the serum levels of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) and anti-inflammatory cytokines (IL-10) were determined by ELISA kit according to the manufacturer's instructions.

The main organs including the heart, liver, spleen, lung, kidney, small intestine, large intestine and stomach were dissected and fixed with 4% paraformaldehyde, and further stained with H&E.

Use GraphPad Prism to perform statistical analysis on the samples. Each experiment is carried out at least 3 times, and the data are expressed as mean±standard deviation. A one-way analysis of variance (ANOVA) test was used to determine statistical significance, unless otherwise stated, p<0.05 was considered significant.

[email protected]’s preparation instructions are shown in Figure 2A. First observe the morphology of [email protected] using SEM and DLS, as shown in Figure 2B and C. Spherical, smooth surface and monodisperse particles can be observed under SEM and DLS, which indicates that the particle size, yield, LE and EE of the successful preparation are shown in Table 2. Through the double emulsion method, about 79.89% of PY can be obtained. The particle size of [email protected] is about 25.12 μm. The LE of SPRC and KETO are 6.47% and 2.64%, respectively, while the EE of SPRC and KETO are 36.27% and 68.28%, respectively. . As shown in Figure 2D and E, [email protected] shows excellent storage stability at 4°C or 25°C and 50% humidity, which shows that [email protected] is stable and SPRC and KETO are in storage conditions . The results of in vitro release are shown in Figure 2F. According to reports, 47 together with our previous experiments (data not shown), 200 mg of KETO can be successfully loaded into PLGA microspheres. However, a final KETO amount of 45 mg was chosen in this study. The reason why we reduce the KETO loading is because a large amount of KETO may cause potential side effects in the body. Further details will be explained below. Table 2 Characterization parameters of [email protected] (n = 3, Mean ± SD) Figure 2 Preparation of [email protected] (A) Schematic diagram of [email protected] preparation (B) [email protected] SEM protected] (scale bar = 10 μm). (C) [email protected] particle size distribution (D and E) [email protected] shows stable particle size and load efficiency when stored at 4°C or 25°C and 50% humidity, and (F) [ email protected]] shows sustained release in vitro.

Table 2 Characteristic parameters of [email protected] (n = 3, Mean ± SD)

Figure 2 Preparation of [email protected] (A) Schematic diagram of preparation of [email protected] (B) SEM of [email protected] (scale bar = 10 μm). (C) [email protected] particle size distribution (D and E) [email protected] shows stable particle size and load efficiency when stored at 4°C or 25°C and 50% humidity, and (F) [ email protected]] shows sustained release in vitro.

According to reports, SPRC can promote the release of H2S in the body, and as far as we know, there is no report that KETO can increase the release of H2S in the body. As shown in Figure 3A, the SPRC group showed an immediate increase in H2S and H2S metabolism within 6 hours after a single injection of SPRC solution, while [email protection] can slowly increase the H2S concentration in the body at least 24 hours after the injection. At the same time, when SPRC and KETO were co-injected, there was no significant change in real-time H2S levels in vivo compared with SPRC alone. However, after 24 hours, as shown in Figure 3B, the H2S concentration in the [email protected] group showed a downward trend. At the same time, the SPRC and SPRC KETO groups no longer produced H2S in the body. Interestingly, 108 hours after the supplement was injected, the H2S levels of all groups remained stable, indicating that the endogenous H2S produced by the supplement was completely metabolized (Figure 3C). It was observed that after the administration of KETO, the same level of H2S in the body as the control group was observed to be stable, indicating that the injection of KETO alone did not promote endogenous H2S (as shown in Figure 3). Figure 3 Real-time H2S concentration in vivo after a single injection of supplement. (A) The first 24 hours after a single injection, (B) 36-96 hours after a single injection, and (C) 108-168 hours after a single injection of supplements. In the figure, * indicates a significant difference compared with the control group. (N = 5, mean ± SD).

Figure 3 Real-time H2S concentration in vivo after a single injection of supplement. (A) The first 24 hours after a single injection, (B) 36-96 hours after a single injection, and (C) 108-168 hours after a single injection of supplements. In the figure, * indicates a significant difference compared with the control group. (N = 5, mean ± SD).

Clinically, RA patients often have physical signs such as swelling, erythema, and fever. The anti-inflammatory experimental design is shown in Figure 4A. According to reports, the establishment of AIA is usually accompanied by weight loss in the AIA model group. 57 This phenomenon can be observed in Figure 4B. Then, the anti-edema effect of [Email Protection] on AIA in rats was evaluated by measuring paw volume and arthritis index. As shown in Figure 4C-D, no significant increase in paw volume and arthritis index was observed in the first 10 days. From the 10th day, a sharp increase in paw volume and arthritis index can be observed in the AIA model group, and [email protection] seems to suppress paw volume and arthritis index. In contrast, SPRC and KETO did not succeed in inhibiting the development of AIA progression, which indicates that the co-delivery of SPRC and KETO has a synergistic effect on anti-edema. In Figure 4E, erosion can be observed in the model group as well as the SPRC and KETO groups, which indicates that the treatment effect of SPRC and KETO alone is lower. However, the [email protection] group showed a significant therapeutic effect. [email protected] The anti-inflammatory effect of [email protected] is also assessed by the measurement of plasma cytokines. As shown in Figure 4F-I, compared with the model group, neither the SPRC group nor the KETO group showed significant anti-inflammatory effects in AIA rats, while the pro-inflammatory cytokines (Figure 4F-H) were significantly reduced, with anti-inflammatory effects Increase. Cytokines can be observed in the [email protection] group (Figure 4I). Figure 4 [Email protection] can reduce paw edema and inflammation in AIA rats. (A) Experimental design for AIA mitigation. (B) Changes in body weight during the 30-day experiment. (C) Measure paw volume and (D) arthritis index to assess the severity of swelling symptoms. (E) Representative 3D reconstructed images of rat paws from different treatment groups. The pro-inflammatory cytokine levels of (F) TNF-a, (G) IL-1b and (H) IL-6 and the anti-inflammatory cytokine levels of (I) IL-10 in rats were measured (n = 5, average Value ± standard deviation). In this figure, * indicates a significant difference compared with the model group.

Figure 4 [Email protection] can reduce paw edema and inflammation in AIA rats. (A) Experimental design for AIA mitigation. (B) Changes in body weight during the 30-day experiment. (C) Measure paw volume and (D) arthritis index to assess the severity of swelling symptoms. (E) Representative 3D reconstructed images of rat paws from different treatment groups. The pro-inflammatory cytokine levels of (F) TNF-a, (G) IL-1b and (H) IL-6 and the anti-inflammatory cytokine levels of (I) IL-10 in rats were measured (n = 5, average Value ± standard deviation). In this figure, * indicates a significant difference compared with the model group.

The Von Frey test was used to study the anti-nociceptive effect, and this test is also an important indicator for evaluating the severity of inflammation in AIA rats. The experimental design and schedule are shown in Figures 5A and B, respectively, and the results are shown in Figure 5C. The rats in the control group showed a stable withdrawal threshold at 15g. At the same time, with the establishment of AIA, a sharp drop in the exit threshold can be observed. However, neither the SPRC group nor the KETO group could prevent the trend of lower withdrawal threshold caused by AIA. Interestingly, the [email protected] group showed a significantly different withdrawal threshold compared with the model group (P <0.05), which indicates that [email protected] has a potential analgesic effect. Figure 5 Anti-nociception experiment. (A) Description of the experimental protocol, and (B) the timetable for this experiment. (C) Changes in the withdrawal threshold s of AIA rats in different treatment groups (n = 5, mean ± SD). In this figure, * indicates a significant difference compared with the model group.

Figure 5 Anti-nociception experiment. (A) Description of the experimental protocol, and (B) the timetable for this experiment. (C) Changes in the withdrawal threshold s of AIA rats in different treatment groups (n = 5, mean ± SD). In this figure, * indicates a significant difference compared with the model group.

Long-term high-dose SPRC usually causes side effects such as organ damage, which may be caused by the persistent high H2S concentration in the body, and long-term exposure to H2S may cause harm to the organism. 59,60 Supplements that assess potential harm to the body, remove the heart, liver, spleen, kidneys and lungs from rats, and examine them by H&E staining. As shown in Figure 6A, compared with the control group, no tissue damage was observed in any experimental group, which indicates that the supplement has lower irritation and toxicity in the body. Figure 6 Evaluation of potential adverse effects of supplements in experimental rats. After 30 days of experiment (scale bar=100μm), (A) heart, liver, spleen, kidney, lung, (B) and stomach, small intestine, and large intestine were examined by H&E staining.

Figure 6 Evaluation of potential adverse effects of supplements in experimental rats. After 30 days of experiment (scale bar=100μm), (A) heart, liver, spleen, kidney, lung, (B) and stomach, small intestine, and large intestine were examined by H&E staining.

In addition, as a non-steroidal anti-inflammatory drug, KETO may also have gastrointestinal side effects in rats. 61-63 In this article, the stomach, small intestine, and large intestine were also removed from the rat and further examined by H&E staining. As shown in Figure 6B, no damage was observed in any experimental group, which indicates that the supplement has a lower stimulation of the gastrointestinal system.

PLGA is a pharmaceutical material with high biocompatibility and biodegradability approved by the FDA. 64 PLGA microsphere preparations have attracted much attention due to their slow release, low toxicity, and stable fluctuations in blood concentration. 65-68 In this study, the release curve of KETO showed a two-stage release curve, namely, the initial burst release phase and the sustained release phase. KETO is encapsulated in the oil phase near the particle surface, while SPRC is in the internal water phase near the particle core, which explains the initial burst of KETO instead of SPRC. In addition to the initial burst release, delayed SPRC can be observed compared with KETO, which further proves that SPRC is distributed near the particle core and KETO is close to the particle surface.

Our previous study used SPRC at a dose of 100 mg kg-1 to treat AIA in rats by subcutaneous injection or oral administration, q72h once, showing a satisfactory therapeutic effect, while the dose of 50 mg kg-1 showed limited treatment effect. 55,58 However, in this study, we have different findings. In the SPRC group, we only used SPRC at a dose of 50 mg kg-1, which is consistent with our previous study and no therapeutic effect was observed. When SPRC and KETO are co-delivered in PLGA microspheres ([email protected]) at a dose equivalent to 50 mg kg-1 SPRC, a significant therapeutic effect on AIA can be observed. The reasons for the improved treatment effect may come from two aspects. On the one hand, compared with SPRC powder, the sustained release of SPRC from [email protected] may increase the H2S concentration in the body to a relevant stable level. 58,69 On the other hand. The co-delivery of NSAID may also enhance the therapeutic efficacy of SPRC.

In the anti-nociception study, the control group rats showed a stable withdrawal threshold during the experiment. However, with the establishment of AIA, it can be observed that the withdrawal threshold of the AIA group is significantly reduced, which is consistent with the reported study. 70,71 In theory, KETO, as an excellent analgesic, should show its anti-nociceptive effect. When injected subcutaneously into rats at a dose of 2–5 mg kg-1 q24h. 72,73 However, although we injected KETO at a dose of 10 mg kg-1, a limited anti-nociceptive effect was observed in this study. It may be due to the change of the dosing interval from q24h to q72h. This result emphasizes the importance of maintaining KETO concentration in the body.

In this study, all of our experimental groups had negligible damage to the main organs of rats, and some reports showed that therapeutic doses of KETO can cause serious gastrointestinal toxicity, such as acute gastrointestinal bleeding, erosion and ulcer. 72 The sustained-release mode of KETO from [Email Protection] (even higher than the therapeutic dose) may be one reason for the reduction of side effects, and the co-delivery of H2S release donors may be another important reason.

In this study, the PLGA microspheres ([email protected]) co-loaded by SPRC and KETO were successfully prepared, [email protected] were monodisperse spheres and sustained release in vitro (up to 15 days). [email protected] can continue to increase the concentration of H2S in the body, and [email protected] also shows therapeutic effects on AIA (anti-inflammatory and anti-nociception) in rats with low stimulation to major organs. Most importantly, it can be inferred that the co-delivery of H2S donors and NSAIDs may promote the anti-inflammatory effects of NSAIDs.

This work was supported by the Macau Science and Technology Development Fund (FDCT) (0067/2018/A2, 033/2017/AMJ, 0007/2019/AKP, 0052/2020/A, 0011/2020/A1, 0030/2018/A1 ), and the National Natural Science Foundation of China (No. 81973320) awarded Zhu Yizhun.

The authors report no conflicts of interest in this work.

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