A cell electrochemical sensor and its use for assessing capsaicin anti-inflammatory activity

Abstract

The present application belongs to the field of biosensing technology, and particularly relates to a cell electrochemical sensor and its use for evaluating the anti-inflammatory activity of capsaicin. The present application first prepares Sb2O4 / rGO, and then synthesizes a DDAB-HIMIMPF6 composite material with stable current response and low biological toxicity through one step, and then combines the DDAB-HIMIMPF6 composite material with Sb2O4 / rGO, and uses the electrochemical sensor to load, and finally obtains a cell electrochemical sensor with high selectivity and high sensitivity, which can be applied to evaluate the anti-inflammatory activity of capsaicin. Moreover, the present application provides a cell electrochemical sensor which can not only realize the use for evaluating the anti-inflammatory effect of capsaicin, but also has the outstanding advantages of low cost, fast detection, simple operation and miniaturization, and has a broad application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biosensing technology, and particularly relates to a cell electrochemical sensor and its use for evaluating the anti-inflammatory effect of capsaicin. BACKGROUND

[0002] Capsaicin (8-methyl-N-vanillyl-6-nonenamide) is a derivative of benzylamine, and is the main compound in the capsaicin family, accounting for nearly 70% of the total number of capsaicin compounds in spices. The content of capsaicin is an important quality index of commercial peppers and other peppers. Many people consider capsaicin as the king of medicinal materials because they believe that it can bring many health benefits. It is reported that capsaicin not only increases the flavor of food, but also has various pharmacological and physiological activities, including analgesia, anticancer, anti-inflammatory, antioxidant, improvement of the gastrointestinal system, weight loss, etc. Among them, the natural anti-inflammatory ability of capsaicin can relieve symptoms such as arthritis, heart disease and diabetes, and reduce the risk of their occurrence. Therefore, testing the anti-inflammatory activity of capsaicin in cells has important clinical and practical significance.

[0003] Capsaicin anti-inflammatory is a newly developed method, and capsaicin anti-inflammatory detection has obvious advantages because it simulates the growth process of cells, including cell absorption and metabolism. At present, A549 cells are widely used as an in vitro model of type II alveolar epithelial cells, and are the preferred cells for establishing an in vitro cell model of acute lung injury (ALI). Endotoxin is an important factor leading to ALI, and its main component, lipopolysaccharide (LPS), plays an important role in ALI. LPS-induced A549 cells can establish an ideal in vitro inflammatory injury model of ALI. Nitric oxide (NO) is selected as a representative of inflammatory factors. Studies have shown that the amount of NO released from cells is proportional to the concentration of LPS, and capsaicin pretreatment can significantly reduce LPS-induced acute lung injury in mice. Therefore, the anti-inflammatory activity of capsaicin is evaluated by detecting the amount of NO released.

[0004] The main methods for detecting nitric oxide include gas chromatography-mass spectrometry, fluorescence method, and spectroscopic analysis method and chemical analysis method. The spectroscopic analysis method includes infrared absorption spectroscopy and ultraviolet absorption spectroscopy. The chemical analysis method mainly uses the determination of the reaction product of nitric oxide and other compounds to indirectly quantify the concentration of NO, and the common methods are Griess reagent method and iron salt method. However, due to the factors such as complicated detection steps, expensive detection reagents and short half-life of NO of traditional methods, it is imperative to select a rapid, simple, low-cost and efficient method for detecting nitric oxide.

[0005] Compared with other detection methods, the electrochemical sensor has small electrode size, non-destructive analysis mode, minimal or no reagent requirement, high sensitivity, simplicity and low cost. Therefore, electrochemical detection is a feasible method for measuring NO released by cells. In the existing technology, the patent "fluorescent probe compound for detecting nitric oxide and preparation method and application thereof" (CN118126073A) discloses a high-stability and high-selectivity nitric oxide fluorescent probe, but the preparation process of the probe is relatively complicated and the preparation time is relatively long. The patent "fluorescent probe capable of separately and simultaneously detecting nitric oxide and nitrosyl and preparation method and application thereof" (CN116655692A) discloses a high-selectivity, non-invasive visual fluorescent detection probe, but the fluorescence is easily quenched and the fluorescence intensity is weak, which also reduces the detection effect. SUMMARY

[0006] In view of the above problems, the present application aims to solve one of the problems; the present application provides a cell electrochemical sensor and its use for evaluating the anti-inflammatory effect of capsaicin, which has the outstanding advantages of low cost, fast detection, simple operation, strong miniaturization capability, etc.

[0007] In order to achieve the above purpose, the present application adopts the following technical solutions:

[0008] A preparation method of a cell electrochemical sensor, the steps are as follows:

[0009] Step one: preparation of Sb2O4 / rGO with flower-shaped nanostructure;

[0010] (1) dissolve antimony trichloride in ethanol to obtain an antimony trichloride ethanol solution; then add a graphene oxide suspension to the antimony trichloride ethanol solution and mix, and then perform ultrasonic treatment to obtain a mixed solution;

[0011] (2) seal the mixed solution obtained in step (1) in a reaction kettle and perform heating reaction, collect the generated black precipitate after heating reaction, then soak in ultrapure water overnight, and finally collect the precipitate after filtration, freeze-dry the precipitate product, and obtain the final product, which is Sb2O4 / rGO;

[0012] Step two: preparation of DDAB-HIMIMPF6 composite material;

[0013] Mix the didodecyldimethylammonium bromide solution with 1-hexyl-3-methylimidazolium hexafluorophosphate to obtain a mixed material, which is denoted as DDAB-HIMIMPF6 composite material (which will become solid at room temperature, therefore, if it is in solid state before use, the composite material needs to be changed to liquid by heating);

[0014] Step three: preparation of an electrochemical sensor;

[0015] (1) The Sb2O4 / rGO prepared in step one was weighed and dissolved in pure water to obtain a mixture, which was then added dropwise to a screen-printed electrode (SPCE) and dried with nitrogen to obtain a treated screen-printed electrode;

[0016] (2) The DDAB-HIMIMPF6 composite material prepared in step two was heated in a water bath to form a liquid state, which was then added dropwise to the treated screen-printed electrode of step 1 and dried at room temperature to obtain a cell electrochemical sensor.

[0017] Preferably, the amount of antimony trichloride, ethanol and graphene oxide suspension in step one (1) is 0.5 mol: 15 ml: 15 ml, wherein the concentration of graphene oxide suspension is 1 mg / ml; the ultrasonic treatment time is 30 min.

[0018] Preferably, the heating reaction temperature in step two (2) is 180℃, and the time is 21 h.

[0019] Preferably, the volume ratio of dodecyl dimethyl ammonium bromide solution to 1-hexyl-3-methyl imidazole hexafluorophosphate in step two is 5:1, wherein the concentration of dodecyl dimethyl ammonium bromide solution is 0.1M.

[0020] Preferably, the amount of Sb2O4 / rGO and pure water in step three (1) is 1 mg: 1 ml, and the amount of the mixture added dropwise is 5 μL.

[0021] Preferably, the water bath heating temperature in step three (2) is 20-40℃, and the amount of dropwise addition is 5 μL.

[0022] The use of the cell electrochemical sensor for evaluating the anti-inflammatory effect of capsaicin is as follows:

[0023] Step 1: Preparation of cell-methyl acrylate gelatin hydrogel (GelMA);

[0024] (1) Gelatin was dissolved in phosphate buffer solution (PBS), after stirring, methacrylate anhydride was added dropwise, stirred uniformly to obtain a mixed solution; then the mixed solution was diluted 3-5 times with phosphate buffer solution to terminate the reaction to obtain a reaction liquid; the reaction liquid obtained in step 1 was dialyzed to obtain a dialyzed liquid; then the dialyzed liquid was filtered, and the obtained filtrate was pre-frozen to obtain a GelMA prepolymer; wherein the amount ratio of gelatin, phosphate buffer solution and methacrylate anhydride is 5g: 50ml: 4-6ml; the temperature for stirring uniformly is 50℃, when the phosphate buffer solution is used for dilution, the temperature of the phosphate buffer solution is 50℃, and the dilution is 1-fold each time; the dialysis temperature is 50℃, and the time is 7-14d; the filtration is performed using a membrane with a pore size of 0.22μm; the pre-freezing temperature is-20℃ or-80℃, and the pre-freezing time is 5-10d.

[0025] (2) GelMA prepolymer was weighed in a brown bottle, then PBS containing a photoinitiator Irgacure 2959 was added, and after warm bath dissolution, filtration was performed to obtain a GelMA solution; wherein the amount ratio of GelMA prepolymer and PBS is 0.25-1g: 5ml; the photoinitiator is Irgacure 2959, and the concentration in PBS is 0.5%(w / v); the warm bath temperature is 40℃, and the filtration is performed using a membrane with a pore size of 0.22μm;

[0026] (3) A549 cells were resuspended with the GelMA solution to obtain a cell-GelMA solution, wherein the cell density was 10 6 cells / ml; then the cell-GelMA solution was added to each well of a 96-well plate, and photocuring was performed under ultraviolet light to form a cell-GelMA hydrogel, which was recorded as A549 / GelMA; wherein the amount of the cell-GelMA solution was 100μL, the ultraviolet light conditions were: 405nm, 50mW / cm 2 , and the photocuring time was 30s;

[0027] Step 2: Establishment of LPS-induced A549 cell inflammation model

[0028] Firstly, LPS solution was prepared in DMEM medium, and the concentration was 0-20μg / ml; then an equal volume of LPS solution was added to A549 / GelMA for induction treatment; after the induction treatment, the supernatant was taken as a test liquid, and a cell electrochemical sensor was used for testing, and DPV method was used to determine the current signal change, and the corresponding current value was recorded as ILPS; the induction treatment time was 24h;

[0029] Step 3: Capsaicin anti-inflammatory activity detection

[0030] (1) First, a capsaicin solution was prepared using DMEM medium with a concentration of 0–180 μM; then, 100 μL of capsaicin solution was added to the A549 / GelMA prepared in step 1 for incubation, with an incubation temperature of 37 °C and a gas environment of 5% CO2; after incubation, 100 μL of LPS solution (concentration of 1 μg / ml) was added for induction for 24 h.

[0031] After induction, the supernatant was used as the test solution, and the cell electrochemical sensor was used for testing. The peak current value of NO released by the cells was detected by the DPV method, and the current value was recorded as ICAP.

[0032] The relative anti-inflammatory capacity of capsaicin was evaluated using formula (I):

[0033]

[0034] Where: ILPS is the peak current value of DPV induced by LPS treatment;

[0035] ICAP is the DPV current value of A549 cells after incubation with capsaicin followed by LPS induction.

[0036] IA is the blank control, i.e., the peak current of DPV without capsaicin and LPS treatment;

[0037] △NO: The amount of NO release reduced.

[0038] By comparing the NO current value released by A549 cells stimulated by LPS with the NO current value released by A549 cells stimulated by LPS after intervention with capsaicin, the reduction in NO release can be calculated according to formula (I).

[0039] The anti-inflammatory ability of capsaicin is assessed by the reduction in NO release (ΔNO). The greater the reduction, the stronger the anti-inflammatory ability of capsaicin.

[0040] The beneficial effects of this invention are:

[0041] On the one hand, the unique, highly networked flower-like nanostructure of Sb₂O₄ / rGO, due to its unique three-dimensional flower-like structure, allows NO to easily enter the reaction center for further capture and adsorption, subsequently forming a crystalline structure within the Sb₂O₄ / rGO nanostructure. 3+ and Sb 4+ Rapid electron transfer occurs between them, and charge transport is enhanced by reducing graphene oxide sheets.

[0042] On the other hand, a novel biocompatible composite membrane consisting of the water-insoluble surfactant bis(dodecyl dimethyl ammonium bromide) (DDAB) and the hydrophobic room-temperature ionic liquid (RTIL) 1-hexyl-3-methylimidazolium hexafluorophosphate (HIMIMPF6) can be used as an electrode coating to eliminate NO.2- , interference of ascorbic acid and uric acid, and improved detection effect of the biosensor on NO gas.

[0043] In summary, the cell electrochemical sensor provided by the application not only has the use of evaluating the anti-inflammatory effect of capsaicin, but also has the outstanding advantages of low cost, fast detection, simple operation and miniaturization. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 SEM image of the GelMA prepolymer.

[0045] Figure 2 NMR hydrogen spectrum of GelMA.

[0046] Figure 3 Standard curve of different concentrations of sodium nitrite solution at 540 nm. DETAILED DESCRIPTION

[0047] The application will be described in more detail by means of the following implementation examples; the following examples are only illustrative, and the application is not limited by these implementation examples.

[0048] Example 1:

[0049] Step one: synthesis method of Sb2O4 / rGO

[0050] Dissolve 0.5 mol of antimony trichloride in 15 ml of ethanol to provide the element antimony, to obtain liquid ①; add 15 ml of 1 mg / ml graphene oxide suspension to liquid ① and mix, and ultrasonically treat for 30 min to obtain liquid ②.

[0051] Seal liquid ② in a polytetrafluoroethylene-lined reaction kettle, and heat at 180℃ for 21 h to reduce the graphene oxide and generate antimony tetroxide; after the heating reaction, collect the generated black precipitate, then soak in ultrapure water overnight to remove impurities, filter to collect the precipitate, and freeze-dry the product to obtain the final product Sb2O4 / rGO.

[0052] Step two: synthesis method of DDAB-HIMIMPF6

[0053] Mix 1 ml of didodecyldimethylammonium bromide solution (0.1 M) with 200 μL of 1-hexyl-3-methylimidazolium hexafluorophosphate to obtain the mixed material, which is denoted as DDAB-HIMIMPF6 composite material (which will become solid at room temperature, so if it is in a solid state before use, the composite material needs to be changed to a liquid by heating) ;

[0054] Step three: preparation of modified nitrogen monoxide electrochemical sensor

[0055] Take 1 mg Sb2O4 / rGO dissolved in 1 ml of pure water and stir until uniform, to obtain a mixture; and take 5 μL of the mixture and drop it on the screen-printed electrode, dry it under nitrogen, to obtain the treated screen-printed electrode;

[0056] Then the DDAB-HIMIMPF6 composite material is heated in a 40℃ water bath, melted into a liquid state, and then 5 μL is taken and added to the screen-printed electrode treated in step 1, and dried at room temperature, to obtain a cell electrochemical sensor.

[0057] Example 2:

[0058] The use of the cell electrochemical sensor to evaluate the anti-inflammatory effect of capsaicin is as follows:

[0059] Step 1: Preparation of cell-methyl methacrylate acylated gelatin hydrogel (GelMA)

[0060] (1) Dissolve 5 g of gelatin in 50 ml of phosphate buffered saline, stirring gently. Then add 4 ml of methacrylate anhydride to the gelatin solution and stir at 50℃ for 2 h. Dilute the solution with 50℃ phosphate buffered saline, 1-fold each time, 5 times to terminate the reaction, to obtain a reaction solution; dialyze the reaction solution obtained in step 1, dialyze at 50℃ for 7 d to remove low molecular weight impurities (molecular weight cut-off: 0-22 kDa), to obtain a dialyzed solution; then filter the dialyzed solution through a 0.22 μm membrane filter, pre-freeze at -80℃, and then freeze for 5 d, to obtain a GelMA prepolymer.

[0061] Figure 1 It is a field emission scanning electron microscope image of GelMA, which has a loose and porous structure, which is conducive to wrapping cells and transporting NO produced by cells. Figure 2 It is a 1H nuclear magnetic resonance spectrum of GelMA, as can be seen from the figure that GelMA is synthesized from gelatin, and after calculation, the synthesized GelMA precursor has a substitution degree as high as 76%.

[0062] (2) Take 0.25 g of GelMA prepolymer in a brown bottle, add 5 ml of 0.5% (w / v) photoinitiator Irgacure 2959 in PBS, dissolve at 40℃, and filter sterilize while hot with a 0.22 μm membrane filter.

[0063] (3) Take A549 cells and resuspend them in the GelMA solution to obtain a cell-GelMA solution, with a cell density of 10 6 cells / ml; then add 100 μL of the cell-GelMA solution to each well of a 96-well plate, and photocure under ultraviolet light (405 nm, 50 m W / cm 2 ) for 30 s to form a cell-GelMA hydrogel, denoted as A549 / GelMA;

[0064] Step 2: Establishment of an LPS-induced A549 cell inflammation model

[0065] Add 100 μL of LPS solutions prepared in DMEM medium at concentrations of 0, 0.4 μg / ml, 0.8 μg / ml, 1 μg / ml, 2 μg / ml, 4 μg / ml, 5 μg / ml, 10 μg / ml, and 20 μg / ml to the A549 / GelMA obtained in step 1 for induction treatment for 24 h.

[0066] After the induction treatment, the supernatant was used as the test solution and tested using a cell electrochemical sensor. The change in current signal was measured by the DPV method and the corresponding current value was recorded as ILPS.

[0067] Step 3: Validation (NO assay kit)

[0068] (1) NO generation level was determined using a NO assay kit based on Griess reaction.

[0069] RAW264.7 cells (100 μL, 10 5 (particles / ml) were induced in 96-well plates with 0, 0.4 μg / ml, 0.8 μg / ml, 1 μg / ml, 2 μg / ml, 4 μg / ml, 5 μg / ml, 10 μg / ml, and 20 μg / ml LPS solutions for 24 h.

[0070] Subsequently, 50 μL of culture supernatant was collected and mixed with 50 μL of Griess reagent I and 50 μL of Griess reagent II in an ELISA plate. After incubation for 15 min, the absorbance at 540 nm was measured using an ELISA reader.

[0071] (2) The standard curve generated by determining the NaNO2 standard solution using the same method is shown below. Figure 3 The detection of nitric oxide can be achieved based on a standard curve, and the results can be compared with the electrochemical results of this embodiment to determine the reliability of the method.

[0072] Step 4: Capsaicin anti-inflammatory activity assay

[0073] First, a capsaicin solution with a concentration of 0–180 μM was prepared using DMEM medium. Then, 100 μL of the capsaicin solution was added to the A549 / GelMA prepared in step 1 for incubation at a temperature of 37 °C and a gas environment of 5% CO2. After incubation, 100 μL of LPS solution (concentration of 1 μg / ml) was added for induction for 24 h.

[0074] The supernatant after induction is taken as a test solution, and the cell electrochemical sensor is used for testing, and the peak current value of NO released by the cells is detected by using DPV method, and the current value is recorded as ICAP.

[0075] The relative anti-inflammatory ability of capsaicin is evaluated by formula (I):

[0076]

[0077] In the formula, ILPS is the DPV peak current value of LPS induction treatment;

[0078] ICAP is the DPV current value of A549 cells after incubation with capsaicin and then LPS induction treatment;

[0079] IA is the DPV peak current of the blank control group without capsaicin and LPS treatment;

[0080] ΔNO: The reduction amount of NO release.

[0081] The NO current value released by LPS-stimulated A549 cells and the NO current value released by A549 cells after intervention with capsaicin and then LPS stimulation are compared; then according to formula (I), the reduction amount of NO release can be calculated;

[0082] The anti-inflammatory ability of capsaicin is evaluated by the reduction amount of NO release, and the greater the reduction amount, the stronger the anti-inflammatory ability of capsaicin.

[0083] Description: The above examples are only used to illustrate the technical solutions described in the present application and do not limit the technical solutions described in the present application; therefore, although the present application has been described in detail with reference to the above-mentioned embodiments, those skilled in the art should understand that the present application can still be modified or replaced by equivalents; all technical solutions and improvements that do not deviate from the spirit and scope of the present application should be covered within the scope of the claims of the present application.

Claims

1. A method for preparing a cell electrochemical sensor, characterized in that, Includes the following steps: Step 1: Preparation of flower-like nanostructured Sb₂O₄ / rGO; (1) Dissolve antimony trichloride in ethanol to obtain an antimony trichloride ethanol solution; then add graphene oxide suspension to the antimony trichloride ethanol solution and mix, and then treat with ultrasound to obtain a mixed solution; (2) The mixed solution obtained in step (1) is sealed in a reaction vessel and heated to react. After the heating reaction, the generated black precipitate is collected and then soaked in ultrapure water overnight. Finally, the precipitate is collected again after filtration. The precipitate product is freeze-dried to obtain the final product, which is Sb2O4 / rGO. Step 2: Preparation of DDAB-HIMIMPF6 composite material; The mixture of bis(dodecyl dimethyl)ammonium bromide solution and 1-hexyl-3-methylimidazolium hexafluorophosphate was denoted as DDAB-HIMIMPF6 composite material. Step 3: Fabrication of the electrochemical sensor; (1) Weigh the Sb2O4 / rGO prepared in step one, dissolve it in pure water and stir until uniform to obtain a mixed solution; then take the mixed solution and drop it onto the screen printing electrode, blow it dry with nitrogen gas to obtain the treated screen printing electrode. (2) The DDAB-HIMIMPF6 composite material prepared in step 2 is heated in a water bath to form a liquid state, and then dropped onto the screen-printed electrode after step 1. After drying at room temperature, the cell electrochemical sensor is obtained.

2. The method for preparing a cell electrochemical sensor according to claim 1, characterized in that, In step one (1), the ratio of antimony trichloride, ethanol and graphene oxide suspension is 0.5 mol: 15 ml: 15 ml, where the concentration of graphene oxide suspension is 1 mg / ml; the ultrasonic treatment time is 30 min.

3. The method for preparing a cell electrochemical sensor according to claim 1, characterized in that, In step two (2), the heating reaction is carried out at a temperature of 180°C for 21 hours.

4. The method for preparing a cell electrochemical sensor according to claim 1, characterized in that, In step two, the volume ratio of the dodecyl dimethyl ammonium bromide solution to 1-hexyl-3-methylimidazolium hexafluorophosphate is 5:1, and the concentration of the dodecyl dimethyl ammonium bromide solution is 0.1M.

5. The method for preparing a cell electrochemical sensor according to claim 1, characterized in that, In step three (1), the ratio of Sb2O4 / rGO to pure water is 1 mg: 1 ml, and the amount of the mixed solution added is 5 μL.

6. The method for preparing a cell electrochemical sensor according to claim 1, characterized in that, In step three (2), the water bath heating temperature is 20-40℃, and the amount added is 5μL.

7. The use of the cell electrochemical sensor prepared by any one of claims 1-6 for evaluating the anti-inflammatory effects of capsaicin.

8. The use according to claim 7, characterized in that, The steps are as follows: Step 1: Preparation of cell-methacrylated gelatin hydrogel; (1) Dissolve gelatin in phosphate buffer, stir, and then add methacrylate anhydride dropwise, stirring until homogeneous to obtain a mixed solution; then dilute the mixed solution 3-5 times with phosphate buffer to terminate the reaction, obtaining a reaction solution; dialyze the reaction solution obtained in step 1 to obtain a dialysate; then filter the dialysate, and pre-freeze the filtrate to obtain GelMA prepolymer; wherein the ratio of gelatin, phosphate buffer, and methacrylate anhydride is 5g:50ml:4-6ml; the stirring temperature is 50℃, and the temperature of the phosphate buffer is 50℃ when diluting with phosphate buffer, and the dilution is 1-fold each time; the dialysis temperature is 50℃, and the time is 7-14 days; filtration is performed using a membrane with a pore size of 0.22μm; the pre-freezing temperature is -20℃ or -80℃, and the pre-freezing time is 5-10 days; (2) Weigh the GelMA prepolymer into a brown bottle, then add PBS containing the photoinitiator Irgacure 2959, dissolve it in a warm bath, and then filter it to obtain a GelMA solution; wherein the ratio of GelMA prepolymer to PBS is 0.25-1g:5ml; the photoinitiator is Irgacure 2959, and the mass-volume percentage in PBS is 0.5%; the temperature of the warm bath is 40℃, and the filtration is performed using a membrane with a pore size of 0.22μm; (3) A549 cells were resuspended in GelMA solution to obtain cell-GelMA solution, in which the cell density was 10-1. 6 Cells / ml; then, cell-GelMA solution was added to each well of a 96-well plate and photocured under UV light to form a cell-GelMA hydrogel, denoted as A549 / GelMA; the volume of cell-GelMA solution used was 100 μL, and the UV light conditions were: 405 nm, 50 mW / cm². 2 The photocuring time is 30 seconds; Step 2: Establishment of an LPS-induced A549 cell inflammation model First, LPS solutions were prepared in DMEM medium at concentrations of 0–20 μg / ml. Then, an equal volume of LPS solution was added to A549 / GelMA for induction treatment. After induction treatment, the supernatant was used as the test solution, and the cell electrochemical sensor was used for testing. The change in current signal was measured using the DPV method, and the corresponding current value was recorded as ILPS. The induction treatment time was 24 h. Step 3: Capsaicin anti-inflammatory activity assay (1) First, a capsaicin solution was prepared using DMEM medium with a concentration of 0–180 μM; then, 100 μL of capsaicin solution was added to the A549 / GelMA prepared in step 1 for incubation, with an incubation temperature of 37 °C and a gas environment of 5% CO2; after incubation, 100 μL of LPS solution with a concentration of 1 μg / ml was added for induction for 24 h. After induction, the supernatant was used as the test solution and tested using a cell electrochemical sensor. The peak current value of NO released by the cells was detected by the DPV method and recorded as ICAP. The relative anti-inflammatory capacity of capsaicin was evaluated using formula (I): Where: ILPS is the peak current value of DPV induced by LPS treatment; ICAP is the DPV current value of A549 cells after incubation with capsaicin followed by LPS induction. IA is the blank control, i.e., the peak current of DPV without capsaicin and LPS treatment; △NO: The amount of NO release reduced; By comparing the NO current value released by A549 cells stimulated by LPS with the NO current value released by A549 cells stimulated by LPS after intervention with capsaicin, the reduction in NO release can be calculated according to formula (I). The anti-inflammatory ability of capsaicin is assessed using the ΔNO value; the larger the ΔNO value, the stronger the anti-inflammatory ability of capsaicin.

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