Application of Tetrandrine in Prevention and Treatment of Salmonella Infection

Abstract

The application discloses application of fangchinoline in prevention and treatment of salmonella infection. The fangchinoline is an isoquinoline alkaloid and is a host-directed therapy natural compound. The application discloses that the fangchinoline has a strong effect on treatment of salmonella infection diseases in vivo and in vitro, and the effect is specifically shown as that the fangchinoline can effectively reduce the bacterial amount of intracellular infection bacteria. The fangchinoline administration can significantly reduce the pathogenic bacteria load in the liver, spleen, colon, cecum and ileum of mice, and reduce the pro-inflammatory factor level of the organism. The safety in vivo and in vitro shows that the fangchinoline has no any toxic side effect under the test dose. In addition, the fangchinoline has significant synergistic antibacterial activity in combination with fluoroquinolones, which is shown as reduction of intracellular salmonella infection bacteria number and reduction of the pathogenic bacteria load in the liver, spleen and colon of mice. The application provides a new perspective for development of a new anti-infection drug, and also provides a new idea for prevention and control of the increasingly serious salmonella infection.

Description

Technical Field

[0001] This invention relates to the application of tetrandrine in the prevention and treatment of Salmonella infection, and belongs to the field of pharmaceutical technology. Background Technology

[0002] Salmonella is a foodborne zoonotic pathogen that causes acute gastroenteritis in humans and animals, accompanied by systemic symptoms such as fever. Severe cases can lead to bacteremia, resulting in high morbidity and mortality. Clinically, treatment of Salmonella infection primarily relies on antibiotics. However, with the spread and prevalence of multidrug-resistant strains, treating Salmonella infection has become a serious challenge. In recent years, host-directed therapy (HDT) with natural compounds has emerged as a promising anti-infective strategy for treating various intracellular bacterial infections. HDT drugs target host immune response pathways, enhancing host defense capabilities and ultimately helping host cells clear pathogens from within. Compared to antibiotics, HDT drugs do not directly exert survival pressure on pathogens, thus reducing the likelihood of inducing drug resistance. This has significant practical implications for the prevention and control of Salmonella infection.

[0003] Therefore, utilizing natural compounds to treat Salmonella infection is a promising antibacterial strategy. Among them, tetrandrine, derived from the rhizomes of plants in the Menispermaceae family, possesses various pharmacological activities, including anti-inflammatory and analgesic effects for treating rheumatism and arthritis; anti-tumor effects, inhibiting the proliferation of breast and bladder cancer cells; antihypertensive effects, which can be used to treat cardiovascular diseases; and anti-HIV-1 effects, effectively inhibiting viral replication. The molecular formula of tetrandrine is C1. 37 H 40 N2O6. The chemical structural formula of fangchinorline is shown in Formula 1:

[0004]

[0005] Tetrandrine, an alkaloid, has immune-enhancing, sedative, and anti-inflammatory effects. However, there is currently no research on its antibacterial applications. Summary of the Invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide the application of tetrandrine in the preparation of drugs for treating Salmonella infection.

[0007] A second objective of this invention is to provide the therapeutic effect of a combination of tetrandrine and fluoroquinolone drugs on Salmonella infection.

[0008] Technical solution: In order to solve the above-mentioned technical problems, the present invention provides the application of tetrandrine in the preparation of drugs for treating Salmonella or Salmonella infection.

[0009] The drug mentioned includes a pharmaceutically acceptable carrier.

[0010] The drugs mentioned also include antibiotics.

[0011] The antibiotics mentioned include fluoroquinolone antibiotics.

[0012] The present invention also includes a tetrandrine compound preparation, wherein the tetrandrine compound preparation comprises tetrandrine and fluoroquinolone antibiotics.

[0013] The mass ratio of the tebufenozide to the fluoroquinolone antibiotic is 1:1.

[0014] The concentration of the tebufenozide is 4–1024 μg / mL, and the fluoroquinolone antibiotics include levofloxacin.

[0015] Preferably, the concentration of the tetrandrine is 1.25-5 μM, and the concentration of the fluoroquinolone antibiotic is 1.25-5 μM.

[0016] When the tetrandrine compound preparation is used to treat Salmonella infection in mice, the concentration of tetrandrine is 5-10 mg / kg mouse.

[0017] Preferably, the concentration of the fluoroquinolone antibiotic is 5–10 mg / kg mouse.

[0018] The present invention also includes the application of the aforementioned tetrandrine compound preparation in drugs for treating Salmonella or Salmonella infection.

[0019] The drugs mentioned include those that reduce cellular inflammation.

[0020] The drug includes drugs that reduce the expression levels of pro-inflammatory factors TNF-α and / or IFN-γ; and drugs that increase the expression levels of IL-4 and / or IL-10.

[0021] This invention demonstrates that tetrandrine can effectively reduce the amount of Salmonella infection in vivo and in vitro.

[0022] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: This invention discloses for the first time that buprofen has a strong anti-Salmonella infection effect both in vivo and in vitro, specifically manifested in its effective reduction of intracellular Salmonella infection load. Buprofen significantly reduces the pathogen load in the liver, spleen, colon, cecum, and ileum of mice, and reduces the level of inflammation in the body. Furthermore, buprofen exhibits significant synergistic bactericidal activity when used in combination with fluoroquinolone drugs. Specifically, it reduces the number of intracellular Salmonella infection bacteria and the pathogen load in the liver, spleen, and colon of mice. Mechanistic studies show that buprofen can help the host clear infecting bacteria by enhancing macrophage lysosomal function and autophagy activity. This invention provides a new perspective for the development of novel drug resistance control strategies and a new method for the control of the increasingly serious Salmonella infection. Attached Figure Description

[0023] Figure 1 Screening for natural compounds to inhibit intracellular replication in Salmonella.

[0024] Figure 2 To evaluate the in vitro efficacy of fentanyl in reducing Salmonella infection, CON represents the blank control and Vehicle represents the DMSO negative control.

[0025] Figure 3 To evaluate the in vivo efficacy of phenoxybenzamine in controlling Salmonella infection, the term "Vehicle" refers to a DMSO negative control.

[0026] Figure 4 For the in vitro and in vivo safety evaluation of fentanyl, "Vehicle" refers to a DMSO negative control.

[0027] Figure 5 The image shows the results of combined treatment of Salmonella infection with tebuconazole and fluoroquinolones. In this image, Vehicle represents the DMSO negative control.

[0028] Figure 6 The image shows the results of using fentanyl to enhance the lysosomal activity of macrophages. In this image, "Vehicle" represents the DMSO negative control.

[0029] Figure 7 To investigate the effect of phenoxyline on the inflammatory level of Salmonella-infected cells, CON represents the blank control and Vehicle represents the DMSO negative control. Detailed Implementation

[0030] The technical solution of the present invention will be further described below with reference to the accompanying drawings. It should be noted that those skilled in the art can make several modifications and improvements without departing from the principle of the present invention, and these should also be considered within the scope of protection of the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were all purchased from conventional biochemical reagent stores. The quantitative experiments in the following embodiments were all performed in triplicate, and the results were averaged.

[0031] The LB broth medium used in the examples was an aqueous solution containing 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl; the MHB broth medium was an aqueous solution containing 2 g / L beef meal, 1.5 g / L soluble starch, and 17.5 g / L acid-hydrolyzed casein; and the DMEM basal medium was a solution containing 2 mM L-glutamic acid, 1 mM sodium pyruvate, glucose, amino acids, inorganic salts, and other basic nutrients.

[0032] Male BALB / c mice were purchased from the Comparative Medicine Center of Yangzhou University.

[0033] Tetrandrine was purchased from Macklin, catalog number F809650.

[0034] The Salmonella strain CMCC(B)50336 (SE50336) used in the examples was a strain donated by Professor Zhu Guoqiang of Yangzhou University and has been published (Positive regulation of Type III secretion effectors and virulence by RyhB paralogs in Salmonella enterica serovar Enteritidis.VetRes.2021Mar 10;52(1):44.doi:10.1186 / s13567-021-00915-z.).

[0035] Example 1: Screening of natural compounds that inhibit intracellular replication of Salmonella (HDT)

[0036] Taking Salmonella enteritidis strain SE50336 as an example, RAW264.7 cells were seeded in 96-well plates, and SE50336 strain was simultaneously inoculated into LB medium for activation overnight to regulate OD. 600=0.2, RAW264.7 cells were infected with a multiplicity of infection (MOI) of 10. One hour after infection, each well was incubated with DMEM (gibco, 11995065) complete medium containing 200 μg / mL gentamicin for 0.5 hours to kill extracellular bacteria. The medium was discarded, and the cells were washed three times with PBS (0.01M, pH=7.4). DMEM complete medium containing 50 μg / mL gentamicin was then added, along with different concentrations of natural compounds (Luteolin (20 μM): Purify, BP0896-20 mg; Diosmetin (20 μM): Purify, BP0506-20 mg; Pectolinarigenin (Luteolin)). Hesperidin (50μM): Purify, BP 1069-20mg; Puerarin (50μM): Purify, BP 1176-20mg; Tangeretin (50μM): Purify, BP 1360-20mg; Kurarinone (50μM): Purify, BP 3452-10mg; Scutellarei (100μM): Purify, BP 1277-20mg; Vitexin (100μM): Purify, BP 1447-20mg;

[0037] Anhydroicaritin (200μM): Purify, BP3070-20mg; Maackiain (50μM): Purify, BP0907-20mg; Fangchinoline (20μM): Macklin, F861275-

[0038] 20mg; Daphnetin (100μM): Macklin, D834977; Aloperine (200μM): Macklin, A873769; Mevastatin (50μM): Macklin, M813346; Artemisinin (100μM): Macklin, A800831; Methylprednisolone (20μM): Macklin, B938440; Rhein (200μM): Macklin, R817294; Bergenin (200μM): Macklin, B890212; Imiquimod (200μM): Macklin, I838239; Berberine Berberine hydrochloride (100 μM): Macklin, B877834) was cultured for 12 h, and the cells were lysed with 0.1% Triton X-100 for 20 min. Colony counts (CFUs) were performed on XLD plates by serial 10-fold dilutions.

[0039] The analysis results are shown below. Figure 1 The results showed that treatment with tetrandrine inhibited the replication of intracellular Salmonella by more than 90%, exhibiting the most significant bactericidal effect.

[0040] Example 2: Evaluation of the in vitro efficacy of tetrandrine in reducing Salmonella infection.

[0041] Intracellular Salmonella infection count: RAW264.7 cells were seeded in 96-well plates, and SE50336 strain was simultaneously inoculated into LB medium for activation overnight, and OD was regulated. 600 =0.2, RAW264.7 cells were infected with a multiplicity of infection (MOI) of 10. One hour after infection, each well was incubated with DMEM complete medium containing 200 μg / mL gentamicin for 0.5 hours to kill extracellular bacteria. The medium was discarded, and the cells were washed three times with PBS. Cells were then cultured in complete medium (DMEM medium containing 10% FBS) containing 50 μg / mL gentamicin and different concentrations of tetrandrine (0, 1.25, 2.5, 5 μM) for different time periods (0, 1, 4, 16 hours). Cells were lysed with 0.1% Triton X-100 for 20 minutes, and colony counts (CFUs) were performed on XLD plates after 10-fold serial dilutions.

[0042] Flow cytometry: The GFP-laden plasmid pXG10SF (a plasmid donated by Professor Zhu Guoqiang of Yangzhou University, Positive regulation of Type III secretion effectors and virulence by RyhB paralogs in Salmonella enterica serovar Enteritidis. Vet Res. 2021 Mar 10;52(1):44. doi:10.1186 / s13567-021-00915-z.) was transformed into the SE50336 strain. RAW264.7 cells were seeded in 6-well plates for 12 h. The GFP-laden SE50336 strain was then infected with the cells at MOI=10 for 1 h. The cells were then treated with complete culture medium (containing 10%...) Incubate with 200 μg / mL gentamicin diluted in FBS (DMEM medium) for 0.5 h, wash 3 times with PBS, add tetrandrine (containing 50 μg / mL gentamicin diluted in complete medium to a final concentration of 5 μM) and treat for 16 h, wash 3 times with PBS, collect cells from each well, filter through a sieve and collect the liquid, and analyze with a CytExpert flow cytometer.

[0043] Confocal microscopy observation: Sample pretreatment was similar to that of flow cytometry. SE50336 strain carrying GFP plasmid pXG10SF was used to infect cells with an MOI of 10. Cells were treated with different final concentrations of tetrandrine (0, 1.25, 2.5, 5 μM) diluted in complete medium containing 50 μg / mL gentamicin for 16 h. Cells were washed three times with PBS, fixed with 4% formaldehyde for 20 min, washed three times with PBS, and incubated with DAPI at a final concentration of 5 μg / mL for 20 min. Cells were washed three times with PBS, and antifluorescence quencher and sealing oil were added to the slides for observation using a CLSM microscope.

[0044] The analysis results are shown below. Figure 2 The results showed that tetrandrine reduced the number of intracellular Salmonella infections in a time- and concentration-gradient manner, and the effect of 5 μM tetrandrine treatment for 16 h was the most significant, as evidenced by a significant reduction in the number of CFUs counted on XLD plates and a decrease in green fluorescent protein (Salmonella) in flow cytometry and co-microscopy results.

[0045] Example 3: Evaluation of the in vivo efficacy of tetrandrine in reducing Salmonella infection.

[0046] Salmonella infection model in mice: Eighteen male BALB / c mice (Comparative Medicine Center of Yangzhou University, 6-8 weeks old, weighing 20±2g) were randomly divided into three groups: Vehicle group, tetrandrine group (FAN, 10mg / kg), and ciprofloxacin group (CPFX, 10mg / kg), with six mice in each group. They were acclimatized for 3 days with free access to food and water, and were fasted for 12 hours before infection. Salmonella SE50336 (1×10⁻⁶) was administered orally via gavage. 8 A mouse model of Salmonella infection was established using CFU / mouse. Twenty-four hours after infection, mice were injected intraperitoneally with 200 μL DMSO (Vehicle), 10 mg / kg tebufenoxanine (FAN), or 10 mg / kg ciprofloxacin (CPFX), respectively. Twelve hours after treatment, blood samples were collected from the eyeballs for routine blood tests. Mice were then euthanized by cervical dislocation, and the liver, spleen, and various segments of the small intestine (colon, cecum, and ileum) were harvested. Salmonella colony counts were performed on XLD plates, and inflammatory factors were detected using an ELISA kit.

[0047] The analysis results are shown below. Figure 3 The results showed that, compared with Vehicle mice, tetrandrine treatment significantly reduced the number of white blood cells and neutrophils in the blood of mice, and significantly reduced the bacterial load in organs of Salmonella-infected mice, decreased the expression of pro-inflammatory factors, and increased the expression of anti-inflammatory factors, indicating that tetrandrine alleviated the inflammatory response in infected mice.

[0048] Example 4: In vitro and in vivo safety evaluation of tetrandrine

[0049] Mouse toxicity experiment: Twenty-four male BALB / c mice (Yangzhou University Comparative Medicine Center, 6-8 weeks old, weighing 20g) were randomly divided into a Vehicle group and a fangchinorhinone group (FAN, 30mg / kg), with six mice in each group. 200μL of DMSO (Vehicle group) and 30mg / kg of fangchinorhinone were injected intraperitoneally into the right side of the mice, respectively. The mice's behavior and weight were observed and recorded daily for 14 days. Blood samples were collected from the mice's eyes on days 2 and 14 for blood biochemical analysis and sodium in vivo. + K + Cl - Concentration detection was performed, and liver and kidney samples were collected after cervical dislocation for HE staining.

[0050] Erythrocyte hemolytic assay: Tebuconazole was diluted (concentrations: 0, 4, 16, 64, 256, 1024 μg / mL) and placed in 96-well plates. Defibrinated blood was centrifuged and resuspended in PBS to prepare an 8% erythrocyte suspension, which was then added to the prepared erythrocyte suspension. The plates were incubated at 37°C for 1 hour, centrifuged at 4°C × 3000g for 10 min, and 100 μL was taken to measure the absorbance at 576 nm. PBS and sterile double-distilled water were used as negative and positive controls, respectively. The hemolysis rate was calculated as follows: Hemolysis rate (%) = [(OD...] 576 Sample-OD 576 (Negative control) / (OD) 576 Positive control - OD 576 [Negative control] × 100%.

[0051] The analysis results are shown below. Figure 4 The results showed that, compared with the Vehicle group, there was no significant difference in body weight among mice treated with tebuconazole, and the survival rate was 100%. There were no significant differences in ALT, AST, UREA, and CREA levels. Na... + K + CL - The concentrations were similar and within the normal range. Liver and kidney HE staining results showed no significant differences, indicating low in vivo toxicity of buspirone. High concentrations of buspirone (1024 μg / mL) did not cause hemolysis of sheep erythrocytes, indicating low in vitro toxicity of buspirone.

[0052] Example 5: Combined therapeutic effect of tetrandrine and levofloxacin on Salmonella

[0053] Salmonella infection model in mice: Twenty-four male BALB / c mice (Comparative Medicine Center, Yangzhou University, 6-8 weeks old, weighing 20±2g) were randomly divided into three groups: Vehicle group, tetrandrine group (FAN, 10mg / kg) + levofloxacin group (LVFX, 10mg / kg), and tetrandrine + levofloxacin group (FAN+LVFX, 5mg / kg each), with six mice in each group. The mice were acclimatized for 3 days with free access to food and water, and were fasted for 12 hours prior to infection. Salmonella SE50336 (1×10⁻⁶) was administered orally via gavage. 8 A Salmonella infection model was established in mice using CFU / mouse. Twenty-four hours after infection, mice were injected intraperitoneally with 200 μL DMSO (Vehicle), 10 mg / kg tetrandrine (FAN), 10 mg / kg levofloxacin (LVFX), 5 mg / kg tetrandrine, and 5 mg / kg levofloxacin (FAN+LVFX), respectively. Twelve hours later, mice were euthanized by cervical dislocation, and their livers, spleens, and cecums were harvested for Salmonella colony counting using XLD plates.

[0054] Combined treatment model of Salmonella cell infection: RAW264.7 cells were seeded in 96-well plates and simultaneously inoculated with SE50336 strain in LB medium for activation overnight, regulating OD. 600 =0.2, RAW264.7 cells were infected with a multiplicity of infection (MOI) of 10. One hour after infection, each well was incubated with DMEM complete medium containing 200 μg / mL gentamicin for 0.5 hours to kill extracellular bacteria. The medium was discarded, and the cells were washed three times with PBS. Cells were then co-cultured with 50 μg / mL gentamicin, 5 μM tetrandrine, and 5 μM of different antibiotics (chloramphenicol CAP, ciprofloxacin CPFX, levofloxacin LVFX, ceftriaxone sodium CTRX), as well as 2.5 μM tetrandrine, 2.5 μM chloramphenicol, 2.5 μM ciprofloxacin, 2.5 μM levofloxacin, and 2.5 μM ceftriaxone sodium for 4 hours and 16 hours, respectively. Cells were lysed with 0.1% Triton X-100 for 20 minutes, and colony counts (CFUs) were performed on XLD plates after 10-fold serial dilutions.

[0055] The analysis results are shown below. Figure 5 The results showed that, compared with the Vehicle group mice, treatment with tetrandrine and levofloxacin significantly reduced the bacterial load in the liver, spleen, and cecum of infected mice. At the same time, the results of the cell infection model also showed that tetrandrine had a combined therapeutic effect with fluoroquinolones, especially levofloxacin.

[0056] Example 6: Tetrandrine promotes intracellular Salmonella clearance by increasing lysosomal activity

[0057] Western blot: RAW264.7 cells were cultured overnight in 24-well plates (1×10⁻⁶ cells / well). 5 SE50336 strain infected cells (1×10⁻⁶ cells / well) with an MOI of 10⁻⁶. 6After 1 hour, each well was incubated with complete medium containing 200 μg / mL gentamicin (DMEM medium containing 10% FBS) for 0.5 hours to kill extracellular bacteria. The medium was discarded, and the cells were washed three times with PBS. Cells were then incubated with complete medium containing 50 μg / mL gentamicin and 5 μM tetrandrine for different time periods (0, 1, 4, 16 hours), washed twice with PBS, and collected. Cells were centrifuged at 4℃ × 1000 rpm for 5 minutes. The supernatant was discarded, and 200 μL of a 1 mM concentration of the protease inhibitor PMSF (Beyotime, ST506) and phosphatase inhibitor (Beyotime, P1045) diluted with RIPA lysis buffer (Beyotime, P0013B) was added to extract proteins. After dilution with 5×SDS loading buffer, the protein was transferred via SDS-PAGE and PVDF membrane wet transfer, blocked with rapid blocking buffer for 10 min, washed three times with TBST, and incubated overnight at 4°C with anti-LAMP1, LAMP2, and ATP6V0D1 antibodies (purchased from Abclonal, catalog numbers A16894, A1961, and A4271, respectively). After washing three times with TBST, the protein was incubated with HRP-goat anti-rabbit secondary antibody on a shaker at room temperature for 1 h. After development with ECL, the protein was photographed, and the grayscale value was measured using ImageJ software.

[0058] Immunofluorescence: RAW264.7 cells were cultured overnight in 24-well plates (1×10⁻⁶ cells / well). 5 Cells were infected with GFP-SE50336 strain at an MOI of 10 (1×10⁻⁶ cells / well). 6 After 1 hour, add 200 μg / mL gentamicin complete medium (DMEM medium containing 10% FBS) to each well and incubate for 0.5 hours to kill extracellular bacteria. Discard the medium and wash three times with PBS. Add 50 μg / mL gentamicin and 5 μM tetrandrine complete medium (DMEM medium containing 10% FBS) and incubate for 16 hours. Wash three times with PBS. Fix cells with 4% formaldehyde for 20 minutes. Wash three times with PBS. Apply 0.1% Trillium permeabilization membrane for 30 minutes. Wash three times with PBS. Block each well with rapid blocking buffer for 10 minutes. Wash three times with PBS. Add anti-LAMP1 antibody and incubate overnight at 4°C. Wash three times with PBS. Cy3 Goat Anti-Rabbit IgG (H+L) red fluorescent secondary antibody (purchased from Abclonal, catalog number AS007) was incubated on a shaker at room temperature for 1 hour, washed three times with PBS, and then DAPI was added to each well for 20 minutes. The mixture was washed three times with PBS. Antifluorescence quencher and sealing oil were added to the slides, and the staining was observed under a fluorescence microscope. Fluorescence values ​​were measured using ImageJ software. After staining, the fluorescence intensity of LAMP1 was observed under a fluorescence microscope, and fluorescence values ​​were measured using ImageJ software.

[0059] Lyso-Tracker-Red Tracing of Acidic Lysosomes: RAW264.7 cell culture was performed as described above. When the cultured cells reached 70%-80% confluence, the original culture medium was replaced with preheated (37°C) medium containing the Lyso-Tracker-Red probe (Beyotime, catalog number C1046). The cells were then incubated with 5 nM Lyso-Tracker-Red probe at 37°C for 30 min. The culture medium was discarded, and 1× Hoechst 33342 staining solution (Beyotime, C1026) was added, followed by incubation at 37°C for 10 min. Finally, the cells were observed using a fluorescence microscope, and the fluorescence value was measured using ImageJ software; the deeper the staining, the stronger the lysosomal activity.

[0060] The analysis results are shown below. Figure 6 The results showed that fangchinorlinine significantly promoted the expression of ATP6V0D1, LAMP1, and LAMP2 proteins in a time-dependent manner. Fangchinorlinine-treated Salmonella-infected cells showed increased LAMP1 fluorescence and enhanced Lyso-Tracker-Red fluorescence, indicating that fangchinorlinine significantly improved lysosomal activity and increased the number of acidic lysosomes.

[0061] Example 7: Tetrandrine reduces inflammation in Salmonella-infected cells.

[0062] Western blot: RAW264.7 cells were cultured overnight in 24-well plates (1×10⁻⁶ cells / well). 5 SE50336 strain infected cells (1×10⁻⁶ cells / well) with an MOI of 10⁻⁶. 6After 1 hour, add 200 μg / mL gentamicin complete medium (DMEM medium containing 10% FBS) to each well and incubate for 0.5 hours to kill extracellular bacteria. Discard the medium and wash three times with PBS. Add 50 μg / mL gentamicin and 5 μM tetrandrine complete medium and incubate for 16 hours. Wash twice with PBS, collect cells, and centrifuge at 4℃ × 1000 rpm for 5 minutes. Discard the supernatant and add 200 μL of a 1 mM protease inhibitor PMSF and a phosphatase inhibitor diluted with RIPA lysis buffer to extract proteins. After dilution with 5×SDS loading buffer, the protein was transferred via SDS-PAGE and PVDF membrane wet transfer, blocked with rapid blocking buffer for 10 min, washed three times with TBST, and incubated overnight at 4°C with anti-IκB (Abclonal, A19714), p-IκB (Abclonal, AP0707), p65 (Abmart, T55034), p-p65 (Abmart, TA2006), NF-κB (Abclonal, A6667), and p-NF-κB (Abclonal, AP1355) antibodies, washed three times with TBST, and incubated with HRP-goat anti-rabbit secondary antibody on a shaker at room temperature for 1 h. After development with ECL, the protein was photographed, and the grayscale value was measured using ImageJ software.

[0063] ELISA assay for inflammatory cytokine levels in Salmonella-infected cells: ELISA kits for inflammatory cytokines were used, including the pro-inflammatory IFN-γ ELISA kit (mlbio, ml002277), the TNF-α ELISA kit (mlbio, ml002095), and the anti-inflammatory IL-4 ELISA kit (mlbio, ml064310) and the IL-10 ELISA kit (mlbio, ml037888). Each kit had three replicate wells. 50 μL of diluted standards and 50 μL of the serum sample to be tested were added to each well, followed immediately by 50 μL of horseradish peroxidase (HRP)-labeled detection antibodies (HRP-IFN-γ, HRP-TNF-α, HRP-IL-4, HRP-IL-10). The plates were covered and incubated at 37°C for 1 h. The liquid in each well was discarded, and each well was filled with diluted 1× wash buffer. The wash buffer was discarded, and the wells were patted dry with absorbent paper. This process was repeated 5 times. Add 50 μL each of substrate A and B from the respective inflammatory factor ELISA kit to each well, gently vortex to mix, and incubate at 37°C in the dark for 10 min. Then add 50 μL of stop solution to each well and measure the absorbance at 450 nm using a microplate reader. Plot a standard curve with the concentration of the standard on the x-axis and the OD value on the y-axis. The corresponding concentration of the sample can be derived from the standard curve based on its OD value.

[0064] See results Figure 7The results showed that the expression of phosphorylated proteins in the NF-κB inflammatory pathway was significantly reduced in infected cells treated with tetrandrine. At the same time, tetrandrine significantly inhibited the expression of the pro-inflammatory factor TNF-α and significantly increased the expression levels of the anti-inflammatory factors IL-4 and IL-10, indicating that tetrandrine can effectively inhibit the inflammatory response induced by Salmonella infection.

[0065] In summary, tetrandrine can be considered as a potential HDT compound for the treatment of intracellular Salmonella infection. It has significant bactericidal effects both in vivo and in vitro, and can significantly inhibit the inflammatory response caused by Salmonella infection.

Claims

1. The use of a combined preparation of tetrandrine and sinomenine for the preparation of a medicament for the treatment of salmonella or salmonella infection, characterized in that, The Sinomenine Noline Compound Preparation is composed of sinomenine noline and levofloxacin, and the medicine further comprises a pharmaceutically acceptable carrier, the mass ratio of sinomenine noline to levofloxacin is 1:1, and the concentration of sinomenine noline is 4-1024 μg / mL.

2. Use according to claim 1, characterized in that, The medicine is a medicine for reducing cell inflammation.

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