Application of fangchinoline in prevention and treatment of salmonella infection

Abstract

The invention discloses an application of fangchinoline in prevention and treatment of salmonella infection. The fangchinoline alkali is an isoquinoline alkaloid, and is a host-oriented treatment natural compound. The fangchinoline disclosed by the invention has a relatively strong effect of treating salmonella infection diseases in vivo and in vitro, and specifically, the fangchinoline can effectively reduce the quantity of intracellular infection bacteria. Administration of the fangchinoline can significantly reduce the load of pathogenic bacteria in the liver, spleen, colon, cecum and ileum of a mouse, and reduce the proinflammatory factor level of an organism. In-vivo and in-vitro safety shows that the fangchinoline has no toxic or side effect below the test dosage. Besides, the fangchinoline alkali and the fluoroquinolone medicine are combined for use, so that the remarkable synergistic antibacterial activity is achieved, and the remarkable synergistic antibacterial activity is shown in reducing the number of salmonella infection bacteria in cells and reducing the load of pathogenic bacteria in the liver, the spleen and the colon of a mouse. The invention provides a new perspective for the development of novel anti-infection drugs, and also provides a new idea for the prevention and control of increasingly serious salmonella infection.

Description

Technical Field

[0001] The invention relates to application of fangchinoline in preventing and treating salmonella infection, and belongs to the technical field of medicine. Background Art

[0002] Salmonella is a foodborne zoonotic pathogen that causes acute gastroenteritis in humans and animals, accompanied by systemic symptoms such as fever. In severe cases, bacteremia occurs, leading to high morbidity and mortality. Clinically, the treatment of Salmonella infection mainly relies on antibiotics. With the spread and prevalence of multidrug-resistant strains, the treatment of Salmonella infection has become a severe challenge. In recent years, host-directed therapy (HDT) natural compounds have become a promising anti-infection strategy for the treatment of a variety of intracellular bacterial infections. HDT drugs enhance host defense capabilities by targeting host immune response pathways, ultimately helping host cells to clear intracellular pathogens. Compared with antibiotics, HDT drugs do not directly exert survival pressure on pathogens, so it is not easy to induce bacterial resistance, which is of great practical significance for the prevention and control of Salmonella infection.

[0003] Based on this, using natural compounds to treat Salmonella infection is a potential antibacterial strategy. Among them, fangchinoline, which comes from the rhizomes of Menispermaceae plants, has multiple pharmacological activities, including anti-inflammatory and analgesic effects, treating rheumatism and arthritis; anti-tumor effects, inhibiting the proliferation of breast cancer and bladder cancer cells; hypotensive effects, which can be used to treat cardiovascular diseases; anti-HIV-1, which can effectively inhibit viral replication, etc. The molecular formula of fangchinoline is C 37 H 40 N2O6. The chemical structural formula of fangchinoline is shown in Formula 1:

[0004]

[0005] As an alkaloid, fangchinoline has the effects of enhancing immunity, sedation, and anti-inflammatory. However, there is no research on the use of fangchinoline in antibacterial aspects. Summary of the invention

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

[0007] The second objective of the present invention is to provide a combined therapeutic effect of fangchinoline and fluoroquinolone drugs on Salmonella infection.

[0008] Technical solution: In order to solve the above technical problems, the present invention provides the use of fangchinoline in the preparation of drugs for resisting Salmonella or Salmonella infection.

[0009] Wherein, the drug includes a pharmaceutically acceptable carrier.

[0010] Wherein, the medicine also includes antibiotics.

[0011] Wherein, the antibiotics include fluoroquinolone antibiotics.

[0012] The present invention also includes a fangchinoline compound preparation, which comprises fangchinoline and fluoroquinolone antibiotics.

[0013] Wherein, the mass ratio of the fangchinoline to the fluoroquinolone antibiotics is 1:1.

[0014] Wherein, the concentration of the fangchinoline is 4-1024 μg / mL, and the fluoroquinolone antibiotics include levofloxacin.

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

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

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

[0018] The present invention also includes the use of the fangchinoline compound preparation in drugs for resisting Salmonella or Salmonella infection.

[0019] Wherein, the drug includes a drug that reduces cellular inflammation.

[0020] Among them, the drugs include drugs that reduce the expression level of pro-inflammatory factors TNF-α and / or IFN-γ; the drugs include drugs that increase the expression level of IL-4 and / or IL-10.

[0021] The invention shows that fangchinoline can effectively reduce the amount of salmonella infection bacteria in vivo and in vitro.

[0022] Beneficial effects: Compared with the prior art, the present invention has the following outstanding and significant advantages: The present invention discloses for the first time that fangchinoline has a strong anti-Salmonella infection effect both in vivo and in vitro, which is specifically manifested in that fangchinoline effectively reduces the amount of intracellular Salmonella infection bacteria. Fangchinoline significantly reduces the pathogen load in the liver, spleen, colon, cecum and ileum of mice, and reduces the body's inflammation level. In addition, fangchinoline has significant synergistic bactericidal activity when used in combination with fluoroquinolones. It is specifically manifested in reducing the number of intracellular Salmonella infection bacteria and reducing the pathogen load in the liver, spleen and colon of mice. Mechanism studies have shown that fangchinoline can help the host clear infected bacteria by improving the lysosomal function and autophagy activity of macrophages. This invention provides a new perspective for the development of new drug resistance prevention and control strategies, and also provides a new method for the prevention and control of increasingly serious Salmonella infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Screening of natural compounds for inhibition of Salmonella intracellular replication.

[0024] Figure 2 This is to evaluate the in vitro effect of fenvalerate in reducing Salmonella infection, where CON means blank control and Vehicle means DMSO negative control.

[0025] Figure 3 This is to evaluate the in vivo effect of fangjinoline in controlling Salmonella infection, where Vehicle means DMSO negative control.

[0026] Figure 4 This is for the in vivo and in vitro safety evaluation of fangchinoline, where Vehicle means DMSO negative control.

[0027] Figure 5 This is a graph showing the results of combined treatment of Salmonella infection with fangchinoline and fluoroquinolones, where Vehicle means DMSO negative control.

[0028] Figure 6 This is a graph showing the results of fangchinoline improving the lysosomal activity of macrophages, where Vehicle means the DMSO negative control.

[0029] Figure 7 To investigate the effect of quinolones on the inflammatory level of Salmonella-infected cells, CON means blank control and Vehicle means DMSO negative control. DETAILED DESCRIPTION

[0030] The technical scheme of the present invention is further described below in conjunction with the accompanying drawings. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several modifications and improvements can also be made, which should also be considered to belong to the protection scope of the present invention. The experimental methods in the following embodiments are conventional methods unless otherwise specified. The experimental materials used in the following embodiments are purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative experiments in the following embodiments are all repeated three times, and the results are averaged.

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

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

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

[0034] The strain Salmonella CMCC(B)50336 (SE50336) used in the example was 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. Vet Res. 2021 Mar 10; 52(1):44. doi:10.1186 / s13567-021-00915-z.).

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

[0036] Taking the Salmonella enteritidis strain SE50336 as an example, RAW264.7 cells were plated in 96-well plates, and the SE50336 strain was inoculated into LB medium for activation overnight. The OD 600=0.2, and RAW264.7 cells were infected with a multiplicity of infection (MOI) of 10. After 1 hour of infection, DMEM (gibco, 11995065) complete medium containing 200 μg / mL gentamicin was added to each well for incubation 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), and DMEM complete medium containing 50 μg / mL gentamicin was added. At the same time, different concentrations of natural compounds (Luteolin (Luteolin, 20 μM): Purify, BP0896-20 mg; Diosmetin (Diosmetin, 20 μM): Purify, BP0506-20 mg; Pectolinarigenin (Luteolin, 20 μM): Purify, BP0506-20 mg; Purify, BP1069-20mg; Puerarin (Puerarin, 50μM): Purify, BP1176-20mg; Tangeretin (Tangeretin, 50μM): Purify, BP1360-20mg; Kurarinone (Kurarinone, 50μM): Purify, BP3452-10mg; Scutellarei (Scutellarin, 100μM): Purify, BP1277-20mg; Vitexin (Vitexin, 100μM): Purify, BP1447-20mg;

[0037] Anhydroicaritin (200 μM): Purify, BP3070-20mg; Maackiain (50 μM): Purify, BP0907-20mg; Fangchinoline (20 μM): 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 The cells were cultured for 12 h with berberine hydrochloride (100 μM): Macklin, B877834), and lysed with 0.1% TritonX-100 for 20 min. The colony counts (CFUs) were performed on XLD plates by 10-fold serial dilutions.

[0039] The analysis results are shown in Figure 1 The results showed that fangchinoline treatment inhibited the replication of intracellular Salmonella by more than 90%, and had the most significant bactericidal effect.

[0040] Example 2 Evaluation of the in vitro effect of fangchinoline on reducing Salmonella infection

[0041] Intracellular Salmonella infection count: RAW264.7 cells were plated in 96-well plates, and SE50336 strain was inoculated into LB medium for activation overnight. OD 600 =0.2, and RAW264.7 cells were infected with a multiplicity of infection (MOI) equal to 10. After 1 hour of infection, DMEM complete medium containing 200 μg / mL gentamicin was added to each well for incubation for 0.5 h to kill extracellular bacteria, the medium was discarded and washed three times with PBS, and complete medium (DMEM medium containing 10% FBS) containing 50 μg / mL gentamicin and different concentrations of fangchinoline (0, 1.25, 2.5, 5 μM) was added for different time (0, 1, 4, 16 h), 0.1% TritonX-100 lysed the cells for 20 min, and colony counts (CFUs) were performed on XLD plates by 10-fold serial dilution.

[0042] Flow cytometry: The GFP plasmid pXG10SF (a gift from 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. 2021Mar 10; 52(1):44. doi:10.1186 / s13567-021-00915-z.) was transferred into the SE50336 strain, and RAW264.7 cells were plated on a 6-well plate for 12 h. The SE50336 strain carrying GFP was infected with an MOI of 10 for 1 h, and complete culture medium (containing 10% The cells were incubated with 200 μg / mL gentamicin diluted in DMEM medium containing FBS for 0.5 h, washed three times with PBS, and treated with 50 μg / mL gentamicin diluted with complete culture medium to a final concentration of 5 μM for 16 h. The cells were washed three times with PBS, and the cells in each well were collected and the liquid was collected after filtering with a filter and detected by CytExpert flow cytometer.

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

[0044] The analysis results are shown in Figure 2 The results showed that fangchinoline reduced the number of intracellular Salmonella infection in a time- and concentration-dependent manner, and the effect was most obvious when the infected cells were treated with 5μM fangchinoline for 16h, as shown by a significant decrease in the number of CFU counted on the XLD plate and a decrease in green fluorescent protein (Salmonella) in flow cytometry and confocal microscopy results.

[0045] Example 3 Evaluation of the in vivo effect of fangchinoline on reducing Salmonella infection

[0046] Mouse Salmonella infection model: 18 male BALB / c mice (Comparative Medicine Center of Yangzhou University, 6-8 weeks old, weighing 20±2g) were randomly divided into Vehicle group, Fangchinoline group (FAN, 10mg / kg) and Ciprofloxacin group (CPFX, 10mg / kg), 6 mice in each group. Adaptive feeding was performed for 3 days, during which time the mice were free to eat and drink water. They were fasted and deprived of water for 12h before infection. Salmonella SE50336 (1×10 8 CFU / mouse) was used to establish a mouse model of Salmonella infection. 200 μL DMSO (Vehicle), 10 mg / kg mouse fangchinoline (FAN), and 10 mg / kg mouse ciprofloxacin (CPFX) were injected into the right abdominal cavity of mice 24 hours after infection. Blood samples were collected from the eyeballs 12 hours after treatment for routine blood tests. The liver, spleen, and small intestine segments (colon, cecum, and ileum) of infected mice were collected after death by cervical dislocation. The colony count of Salmonella in organs was performed using XLD plates, and the inflammatory factors of mice were detected using ELISA kits.

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

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

[0049] Mouse toxicity experiment: 24 male BALB / c mice (Comparative Medicine Center of Yangzhou University, 6-8 weeks old, weighing 20g) were randomly divided into a Vehicle group and a Fangchinoline group (FAN, 30mg / kg), with 6 mice in each group. 200μL DMSO (Vehicle group) and 30mg / kg Fangchinoline were injected into the right side of the mouse abdominal cavity. The behavioral state of the mice was observed and the body weight was recorded every day for 14 days. Blood was collected from the mouse eyeballs on the 2nd and 14th days for blood biochemical tests and Na in the body. + , K + , Cl - Concentration detection, liver and kidney samples were taken after cervical dislocation for HE staining.

[0050] Erythrocyte hemolytic test: dilute fangchinoline (concentrations are: 0, 4, 16, 64, 256, 1024 μg / mL) and place it in a 96-well plate. After centrifugation of the defibrinated blood, resuspend it in PBS to make an 8% red blood cell suspension and add it to the prepared drug solution. Incubate at 37°C for 1 hour, centrifuge at 4°C × 3,000g for 10 minutes, take 100 μL and measure the absorbance at 576nm. Use PBS and sterile double distilled water as negative and positive controls. The hemolysis rate is 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 in Figure 4 The results showed that compared with the Vehicle group, there was no significant difference in the body weight of mice treated with fangchinoline, the survival rate was 100%, and there was no significant difference in ALT, AST, UREA, and CREA tests. + , K + , CL - There was no difference in the content, and both were within the normal range. There was no significant difference in the results of liver and kidney HE staining, indicating that fangchinoline has low toxicity in vivo. High concentrations of fangchinoline (1024μg / mL) will not cause hemolysis of sheep red blood cells, indicating that fangchinoline has low toxicity in vitro.

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

[0053] Mouse Salmonella infection model: 24 male BALB / c mice (Comparative Medicine Center of Yangzhou University, 6-8 weeks old, weighing 20±2g) were randomly divided into Vehicle group, Fangchinoline group (FAN, 10mg / kg) Levofloxacin group (LVFX, 10mg / kg), Fangchinoline and Levofloxacin group (FAN+LVFX, 5mg / kg each), 6 mice in each group. Adaptive feeding for 3 days, free access to food and water during the period, fasting and water deprivation for 12h before infection. Salmonella SE50336 (1×10 8 CFU / mouse) was used to establish a mouse model of Salmonella infection. 24 hours after infection, 200 μL DMSO (Vehicle), 10 mg / kg fangchinoline (FAN), 10 mg / kg levofloxacin (LVFX), 5 mg / kg fangchinoline and 5 mg / kg levofloxacin (FAN+LVFX) were injected into the right abdominal cavity of mice respectively. 12 hours later, the infected mice were killed by cervical dislocation, and the liver, spleen and cecum of the infected mice were collected, and the Salmonella colony counts were performed using XLD plates.

[0054] Combined treatment of Salmonella cell infection model: RAW264.7 cells were plated in 96-well plates, and SE50336 strains were inoculated into LB medium for activation overnight. The OD 600 =0.2, and RAW264.7 cells were infected with a multiplicity of infection (MOI) equal to 10. After 1 hour of infection, DMEM complete medium containing 200 μg / mL gentamicin was added to each well for incubation for 0.5 h to kill extracellular bacteria, the medium was discarded and washed three times with PBS, and 50 μg / mL gentamicin and 5 μM fangchinoline and 5 μM different antibacterial drugs (chloramphenicol CAP, ciprofloxacin CPFX, levofloxacin LVFX, ceftriaxone sodium CTRX) and 2.5 μM fangchinoline and 2.5 μM chloramphenicol, 2.5 μM ciprofloxacin, 2.5 μM levofloxacin, 2.5 μM ceftriaxone sodium were added and co-cultured for 4 h and 16 h, respectively, and 0.1% TritonX-100 was used to lyse the cells for 20 min, and colony counts (CFUs) were performed on XLD plates by 10-fold serial dilutions.

[0055] The analysis results are shown in Figure 5 The results showed that compared with the mice in the Vehicle group, treatment with fangchinoline 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 fangchinoline and fluoroquinolones, especially levofloxacin, had a combined therapeutic effect.

[0056] Example 6 Fangchinoline promotes the clearance of intracellular Salmonella by increasing lysosomal activity

[0057] Western blotting: RAW264.7 cells were cultured overnight in 24-well plates (1×10 5 SE50336 strain was infected with cells at MOI=10 (1×10 6) for 1 hour, add complete medium (DMEM medium containing 10% FBS) containing 200μg / mL gentamicin to each well and incubate for 0.5h to kill extracellular bacteria, discard the medium and wash with PBS three times, add complete medium containing 50μg / mL gentamicin and 5μM fangchinoline for different time (0, 1, 4, 16h), wash twice with PBS, collect cells, centrifuge at 4℃×1000rpm for 5 minutes. Discard the supernatant, add 200μL of protease inhibitor PMSF (Beyotime, ST506) and phosphatase inhibitor (Beyotime, P1045) diluted with RIPA lysis buffer (Beyotime, P0013B) to extract protein. The protein was diluted with 5×SDS loading buffer, passed through SDS-PAGE, wet-transferred to PVDF membrane, blocked with rapid blocking solution for 10 min, and then washed three times with TBST. It was incubated with anti-LAMP1, LAMP2, and ATP6V0D1 antibodies (purchased from Abclonal, with catalog numbers A16894, A1961, and A4271, respectively) at 4°C overnight, washed three times with TBST, and incubated with HRP-goat anti-rabbit secondary antibody on a shaker at room temperature for 1 h. The membrane was photographed after color development with ECL solution, and the grayscale value was measured using ImageJ software.

[0058] Immunofluorescence: RAW264.7 cells were cultured overnight in 24-well plates (1×10 5 cells / well), and GFP-SE50336 strain was infected with MOI=10 (1×10 6 ) for 1h, complete medium containing 200μg / mL gentamicin (DMEM medium containing 10% FBS) was added to each well and incubated for 0.5h to kill extracellular bacteria, the medium was discarded and washed three times with PBS, complete medium containing 50μg / mL gentamicin and 5μM fangchinoline (DMEM medium containing 10% FBS) was added and cultured for 16h, washed three times with PBS, fixed the cells with 4% formaldehyde for 20min, washed three times with PBS, permeabilized with 0.1% triton for 30min, washed three times with PBS, blocked with rapid blocking solution for 10min in each well, washed three times with PBS, added anti-LAMP1 antibody and incubated overnight at 4℃, washed three times with PBS, Cy3 Goat Anti-RabbitIgG (H+L) red fluorescent secondary antibody (purchased from Abclonal, catalog number AS007) was incubated on a shaker at room temperature for 1 hour, washed 3 times with PBS, and DAPI with a final concentration of 5 μg / mL was added to each well for incubation for 20 minutes, washed 3 times with PBS, and anti-fluorescence quencher and sealing oil were added to the slides, observed under a fluorescence microscope, and the fluorescence value was measured using ImageJ software. After staining, the fluorescence intensity of LAMP1 was observed under a fluorescence microscope, and the fluorescence value was measured using ImageJ software.

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

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

[0061] Example 7 Fangchinoline reduces inflammation in Salmonella-infected cells

[0062] Western blotting: RAW264.7 cells were cultured overnight in 24-well plates (1×10 5 SE50336 strain was infected with cells at MOI=10 (1×10 6) for 1 hour, add complete medium containing 200μg / mL gentamicin (DMEM medium containing 10% FBS) to each well and incubate for 0.5h to kill extracellular bacteria, discard the medium and wash with PBS three times, add complete medium containing 50μg / mL gentamicin and 5μM fangchinoline for 16h, wash twice with PBS, collect cells, centrifuge at 4℃×1000rpm for 5 minutes. Discard the supernatant, add 200μL of protease inhibitor PMSF and phosphatase inhibitor diluted with RIPA lysis buffer to a final concentration of 1mM to extract protein. The protein was diluted with 5×SDS loading buffer, passed through SDS-PAGE, wet-transferred to PVDF membrane, blocked with rapid blocking solution for 10 min, and then washed three times with TBST. The membrane was incubated 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 at 4°C overnight, washed three times with TBST, incubated with HRP-goat anti-rabbit secondary antibody on a shaker at room temperature for 1 h, and photographed after ECL solution development. The grayscale value was measured using ImageJ software.

[0063] ELISA method was used to determine the level of inflammatory factors in Salmonella-infected cells: Inflammatory factor ELISA detection kits were used, including the pro-inflammatory factor IFN-γ ELISA kit (mlbio, ml002277), TNF-α ELISA kit (mlbio, ml002095), the anti-inflammatory factor IL-4 ELISA kit (mlbio, ml064310), and the IL-10 ELISA kit (mlbio, ml037888). Three replicate wells were set up in each group, and 50 μL of the diluted standard and 50 μL of the serum sample to be tested were added to the reaction wells, followed by the immediate addition of 50 μL of the detection antibody labeled with horseradish peroxidase (HRP) (HRP-IFN-γ, HRP-TNF-α, HRP-IL-4, HRP-IL-10). Cover the membrane plate and incubate at 37°C for 1 hour. Discard the liquid in the wells, fill each well with the diluted 1× washing buffer, shake off the washing solution, and pat dry with absorbent paper. Repeat this operation 5 times. Add 50 μL of each substrate A and B solution in the respective inflammatory factor ELISA detection kit to each well, gently shake and mix, and incubate at 37°C in the dark for 10 minutes. Then add 50 μL of stop solution to each well, and use an enzyme reader to measure the absorbance at a wavelength of 450 nm. Draw a standard curve with the concentration of the standard as the horizontal axis and the OD value as the vertical axis, and derive the corresponding concentration from the standard curve according to the OD value of the sample.

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

[0065] In summary, fangchinoline 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 fangchinoline in the preparation of drugs for resisting Salmonella or Salmonella infection.

2. The use according to claim 1, characterized in that: The medicine includes a pharmaceutically acceptable carrier.

3. The use according to claim 1, characterized in that: The drugs also include antibiotics.

4. The use according to claim 1, characterized in that: The antibiotics include fluoroquinolone antibiotics.

5. A fangchinoline compound preparation, characterized in that: The fangchinoline compound preparation comprises fangchinoline and fluoroquinolone antibiotics.

6. The fangchinoline compound preparation according to claim 5, characterized in that: The mass ratio of fangchinoline to fluoroquinolone antibiotics is 1:

1.

7. The fangchinoline compound preparation according to claim 5, characterized in that: The concentration of the fangchinoline is 4-1024 μg / mL, and the fluoroquinolone antibiotics include levofloxacin.

8. Use of the fangchinoline compound preparation according to any one of claims 5 to 7 in drugs for resisting Salmonella or Salmonella infection.

9. The use according to claim 8, characterized in that: The drugs include drugs that reduce cellular inflammation.

10. The use according to claim 9, characterized in that: The drugs include drugs that reduce the expression level of pro-inflammatory factors TNF-α and / or IFN-γ; and the drugs include drugs that increase the expression level of IL-4 and / or IL-10.

Images