CN111184714A

CN111184714A - Application of brucea javanica picrol in preparation of medicines for preventing or treating inflammatory diseases

Info

Publication number: CN111184714A
Application number: CN202010096435.6A
Authority: CN
Inventors: 蒋雪梅; 杨星; 罗洋; 邓博文
Original assignee: Chongqing University
Current assignee: Chongqing University
Priority date: 2020-02-17
Filing date: 2020-02-17
Publication date: 2020-05-22

Abstract

The brucea javanica picrol is applied to the medicines for treating related diseases, and can obviously reduce the over-expression of cytokines such as IL-6, IL-8, IL-1 β -1 α - α, ICAM-1 and the like by inhibiting a signal transduction path of NF-kB, improve the inflammatory reaction of skin, reduce the skin damage degree, relieve the symptoms such as erythema, scale, infiltration and the like of the skin, ensure that an epidermal layer is relatively complete and reduce the thickness of the skin damage.

Description

Application of brucea javanica picrol in preparation of medicines for preventing or treating inflammatory diseases

Technical Field

The invention belongs to the technical field of biological medicines, and relates to application of brucea javanica picrol in preparation of medicines for preventing or treating inflammatory diseases.

Background

Psoriasis (Psoriasis), commonly known as Psoriasis, is a common chronic, recurrent, inflammatory skin disease. The clinical manifestations are mainly erythema and scale, the skin damage is clear, the scale accumulation can be higher than the skin surface, the scale accumulation is usually found on the scalp and joints of four limbs of a patient, and the scale accumulation can be widely spread on the skin of the whole body in severe cases. The disease course is long, the disease is easy to recur, some cases are not cured for life, and various complications such as hypertension, diabetes, cardiovascular diseases and the like are accompanied. The disease is stubborn and difficult to treat, seriously affects the life quality of patients and brings great harm to the body and the mind of the patients. Severe psoriasis not only affects the appearance and social activities of the skin of a patient, but also endangers the life of the patient when being treated improperly. According to the report of Word Psoriasis Day consortium: psoriasis is common all over the world, is influenced by various factors such as species, geographical location, environment and the like, and has obvious difference (0-11.8%) in prevalence rate among different people all over the world. National epidemiological survey of psoriasis conducted in 1984 and 2008 shows that: the prevalence rate of psoriasis in China in 1984 is 0.123%, and the prevalence rate in 2008 is 0.47%. At present, the number of psoriasis patients in China is about 600 thousands, and most of psoriasis patients are young and old. The data show that the prevalence rate of psoriasis in China shows a trend of rising year by year, and the development of research on the protection and treatment of psoriasis is significant in scientific significance and social value.

Because the cause of psoriasis is unknown, the pathological manifestations are skin parakeratosis, hyperproliferation, inflammatory infiltration and the like, are related to factors such as heredity, immunity, medicines, environment and the like, the pathogenesis is complex, and specific medicines are lacked. Therefore, the pathogenesis and treatment of the skin disease are always the key and difficult points of research in the skin field. There are many ways to treat psoriasis clinically, including various external treatments, systemic treatments, physical treatments, etc. The preparation method achieves a good progress to a certain extent and relieves various moderate and mild psoriasis. But also has a number of side effects. Due to the complex pathogenesis of psoriasis, the treatment of psoriasis in various ways is more or less limited. For example, topical glucocorticoids can cause adverse reactions such as skin atrophy, pigmentation, vasodilatation and the like after long-term use; topical retinoic acid causes severe irritant dermatitis; the primary treatment with vitamin D3 is significant, but the recurrence is very rapid and more severe after the recurrence. Systemic treatment immunosuppressants represent the drug methotrexate, which causes side effects such as gastrointestinal irritation, bone marrow suppression and aplastic anemia. Physical therapy includes medium wave Ultraviolet (UVB), Psoralen ultraviolet therapy (PUVA), and narrow band medium wave ultraviolet (NB-UVB). However, various biochemical reactions occur in the skin by the irradiation of ultraviolet rays, thereby inducing apoptosis of T cells, and inhibiting KC proliferation and release of inflammatory factors. Although many researchers have devoted themselves to the search for etiology and new drugs, tretinoin and biological agents

Disclosure of Invention

In view of the above, the present invention aims to provide a new medical application, and provides an application of brucea javanica picrol in preparation of a drug for preventing or treating inflammatory diseases, especially in preparation of a drug for treating psoriasis.

In order to achieve the purpose, the invention provides the following technical scheme:

1. application of bruceol (hereinafter referred to as BR) in preparing medicine for preventing and/or treating inflammatory diseases.

Further, the inflammatory disease is psoriasis, rheumatoid arthritis or acute lung injury.

Further, brucea javanica picrol is used for inhibiting the signal transduction pathway of NF-kB.

Brucea javanica picrol pretreatment obviously inhibits NF-kB from transferring from cytoplasm to nucleus in a dose-dependent mode, so that the activation of NF-kB is prevented, and related cytokines such as IL-1, IL-6, IL-8, TNF- α and the like regulated by an NF-kB signal channel are inhibited.

Furthermore, the active dose of the brucea javanica picrol is 1mg/kg/d-10 mg/kg/d.

Furthermore, the active dose of the brucea javanica picrol is 2mg/kg/d-4 mg/kg/d.

Further, the brucea javanica picrol is used for inhibiting cell proliferation.

Further, the cells are human epidermal keratinocytes.

Further, the brucea javanica picrol is used for reducing or inhibiting over-expression of one or more cytokines of IL-6, IL-8, IL-1 β -1 α - α and ICAM-1.

Furthermore, the dosage form of the medicine is any clinically acceptable oral administration dosage form, injection administration dosage form or external administration dosage form.

Further, the medicament is in the form of tablets, granules, pills, liquid preparations, dispersing agents, syrups, gels, aerosols or patches.

2. Application of brucea javanica picrol in preparation of medicines for inhibiting excessive proliferation of human epidermal keratinocytes.

The invention has the beneficial effects that brucea javanica picrol is proved by a large number of experiments to be capable of inhibiting the activity and proliferation of human immortalized keratinocytes, inhibiting a signal transduction pathway of NF-kB, remarkably inhibiting the transfer of NF-kB from cytoplasm to nucleus in a dose-dependent manner, reducing the cytokine over-expression of IL-6, IL-8, IL-1 β -1 α - α, ICAM-1 and the like, and after brucea javanica picrol is injected into an abdominal cavity for treating an imiquimod psoriasis model in a proper dose, the skin damage degree of a low-dose and high-dose drug treatment group at 7 days is between a blank control group and a model group, wherein the high-dose drug treatment slightly has symptoms of erythema, scale, infiltration and the like, the skin damage degree is remarkably lower than that of the model group, the skin damage degree is almost the same as that of the positive control MTX group, the staining result shows that the high-dose brucea javanica picrol group and the positive control MTX group have relatively complete epidermis layer, only slightly excessive treatment, compared with other mice, IFN has relatively small toxic and obvious IFN-induced by the effect of reducing the IMQ-induced by high brucea javanica cell expression, and the high-induced by the statistic effect of the high-induced by the high-dose of the high-IFN-induced brucea javanica-induced by the high-induced by the statistic effect of the brucea javanica-induced by the IMQ-induced by the high-dose of the high-IFN-rat-induced.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 is a graph of the effect of varying concentrations of brucellol on cell viability.

FIG. 2 is a graph of the effect of brucellol on TNF- α -induced proliferation of HaCaT cells.

FIG. 3 is a graph of the effect of brucellol on TNF- α -induced HaCaT cell cycle, in which the A flow cytocycle is plotted and the percentage of B cell cycle is distributed.

FIG. 4 is the expression of pro-inflammatory mediators in HaCaT stimulated by brucellol-treated TNF- α.

FIG. 5 is a graph of immunofluorescence staining of HaCaT cells stimulated with brucellol-treated TNF- α.

FIG. 6 is a Western blot of nucleoprotein and cytoplasmic protein in HaCaT cells stimulated by bruceol treatment with TNF- α.

Figure 7 is a graph of the effect of BR on IMQ-induced changes in skin lesions in psoriasis-like mice.

FIG. 8 is a pathological section of dorsal skin tissue of mice in each group (HE staining, scale: 200 μm) showing the effect of BR on IMQ-induced histological changes in skin lesions in psoriasis-like mice.

FIG. 9 is a representative image of the effect of BR on ear and spleen sites of IMQ-induced psoriasis-like mice, wherein (A) spleen (left) and right ear (right) of each group of mice 7 days after treatment; (B) thickness change of the right ear for 7 consecutive days; (C) body weight of each group of mice on day 7; (D) spleen index (spleen/body weight).

FIG. 10 shows the effect of BR on IMQ-induced secretion of IFN-. gamma., IL-4, and IL-17 from splenic lymphocytes of psoriasis-like mice by ELISA kits in culture supernatants after 72h incubation of splenic lymphocytes.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or under conditions recommended by the manufacturers. Each experiment was independently repeated three times, the experimental data were expressed as mean ± Standard Deviation (SD), and statistical analysis was performed on all experimental data using GraphPad prism7.0 software. The difference between the two groups of experimental data is analyzed by t-test, the difference between the groups of experimental data is analyzed by one-way ANOVA, and the experimental data is considered to have significant difference when P is less than 0.05 and extremely significant difference when P is less than 0.01.

Experimental cells: human immortalized keratinocytes HaCaT (sourced from the dermatology gift of the national hospital, Chongqing).

Experimental reagent:

brucea javanica picrol (Brusatol, hereinafter BR for short, CAS: 14907-98-3, purity > 98%), all of which are planted in Standard pure Biotechnology Co., Ltd., China;

recombinant Human TNF- α, Peprotech Inc., USA;

imiquimod (IMQ), chuanmingxin pharmaceuticals ltd, china;

mouse IFN-. gamma.Elisa kit, Mouse IL-4Elisa kit, Mouse IL-17Elisa kit, Ph.Dr. Biotechnology engineering Co., Ltd., China.

Preparing an experimental reagent:

① brucea javanica picrol (BR) stock solution is prepared by calculating the mass of 1mM brucea javanica picrol according to the relative molecular mass (520.5g/mol) of brucea javanica picrol, and precisely weighing brucea javanica picrol 2.08 × 10^-3g, fully dissolved in 4mL DMSO, then with 0.22 μm filter membrane filtration, the filtrate as stock solution as dark place in-20 degrees C, when clinical use with cell culture medium diluted to the desired concentration, wherein the solution of DMSO concentration is less than 0.01%, neither cell nor mouse has toxic effect. DMSO is added only to increase the solubility of brucea javanica picrol, and other cosolvents can be used to help fully dissolve BR, so that the injection has no crystalline lens and is transparent (no suspension).

② TNF- α solution 10ug TNF- α standard is dissolved in 100ul sterile triple distilled water to obtain 0.1mg/ml TNF- α standard stock solution, which is stored at-20 deg.C in dark and diluted to required concentration when it is used.

Example 1

Effect of different concentrations of BR on HaCaT cell viability

After treating HaCaT cells with 0, 10, 20, 40, 80, 160, 320nM BR for 24h, cell viability was measured by MTS.

① inoculation of cells HaCaT cells in logarithmic growth phase, trypsinization, centrifugation, resuspension, plating the cells in 96-well plates at 5000 cells/well, placing in 5% CO at 37 deg.C₂The culture medium of (3) was cultured for 12 hours.

② drug treatment-removing the culture medium, adding 100. mu.L of brucellol at different concentrations to a 96-well plate, repeating the technique 5 times at each concentration, and culturing in an incubator for 24 h.

③ MTS assay cell culture Medium and CellTiter

The AQueous One Solution cell promotion Assay (MTS) was mixed in a 10:1 ratio protected from light. mu.L of the mixture was added to each well and the culture was continued for 2 hours.

④ Activity detection, wherein an enzyme-labeling instrument is used for reading the absorbance value (OD value) of each hole of cells under 490nm wavelength, and the cell activity and the OD value are in positive correlation.

The results are shown in fig. 1, and show that the activity of HaCaT cells is inhibited to different degrees with the increase of drug concentration, and the cell activity is reduced to 44.5% of the control group (P <0.001) when the BR concentration reaches above 160nM, so in order to avoid the strong toxicity of the drug, BR concentrations of 20nM, 40nM and 80nM are selected for the subsequent experiment (at these concentrations, although the proliferation of cells can be inhibited, the cell death is not caused).

Example 2

Further, TNF- α was used to construct a psoriasis-like inflammatory proliferative cell model, and HaCaT cells were co-cultured with three different concentrations of BR and TNF- α (20ng/mL) at 20nM, 40nM and 80nM for 24h, 48h and 72h, as shown in FIG. 2, which showed that HaCaT cell proliferation was significantly accelerated under TNF- α alone, while BR significantly reduced the abnormal proliferation of HaCaT cells caused by TNF- α when co-cultured with TNF- α and was time and dose dependent.

Example 3

To further explore the potential mechanism of brucellol inhibition of TNF- α -induced HaCaT cell proliferation, changes in cell cycle under different treatment conditions were examined by flow cytometry, as shown in fig. 3, a in fig. 3 is a flow cytocycle plot and B is the percentage of cell cycle distribution, and the results are shown as mean ± standard deviation of three independent experiments, P <0.05, P <0.01 and P <0.001, ns is no statistical difference.

From the results in FIG. 3, it is clear that the proportion of cells in S phase in the group treated with TNF- α alone reached 45.17. + -. 3.56%, which was significantly higher than 40.49. + -. 2.88% in the control group, with significant difference (P <0.05), when TNF- α was co-cultured with brucellol at 20nM, 40nM, 80nM, respectively, the proportion of cells in S phase in each group was 41.56. + -. 2.03%, 36.59. + -. 4.64%, 30.44. + -. 2.79%, respectively, compared to TNF- α, with significant difference (P <0.05), it is clear that treatment with TNF- α accelerated the transition of cell cycle from G1 to S phase, promoting the proliferation of cells, while brucellol greatly inhibited this transition from G1 to S phase, and thus inhibiting the proliferation of keratinocytes, these results indicate that the proliferation of epidermal keratinocytes can be inhibited by mediating the proliferation arrest of brucellol proliferation of the brucelloid cell cycle, thus inhibiting epidermal proliferation of psoriasis.

Example 4

HaCaT cells were pretreated with or without 40nM brucellol for 6h, respectively, then stimulated with TNF- α (20ng/mL) for 20h total RNA was extracted and purified, and the mRNA levels of IL-6, IL-8, IL-1 β -1 α - α and ICAM-1 were further measured by real-time quantitative PCR.

Real-time quantitative PCRThe kit is a reverse transcription kit,

Premix Ex TaqTM II (Takara Co.),

in this example, all the quantitative PCR primers were derived from PrimerBank primer design library and synthesized by Beijing Engineers, New industries, Inc., and the specific primer sequences are detailed in Table 1.

TABLE 1 qRT-PCR primer sequences

Results are shown as mean ± sd of three independent experiments with P <0.05, > P <0.01, > P <0.001 compared to the TNF- α treated group alone, it is clear from figure 4 that BR is able to inhibit TNF- α -induced release of inflammatory mediators, indicating that psoriasis is induced by the acceleration of inflammatory cell infiltration into the epidermis by overproducing inflammatory cytokines.

Example 5

HaCaT cells are pretreated by bruceol (20nM, 40nM and 80nM) for 24h, then treated by TNF- α of 10ng/mL for 1h, and then subjected to immunofluorescence staining, the expression level (red) of NF-kappa B P65 protein is captured by a laser confocal microscope (CLSM), cell nuclei (blue) are stained by DAPI, and the scale bar is 100 μm, as a result, as shown in FIG. 5, NF-kappa B (P65) mainly exists in cell plasma and is hardly expressed in the cell nuclei in a resting state, and after HaCaT cells are treated by TNF- α for 1h, P65 is found to be transferred from the cell plasma to the cell nuclei, and the pretreatment of BR remarkably inhibits the transfer in a dose-dependent manner, so that the activation of NF-kappa B is prevented, and related cytokines such as IL-1, IL-6, IL-8 and TNF- α regulated by NF-kappa B signal pathways are inhibited.

Further, cytoplasmic proteins and nuclear proteins were separated using a cytoplasmic nuclear extraction kit, followed by assay using immunoblotting to measure the protein expression levels of NF- κ B, ikb α and P-ikb α. the results are shown in fig. 6, B being total proteins, C being nucleoprotein and D being cytoplasmic proteins, and the results are shown as mean ± standard deviation of three independent experiments, P <0.05, > P <0.01, > P <0.001 similar to the results in fig. 5, when TNF- α induced HaCaT cells, expression of P65 in the nucleus was clearly observed, whereas after pretreatment with BR, expression of P65 in the nucleus was clearly inhibited, and the results also show that BR was able to effectively alleviate the extent of degradation of ikb α induced by TNF- κ 7 and inhibit phosphorylation of ikb α in the cytoplasm of ikb α in cells induced by TNF-5631 (fig. 7 shows that inhibition of inflammatory cell infiltration of NF-TNF-B in cells and NF-TNF-B9 in psoriasis was induced by epithelial cell infiltration of epithelial cells).

Example 6

Imiquimod (IMQ) belongs to imidazole quinoline compounds, and is a small molecular immunomodulator. Leslie et al applied 5% IMQ to the bare skin of the back of female BALB/c mice each day and after several consecutive days had developed psoriasis-like lesions and pathological manifestations including thickening of the skin epidermis, scaling, infiltration of large numbers of neutrophils in the lesions, all of which were highly similar to the human spontaneous psoriasis-like lesions. Female BALB/c mice (35, 6-8 weeks old) were purchased from Chongqing university of medicine animal laboratories, and housed in the Chongqing university animal laboratories SPF-scale laboratory. All the animal studies related to the present invention were performed according to the regulations on the affairs management of laboratory animals approved by the State Council of the people's republic of China. Meanwhile, all animal experiments were performed according to the regulations of the animal ethics and experimental committee of Chongqing university.

35 BALA/c mice (6-8 weeks) were randomly divided into five groups of 7 mice each, and then the mice were anesthetized by intraperitoneal injection with 80mg/kg sodium pentobarbital and the mice were shaved for hair in an area of about 3X 4cm in size. After 3 days of acclimatization, the treatment was divided into groups for each day as follows, and was continued for 7 days.

1) Blank Control (labeled Control): the exposed area of the back of the mouse is coated with 62.5mg of vaseline cream every day, and simultaneously 0.1mL of physiological saline is injected into the abdominal cavity.

2) Model set (labeled IMQ): the exposed area of the back and the right ear of the mouse are smeared with 62.5mg/d of imiquimod ointment, and simultaneously, 0.1mL of physiological saline is injected into the abdominal cavity.

3) Positive control group (labeled MTX): the mice were applied with imiquimod ointment 62.5mg/d per day on the bare back and on the right ear, and were injected intraperitoneally with 0.1mL of 1mg/kg Methotrexate (MTX).

4) Experimental drug low dose group (labeled L-BR): the exposed area of the back and the right ear of the mouse are smeared with 62.5mg/d of imiquimod ointment, and simultaneously, 0.1mL of brucea javanica picrol of which the weight is 2mg/kg/d is injected into the abdominal cavity according to the weight of the mouse.

5) Experimental drug high dose group (labeled H-BR): the exposed area of the back and the right ear of the mouse are smeared with 62.5mg/d of imiquimod ointment, and simultaneously 0.1mL of brucea javanica picrol of 4mg/kg/d is injected into the abdominal cavity according to the body weight of the mouse.

1. During the modeling process, the skin lesions of the skin on the back of the mice were scored daily according to the Psoriasis Area and Severity Index (PASI) criteria. The method comprises three indexes of erythema, scale and infiltration (each index is 0-4 points), and the sum of the three indexes is the total integral. 0 minute: none; 1 minute: mild; and 2, dividing: moderate; and 3, dividing: (ii) severe; and 4, dividing: is extremely severe. An integral trend line was drawn from PASI integrals to evaluate the dynamic changes in the skin lesions on the back of mice. The body weight and the thickness of the right ear of each group of mice were measured daily and recorded. Fig. 7 is a graph showing the effect of BR on IMQ-induced changes in skin lesions in psoriasis-like mice after 7 days of experiment, as shown in a in fig. 7, the skin on the back of placebo mice was smooth, shiny, and free of erythema, scaling, and infiltration. The model group gradually appears large-area dark red plaques with the increase of time, and the plaques are rich in a large amount of silvery white scales and obviously infiltrated. And peaks at days 4-5, forming typical psoriasis-like symptoms. The skin damage started to improve from day 6, the scale began to shed, and then continued IMQ treatment, the severity of the skin damage did not increase substantially. The positive control group showed only slight changes in erythema, scaling, infiltration, etc. with time and began to regress at 6 days. The skin damage degree of the low-dose and high-dose drug treatment groups is between that of the blank control group and that of the model group, wherein the high-dose drug treatment groups slightly have symptoms of erythema, scales, infiltration and the like, and the skin damage degree is obviously lower than that of the model group and is almost the same as that of the positive group. In fig. 7, B is an integral trend line drawn according to PASI score standards, the score indexes include erythema, scales, and infiltrates (each index score is 0 to 4 points), the sum of the three is a total integral (0 to 12 points), and the integral of the blank control group is close to zero, indicating that there is no skin damage area. The scores of the four other groups showed a trend of increasing first and then decreasing, and the scores reached the highest at 4-5 days. The positive control group and the low/high dose drug treatment group showed significant statistical differences (P <0.001) compared with the model group.

2. After 7 days of molding, each mouse was sacrificed by removing the neck, and the skin of the skin lesion was carefully removed with scissors and cut into small pieces. Paraffin sections are made to carry out HE staining to observe pathological changes of the skin lesion. HE staining results are shown as a in fig. 8, scale: 200 μm, showing the effect of BR on IMQ-induced histological changes in skin lesions in psoriasis-like mice. From the images, it can be seen that the cortex of the blank control group is thinner, and 2-3 layers of normal cells can be seen. The epidermal acanthocyte layer of the model group is obviously thickened, the phenomenon of obvious hyperkeratosis with parakeratosis appears, the vascular hyperplasia is obviously expanded, and the model group shows typical psoriasis-like symptoms. Compared with the model group, the epidermal layer of the skin of the mice in the high-dose brucellol group and the positive control MTX group is relatively complete, has slight hyperkeratosis, and has a remarkable statistical difference (P < 0.001). The pathological change degree of the skin tissues of the low-dose brucea javanica picrol treatment group is between that of the model group and that of the blank control group, the pathological change degree is shown as a small amount of parakeratosis cells, and the thickness of the epidermis is obviously lower than that of the model group (P < 0.01). Meanwhile, 5 representative sites on the section are selected, the thickness of the section is measured, and the average value of the 5 sites is taken as the thickness of the epidermis. As shown in B in FIG. 8, the skin thickness of the blank control group, the model group, the MTX group, the low dose brucea fruit picrol treatment group and the high dose brucea fruit picrol treatment group were 31.33. + -. 1.44. mu.m, 95.67. + -. 4.57. mu.m, 45.54. + -. 2.78. mu.m, 65.63. + -. 3.44. mu.m and 51.26. + -. 3.78. mu.m, respectively. The epidermis of each group of mice was statistically significantly different from that of the model group (P < 0.01).

Thickness of the right ear of the mice was measured daily during 7 days of animal molding, and figure 9 is the effect of BR on IMQ-induced psoriasis-like mouse ear and spleen sites. Wherein a is a representative image of the spleen (left) and right ear (right) of each group of mice 7 days after treatment; b is the change in thickness of the right ear for 7 consecutive days; c is the body weight of each group of mice on day 7; d is spleen index (spleen/body weight). As shown in B in fig. 9, the thickness of the ear layer of each group of mice increased from day 2 onward, and the degree of increase reached a peak at days 4 to 5, and then decreased several days later, except for the control group. The growth rate was the fastest among these in the model group, with significant statistical differences (P <0.001) compared to the MTX group, low/high dose brucellol treated group. On day 7, mice in each group were weighed and lost weight (P <0.01) compared to the blank control group, and the remaining groups showed no significant difference in weight (P >0.05) compared to the IMQ group, indicating that MTX and brucellol at this dose had no significant toxic side effects on the mice (C in figure 9). Further, spleen indices (spleen weight/body weight) of mice were calculated, and as shown in D in FIG. 9, the spleen indices (11.34. + -. 2.87mg/g) of the IMQ group were significantly higher than those of the blank control group (P <0.001), the MTX group, the low and high dose brucea javanica alcohol-treated groups were 6.98. + -. 2.04mg/g, 8.03. + -. 2.94mg/g, and 7.34. + -. 2.45mg/g, respectively, and were significantly different from those of the IMQ group (P < 0.01).

3. And (3) taking out the spleen of each mouse in the step (2), taking spleen lymphocytes, culturing the spleen lymphocytes for 72 hours, and detecting the expression of IFN-gamma, IL-4 and IL-17 in the spleen lymphocytes by an ELISA method.

Psoriasis is considered a T cell mediated immune disease in which there is a notion of Th cell dysfunction. Numerous studies have shown that there is a disturbance of the Th1/Th2 balance in the immune system of psoriasis patients, manifested as a classical Th1 dominant response. Th17 is a novel Th cell subset, and its secreted cytokine IL-17 is an important inflammation factor, playing a crucial role in primary and acquired immune response. The spleen is the largest immune organ of the body, contains a large number of lymphocytes and is the center of humoral immunity and cellular immunity. In this example, the expression of IFN-. gamma.IL-4 and IL-17 in the culture supernatant of splenic lymphocytes of mice was detected by ELISA kit, and the results are shown in FIG. 10. The result of the fact that the secretion levels of IFN-gamma (a cytokine representative of Th1 type cells) and IL-17 (a cytokine representative of Th17 type cells) in spleen lymphocytes of mice in the IMQ group are obviously increased, and the expression level of IL-4 (a cytokine representative of Th2 type cells) is hardly changed proves that the balance of Th1/Th2 is disturbed and Th1 has a dominant response in psoriasis patients. Compared with the IMQ group, the expression level of IFN-gamma and IL-17 in the MTX group and the low/high dose brucellol treatment group is obviously reduced, and the statistical difference is obvious (P is less than 0.01), wherein the improvement capability of the high dose brucellol is almost the same as that of the MTX. The treatment of the brucellol can reduce the capacity of mouse spleen lymphocytes to secrete IFN-gamma and IL-17 cytokines caused by IMQ induction, and the brucellol can be used for treating or preventing psoriasis.

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the foregoing preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

Claims

1. Application of brucea javanica picrol in preparation of medicines for preventing or/and treating inflammatory diseases.

2. Use according to claim 1, wherein the inflammatory disease is psoriasis, rheumatoid arthritis or acute lung injury.

3. Use according to claim 1 or 2, characterized in that brucellol is used to inhibit the signal transduction pathway of NF- κ B.

4. Use according to claim 3, wherein the active dose of bruceol is between 1mg/kg/d and 10 mg/kg/d.

5. Use according to claim 3, characterized in that the active dose of bruceol is comprised between 2mg/kg/d and 4 mg/kg/d.

6. Use according to claim 3, wherein the brucellol is used to inhibit cell proliferation.

7. Use according to claim 3, characterized in that brucellol is used to reduce or inhibit the over-expression of one or more cytokines of IL-6, IL-8, IL-1 β -1 α - α and ICAM-1.

8. The use of claim 3, wherein the medicament is in any one of clinically acceptable oral administration, injection administration or external administration.

9. Application of brucea javanica picrol in preparation of medicines for inhibiting excessive proliferation of human epidermal keratinocytes.