Application of CYP1B1 and AKR1C3 as drug targets for detecting or treating cystitis

Abstract

The invention discloses application of CYP1B1 and AKR1C3 as drug targets for detecting or treating cystitis. The application is specifically as follows: 1) application of CYP1B1 and AKR1C3 as treatment targets in treatment of cystitis; and 2) application as a drug target in detection of cystitis. CYP1B1 and AKR1C3 which are found for the first time and are derived from human can be used as new molecular drug targets for detecting or treating the cystitis, and the drug targets are utilized to realize the purpose of detecting or treating the cystitis, so that a new thought and a gene resource are provided for preventing and treating the cystitis.

Description

Technical Field

[0001] The invention belongs to the technical field of biotechnology and bladder inflammation, and specifically relates to an application of CYP1B1 and AKR1C3 as drug targets for detecting or treating bladder inflammation. Background Art

[0002] Bladder inflammation is a common urinary system disease that can cause urinary tract irritation symptoms. Long-term chronic inflammation can lead to bladder fibrosis, promote bladder trabecular hyperplasia, and cause pyelonephritis or renal abscesses. It also increases the risk of bladder cancer, seriously affecting the patient's quality of life. Existing treatments mainly focus on antibiotic treatment and symptom management, but these methods have problems such as drug resistance and side effects. Therefore, there is an urgent need to develop new therapeutic targets and methods.

[0003] Cytochrome P450 (CYPs) is a group of enzymes composed of large heme pseudogroups that can catalyze the oxidation reactions of a variety of organic substrates. CYP1B1 is expressed in most human organs and has been shown to play an important role in inflammations such as membranous nephropathy, neuroinflammation, and airway inflammation. AKR1C3 is a member of the aldehyde-keto reductase protein superfamily. It is mainly a reductase in the body that promotes the production of high-efficiency testosterone. AKR1C3 is also closely related to inflammation and plays an important role in inflammations such as atopic dermatitis, rheumatoid arthritis, and airway inflammation. However, whether CYP1B1 and AKR1C3 play a role in the occurrence and development of bladder inflammation, as well as the treatment of cystitis, has not been reported. Summary of the invention

[0004] In view of the shortcomings of the above-mentioned prior art, the present invention provides an application of CYP1B1 and AKR1C3 as drug targets for detecting or treating bladder inflammation. CYP1B1 and AKR1C3 derived from humans can provide new drug targets for detecting or treating bladder inflammation, and provide new ideas and gene resources for the prevention and treatment of bladder inflammation.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] A first aspect of the present invention provides an application of CYP1B1 and AKR1C3 as drug targets for detecting or treating bladder inflammation.

[0007] Optionally, CYP1B1 and AKR1C3 are drug targets for detecting gene expression levels in bladder mucosal epithelium; the gene expression level includes RNA level and protein level.

[0008] Optionally, the gene expression level in the bladder mucosal epithelium is detected by PCR and flow cytometry on a bladder mucosal epithelial inflammation model; the bladder mucosal epithelial inflammation model is constructed by inducing inflammation in a normal bladder epithelial cell line SV-HUC-1 by lipopolysaccharide LPS.

[0009] The present invention provides an application of CYP1B1 and AKR1C3 as drug targets for detecting or treating bladder inflammation. By using the first discovered human-derived CYP1B1 and AKR1C3 as new molecular drug targets for detecting or treating bladder inflammation, the purpose of detecting or treating bladder inflammation using the drug targets is achieved, thereby providing new ideas and gene resources for the prevention and treatment of bladder inflammation.

[0010] The second aspect of the present invention provides a drug target for treating bladder inflammation, including CYP1B1 and AKR1C3 as described in the first aspect and various embodiments as drug targets for treating bladder inflammation.

[0011] Alternatively, specific siRNAs for CYP1B1 and AKR1C3 can downregulate the activity of SV-HUC-1 inflammatory cells induced by bladder mucosal epithelial cells and reduce the expression of inflammatory factors in SV-HUC-1 inflammatory cells.

[0012] Alternatively, siRNA specific for CYP1B1 and AKR1C3 can downregulate the expression of CYP1B1 and AKR1C3 in inflammatory cells.

[0013] The present invention provides a drug target for treating bladder inflammation. By utilizing the fact that bladder inflammation may be positively correlated with the expression levels of CYP1B1 and AKR1C3, CYP1B1 and AKR1C3 are used as drug targets for preventing and treating bladder inflammation, thereby achieving the purpose of treating bladder inflammation using the drug target.

[0014] The third aspect of the present invention provides a kit, comprising reagents for detecting the gene expression of CYP1B1 and AKR1C3 as drug targets for detecting bladder inflammation in the first aspect and various embodiments.

[0015] Optionally, primers for amplifying CYP1B1 and AKR1C3 are also included.

[0016] Optionally, the primers used to amplify CYP1B1 are: 5'-TGAGTGCCGTGTGTTTCGG-3' and 5'-GTTGCTGAAGTTGCGGTTGAG-3'; the primers used to amplify AKR1C3 are: 5'-GTCATCCGTATTTCAACCGGAG-3' and 5'-CCACCCATCGTTTGTCTCGTT-3'.

[0017] Optionally, reagents for detecting protein expression levels of IL6 and TNF-α, and antibodies for detecting IL6 and TNF-α are also included.

[0018] The kit provided by the present invention utilizes the characteristic that the gene levels detected by using CYP1B1 and AKR1C3 as drug targets in the bladder mucosa corresponding to bladder inflammation are significantly increased compared with normal bladder mucosa, so that the drug targets can achieve the purpose of detecting bladder inflammation.

[0019] Other features and advantages of the present invention will be described in the following description, and partly become apparent from the description, or understood by practicing the present invention. The purpose and other advantages of the present invention can be realized and obtained by the structures particularly pointed out in the written description and the accompanying drawings.

[0020] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The relative expression levels of TNF-α and IL6 in the bladder mucosa of cystitis (Cystitis group) and the normal bladder mucosa (Normal group) using ELISA method provided in the exemplary embodiment of the present invention. *: P<0.05.

[0022] Figure 2 The exemplary embodiment of the present invention provides HE staining and multiple fluorescent immunohistochemistry to measure the protein expression levels of TNF-α and IL6 in bladder mucosa with cystitis (Cystitis group) and normal bladder mucosa (Normal group).

[0023] Figure 3 are the corresponding expression levels of CYP1B1 and AKR1C3 in normal bladder mucosa (Normal group) and cystitis bladder mucosa (Cystitis group) provided in the exemplary embodiment of the present invention. *: P<0.05.

[0024] Figure 4 The relative expression levels of IL6 and TNF-α in the bladder mucosal epithelial inflammation model (LPS group) and the normal bladder epithelial cell line SV-HUC-1 (NC group) were detected by ELISA in an exemplary embodiment of the present invention. *: P<0.05.

[0025] Figure 5The exemplary embodiment of the present invention provides the use of immunocytofluorescence flow cytometry to detect the relative expression levels of IL6 and TNF-α in the bladder mucosal epithelial inflammation model (LPS group) and the normal bladder epithelial cell line SV-HUC-1 (NC group).

[0026] Figure 6 The expression levels of CYP1B1 and AKR1C3 corresponding to the normal bladder epithelial cell line SV-HUC-1 (NC group) and the bladder mucosal epithelial inflammation model (LPS group) provided in the exemplary embodiment of the present invention. *: P<0.05.

[0027] Figure 7 The exemplary embodiment of the present invention provides that the use of CYP1B1 and AKR1C3 specific siRNA can downregulate the expression levels of CYP1B1 and AKR1C3 in inflammatory cells. *: P<0.05.

[0028] Figure 8 The exemplary embodiment of the present invention provides that the use of CYP1B1 and AKR1C3 specific siRNA can down-regulate the expression levels of TNF-α and IL6 in inflammatory cells. *: P<0.05.

[0029] Fig. 9 The exemplary embodiment of the present invention provides that the use of CYP1B1 and AKR1C3 specific siRNA can reduce the activity of inflammatory cells. *: P<0.05. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0031] The experimental methods described in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. described in the following examples are all commercially available unless otherwise specified.

[0032] The cystitis bladder mucosa and normal bladder mucosa in this application were obtained from 8 patients with prostatic hyperplasia (cystitis group, Cystitis group) admitted to the First Affiliated Hospital of China Medical University from July 2023 to December 2023, aged (66.15±7.33) years old; among them, all patients underwent transurethral resection of the prostate, and cystitis bladder mucosa (cystitis group, Cystitis group) was obtained. In addition, 8 patients (normal group, Normal group) who underwent radical cystectomy during the same period were selected, aged (63.62±5.28) years old, and normal bladder mucosa was obtained. This study was approved by the hospital ethics committee and the patients gave informed consent.

[0033] The first aspect of the present invention provides an application of CYP1B1 and AKR1C3 as drug targets for detecting or treating bladder inflammation.

[0034] Among them, CYP1B1 is cytochrome P450 family 1B1, and AKR1C3 is aldehyde-keto reductase family 1 member C3.

[0035] The present invention discovers for the first time the difference in the expression levels of inflammatory factors in cystitis bladder mucosa and normal bladder mucosa, the expression levels of inflammatory factors include the protein expression levels of IL6 and TNF-α, and / or the relative expression amounts of IL6 and TNF-α, and the expression of CYP1B1 and AKR1C3 in bladder inflammation mucosa increases. Therefore, the inventor detects whether the patient suffers from bladder inflammation by using CYP1B1 and AKR1C3 as drug targets to detect the gene expression levels in the bladder mucosal epithelium of the patient. Therefore, CYP1B1 and AKR1C3 can be used as drug targets for detecting or treating bladder inflammation, and CYP1B1 and AKR1C3 derived from humans can be used as new molecular drug targets for detecting or treating bladder inflammation, and the targets can be used to achieve the purpose of detecting or treating bladder inflammation, providing new ideas and gene resources for the prevention and treatment of bladder inflammation.

[0036] It should be noted that the inventors used immunohistochemistry to analyze the differences in inflammatory expression between the bladder mucosa of cystitis and the normal bladder mucosa, and then combined it with the ELISA method to prove that the expression of inflammatory factors in the bladder mucosa of cystitis was higher than that in the normal bladder mucosa, thereby proving that the bladder mucosa of cystitis showed inflammation.

[0037] See also Figure 1, it can be analyzed that: the relative expression of IL6 corresponding to the cystitis bladder mucosa (Cystitis group) detected by ELISA method is higher than the relative expression of IL6 corresponding to the normal bladder mucosa (Normal group), and the relative expression of TNF-α corresponding to the cystitis bladder mucosa (Cystitis group) detected by ELISA method is higher than the relative expression of TNF-α corresponding to the normal bladder mucosa (Normal group). Among them, when performing ELISA detection, the tissue samples corresponding to the cystitis bladder mucosa and the normal bladder mucosa can be extracted into solutions according to the RIPA instructions, and the protein concentration in the above solution can be measured.

[0038] In addition, the present application also uses HE staining and multiple fluorescent immunohistochemistry methods to sequentially slice and stain the cystitis bladder mucosa and the normal bladder mucosa, see Figure 2 It can be analyzed that the protein expression levels of IL6 and TNF-α corresponding to the bladder mucosa of cystitis are higher than those of the normal bladder mucosa.

[0039] Here, Prism software was used for statistical analysis to perform one-way ANOVA on the cystitis bladder mucosa and the normal bladder mucosa. The independent sample t test was used to compare the cystitis bladder mucosa (Cystitis group) and the normal bladder mucosa (Normal group). P < 0.05 was considered to be significantly different. The analysis results were described using mean ± standard deviation.

[0040] Based on the data obtained by the above-mentioned ELISA method, as well as the data obtained by HE staining and multiple fluorescent immunohistochemistry, it can be analyzed that the expression levels of inflammatory factors corresponding to the bladder mucosa of cystitis (Cystitis group) (protein expression levels of IL6 and TNF-α, and the relative expression levels of IL6 and TNF-α) were significantly increased compared with the expression levels of inflammatory factors corresponding to the normal bladder mucosa (Normal group) (protein expression levels of IL6 and TNF-α, and the relative expression levels of IL6 and TNF-α) (all P < 0.05), which can indicate that the bladder mucosa of cystitis (Cystitis group) presents an inflammatory response.

[0041] In some possible embodiments, CYP1B1 and AKR1C3 are used as drug targets for detecting gene expression levels in bladder mucosal epithelium; gene expression levels include RNA levels and protein levels.

[0042] Here, Real-time PCR was used to detect the gene expression levels of CYP1B1 and AKR1C3 as drug targets in cystitis bladder mucosa (Cystitis group) and normal bladder mucosa (Normal group), respectively.

[0043] The steps of detecting the gene expression levels in the cystitis bladder mucosa (Cystitis group) and the normal bladder mucosa (Normal group) included: extracting tissue RNA according to the instructions of TRIzol reagent, and detecting the purity of RNA concentration by UV spectrophotometer; the primers were synthesized by BGI, and the primers for CYP1B1 were: 5'-TGAGTGCCGTGTGTTTCGG-3' and 5'-GTTGCTGAAGTTGCGGTTGAG-3', which were used to amplify the AKR1C3 The primers were: 5'-GTCATCCGTATTTCAACCGGAG-3' and 5'-CCACCCATCGTTTGTCTCGTT-3'; the total RNA weight was 1ug, and the TAKARA reverse transcription kit was used for reverse transcription and translation; the reaction conditions were: 5 min at 95°C, 15 s at 95°C, 15 s at 55°C, and 30 s at 72°C, for a total of 40 cycles; each sample was repeated 3 times, and the gene expression levels were counted and calculated.

[0044] Here, the statistical analysis was performed using Prism software for one-way analysis of variance, and the independent sample t-test was used for comparison between the two groups. P < 0.05 was considered to be significantly different; the results were described using mean ± standard deviation.

[0045] See also Figure 3 Compared with the normal bladder mucosa (Normal group), the gene levels detected with CYP1B1 and AKR1C3 as drug targets in the cystitis bladder mucosa (Cystitis group) were significantly increased (all P < 0.05). Therefore, it can be explained that bladder inflammation may be positively correlated with the expression levels of genes corresponding to CYP1B1 and AKR1C3. Therefore, CYP1B1 and AKR1C3 can be used as drug targets for the detection and treatment of bladder inflammation.

[0046] In some possible embodiments, the gene expression level in the bladder mucosal epithelium is obtained by detecting the bladder mucosal epithelium inflammation model through PCR and flow cytometry; the bladder mucosal epithelium inflammation model is constructed by inducing inflammation in the normal bladder epithelial cell line SV-HUC-1 by lipopolysaccharide LPS.

[0047] Here, lipopolysaccharide LPS is used to induce inflammation in the normal bladder epithelial cell line SV-HUC-1 to construct a bladder mucosal epithelial inflammation model, which may include the following steps:

[0048] Step S101, using F12K (10% FBS, 1% penicillin-streptomycin) to culture the normal bladder epithelial cell line SV-HUC-1, wherein the culture conditions are 37° C. and a CO2 concentration of 5%. When the cell density of the normal bladder epithelial cell line SV-HUC-1 is 80%, the normal bladder epithelial cell line SV-HUC-1 is digested with 0.25% trypsin to obtain digested SV-HUC-1 cells; SV-HUC-1 is a cell in the normal bladder epithelial cell line.

[0049] Step S102, inducing SV-HUC-1 cells in step S101 with lipopolysaccharide LPS at a concentration of 1000 nmol / L for 48 h, so as to complete lipopolysaccharide LPS-induced inflammation in the normal bladder epithelial cell line SV-HUC-1 and obtain a bladder mucosal epithelial inflammation model.

[0050] Furthermore, the detection of inflammation in the bladder mucosal epithelial inflammation model may include the following steps:

[0051] Step S201, extracting the bladder mucosal epithelial inflammation model and the normal bladder epithelial cell line SV-HUC-1 into solutions according to the RIPA instructions, and measuring the protein concentrations corresponding to the above solutions.

[0052] In this step, the ELISA method was used to detect the relative expression levels of IL6 and TNF-α in the bladder mucosal epithelial inflammation model and the normal bladder epithelial cell line SV-HUC-1. Figure 4 It was possible to analyze that the expression levels of IL6 and TNF-α in the bladder mucosal epithelial inflammation model (LPS group) were significantly increased compared with the normal bladder epithelial cell line SV-HUC-1 (NC group).

[0053] Step S202: using immunocytofluorescence flow cytometry to detect the relative expression levels of IL6 and TNF-α in the normal bladder epithelial cell line SV-HUC-1 (NC group) and the bladder mucosal epithelial inflammation model (LPS group).

[0054] In this step, immunocytofluorescence flow cytometry was used to detect the relative expression levels of IL6 and TNF-α in the bladder mucosal epithelial inflammation model and the normal bladder epithelial cell line SV-HUC-1. Figure 5, it can be analyzed that compared with the normal bladder epithelial cell line SV-HUC-1 (NC group), the corresponding expression levels of IL6 and TNF-α in the bladder mucosal epithelial inflammation model (LPS group) were significantly increased. The normal bladder epithelial cell line SV-HUC-1 (NC group) and the bladder mucosal epithelial inflammation model (LPS group) were fixed, permeabilized, blocked, and stained with primary and secondary antibodies, and the corresponding expression levels of the two factors were detected on a flow cytometer. The above test results were then analyzed by median analysis using Flowjo software, see Figure 5 .

[0055] Step S203, one-way analysis of variance was performed by using Prism software for statistical analysis, and independent sample t-test was used to compare the normal bladder epithelial cell line SV-HUC-1 (NC group) and the bladder mucosal epithelial inflammation model (LPS group).

[0056] Here, P < 0.05 was considered to be significantly different; the results were described using mean ± standard deviation.

[0057] In this step, compared with the normal bladder epithelial cell line SV-HUC-1 (NC group), the expression levels of IL6 and TNF-α in the bladder mucosal epithelial inflammation model (LPS group) were significantly increased (P < 0.05) (see Figure 4 , Figure 5 ), which shows that lipopolysaccharide LPS can cause normal bladder epithelial cells to express inflammatory factors, and the obtained bladder mucosal epithelial inflammation model shows inflammatory response.

[0058] Since the bladder mucosal epithelial inflammation model here shows an inflammatory response, there are differences in the expression levels of inflammatory factors in the bladder mucosal epithelial inflammation model and the normal bladder mucosa. Therefore, the bladder mucosal epithelial inflammation model has increased expression of CYP1B1 and AKR1C3 compared with the normal bladder mucosa. Therefore, PCR can detect that the CYP1B1 and AKR1C3 in the bladder mucosal epithelial inflammation model have increased expression at the gene level compared with the CYP1B1 and AKR1C3 in the normal bladder mucosa.

[0059] A drug target for treating bladder inflammation provided in an embodiment of the second aspect of the present invention includes CYP1B1 and AKR1C3 provided in the first aspect and various embodiments as drug targets for treating bladder inflammation.

[0060] Since, compared with normal bladder mucosa, the gene levels detected using CYP1B1 and AKR1C3 as drug targets in the bladder mucosa corresponding to bladder inflammation were significantly increased, it can be explained that bladder inflammation may be positively correlated with the expression levels of genes corresponding to CYP1B1 and AKR1C3, so CYP1B1 and AKR1C3 can be used as drug targets for the treatment of bladder inflammation.

[0061] Here, the specific upregulation of CYP1B1 and AKR1C3 expression in inflammatory cells corresponding to bladder inflammation is analyzed, and the analysis process may include the following steps:

[0062] Step S301, inoculating logarithmic phase cells into a six-well plate, 4×105 cells per well, and culturing overnight; using LPS to induce a bladder mucosal epithelial inflammation model;

[0063] Step S302, RNA from the bladder mucosal epithelial inflammation model and normal bladder mucosal cells were extracted according to the instructions of TRIzol reagent, and the RNA concentration and purity were detected by ultraviolet spectrophotometer; wherein, 1ug ​​of total RNA was taken and reverse transcribed and translated using TAKARA's reverse transcription kit; the reaction conditions were as follows: reaction at 95°C for 5min, reaction at 95°C for 15s, reaction at 55°C for 15s, reaction at 72°C for 30s, for a total of 40 cycles; each sample was repeated 3 times, and the gene expression was counted and calculated;

[0064] Step S303, one-way analysis of variance was performed using Prism software for statistical analysis, and independent sample t-test was used to compare the two groups of bladder mucosal epithelial inflammation model and normal bladder mucosal cells, with P<0.05 indicating a significant difference; the results were described using mean±standard deviation.

[0065] Compared with the normal cell group (NC group), the expression levels of CYP1B1 and AKR1C3 in the bladder mucosal epithelial inflammation model (LPS group) were significantly increased (P < 0.05) ( Figure 6 ), indicating that bladder inflammation may be positively correlated with the expression levels of CYP1B1 and AKR1C3, and CYP1B1 and AKR1C3 can be used as drug targets for the prevention and treatment of bladder inflammation.

[0066] In some possible embodiments, the specific siRNA of CYP1B1 and AKR1C3 can downregulate the expression of CYP1B1 and AKR1C3 in inflammatory cells.

[0067] Here, the experimental method for downregulating the expression of inflammatory factors in SV-HUC-1 inflammatory cells using specific siRNA of CYP1B1 and AKR1C3 may include the following steps:

[0068] Step S401, select the CYP1B1-specific siRNA and the AKR1C3-specific siRNA designed and synthesized by Jintuosi (Wuhan) Biotechnology Co., Ltd. The CYP1B1-specific siRNA sequence is: 5'-CCCACAGCATGATGCGCAACTTCTT-3'; the AKR1C3-specific siRNA sequence is: 5'-CAGGACTCAAGTACAAGCCTGTCTG-3'.

[0069] Step S402, using F12K (10% FBS, 1% penicillin-streptomycin) to culture human normal bladder epithelial cells SV-HUC-1, the culture conditions are 37°C, CO2 concentration is 5%, and when the cell density reaches 80%, it is digested with 0.25% trypsin for subsequent experiments. Inducing inflammatory cells with LPS to obtain SV-HUC-1 inflammatory cells;

[0070] Step S403: After treating SV-HUC-1 inflammatory cells with CYP1B1-specific siRNA and AKR1C3-specific siRNA for 48 h, the knockdown efficiency was detected by immunocytometry or Western blot according to the characteristics of the two-factor antibodies (P<0.05) ( Figure 7 ), obtain cell samples;

[0071] Step S404: The cell samples obtained in step S403 and the SV-HUC-1 inflammatory cells were used to detect the expression of IL6 and TNF-α by immunocytofluorescence flow cytometry; the cell samples and SV-HUC-1 inflammatory cells were fixed, permeabilized, blocked, and stained with primary and secondary antibodies, and the expression of the two factors was detected on a flow cytometer. The results were analyzed by median analysis using Flowjo software, indicating that the specific siRNA of CYP1B1 and the specific siRNA of AKR1C3 can inhibit the expression of inflammatory factors and reduce the inflammatory response ( Figure 8 ).

[0072] Step S405: Statistical analysis: One-way ANOVA was performed using Prism software, and the comparison between the two groups of cell samples and SV-HUC-1 inflammatory cells was performed using independent sample t-test, with P<0.05 indicating a significant difference. The results were described using mean±standard deviation.

[0073] The analysis showed that compared with the SV-HUC-1 inflammatory cells in the bladder mucosal epithelial inflammation model (LPS group), the expression levels of CYP1B1 and AKR1C3 were significantly downregulated in the SV-HUC-1 inflammatory cells (knockdown group) treated with CYP1B1-specific siRNA and AKR1C3-specific siRNA for 48 hours (P<0.05, Figure 7), while the expression levels of IL6 and TNF-α were significantly downregulated (P<0.05, Figure 8 ), indicating that CYP1B1-specific siRNA and AKR1C3-specific siRNA can inhibit the expression of CYP1B1 and AKR1C3 in bladder inflammatory cells, thereby inhibiting the expression of inflammatory factors and reducing the inflammatory response.

[0074] In some possible embodiments, specific siRNAs of CYP1B1 and AKR1C3 can downregulate the activity of SV-HUC-1 inflammatory cells in the bladder mucosal epithelium and reduce the expression of inflammatory factors in SV-HUC-1 inflammatory cells.

[0075] Taking SV-HUC-1 inflammatory cells in a bladder mucosal epithelial inflammation model as an example, an experimental method for downregulating the activity of SV-HUC-1 inflammatory cells in a bladder mucosal epithelial inflammation model using specific siRNAs of CYP1B1 and AKR1C3 may include the following steps:

[0076] Step S501, after treating SV-HUC-1 inflammatory cells in the bladder mucosal epithelial inflammation model with CYP1B1-specific siRNA and AKR1C3-specific siRNA for 48 hours, a knockdown group corresponding to CYP1B1 (LPS+si-CYP1B1 group) and a knockdown group corresponding to AKR1C3 (LPS+si-AKR1C3 group) were obtained.

[0077] Step S502, the logarithmic phase cells of the bladder mucosal epithelial inflammation model (LPS group), the knockdown group corresponding to CYP1B1 (LPS+si-CYP1B1 group), and the knockdown group corresponding to AKR1C3 (LPS+si-AKR1C3 group) were inoculated into 96-well plates, with 1×104 cells per well, and 0h, 24h, 48h, and 72h groups were set up for culture, respectively. The old culture medium was discarded at the corresponding time, and 100 μL of F12K culture medium containing 10 μL of CCK8 reagent was added to each well, and the wells were placed in an incubator for further incubation for 1.5 h. The cell absorbance OD value was detected at a wavelength of 450 nm on a microplate reader, and the relative cell viability was calculated; wherein, the calculation formula is as follows: relative cell viability (%) = OD experimental group / OD control group × 100%.

[0078] Step S503: Statistical analysis was performed using Prism software for one-way analysis of variance, and t-test was used for comparison between groups. P<0.05 was considered to be significantly different. The results were described using mean±standard deviation.

[0079] The results showed that the proliferation of SV-HUC-1 inflammatory cells in the bladder mucosal epithelial inflammation model could be significantly inhibited by using CYP1B1-specific siRNA and AKR1C3-specific siRNA to treat the SV-HUC-1 inflammatory cells in the bladder mucosal epithelial inflammation model (P<0.05, Fig. 9 ), thereby reducing the activity of SV-HUC-1 inflammatory cells to achieve the purpose of inhibiting inflammation. That is, CYP1B1 and AKR1C3 can be used as new molecular drug targets for detecting or treating bladder inflammation. This target can be used to detect bladder inflammation or screen drugs, thereby achieving the effect of preventing or treating bladder inflammation. This provides new ideas and gene resources for the prevention and treatment of bladder inflammation.

[0080] A kit provided in an embodiment of the third aspect of the present invention includes reagents for detecting the gene expression of CYP1B1 and AKR1C3 as drug targets for detecting bladder inflammation in the embodiment of the first aspect and various implementations.

[0081] Since, compared with normal bladder mucosa, the gene levels detected by using CYP1B1 and AKR1C3 as drug targets in the bladder mucosa corresponding to bladder inflammation are significantly increased, it can be explained that bladder inflammation may be positively correlated with the expression levels of genes corresponding to CYP1B1 and AKR1C3. Therefore, CYP1B1 and AKR1C3 can be used as drug targets for detecting bladder inflammation. Therefore, when the kit includes CYP1B1 and AKR1C3 as reagents for detecting the gene expression of drug targets for bladder inflammation, it can detect bladder inflammation.

[0082] In some possible embodiments, primers for amplifying CYP1B1 and AKR1C3 are also included.

[0083] Here, the primers used to amplify CYP1B1 and AKR1C3 were synthesized by BGI.

[0084] Optionally, the primers used to amplify CYP1B1 are: 5'-TGAGTGCCGTGTGTTTCGG-3' and 5'-GTTGCTGAAGTTGCGGTTGAG-3'; the primers used to amplify AKR1C3 are: 5'-GTCATCCGTATTTCAACCGGAG-3' and 5'-CCACCCATCGTTTGTCTCGTT-3'.

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of CYP1B1 and AKR1C3 as drug targets for detecting or treating bladder inflammation.

2. The use according to claim 1, characterized in that: The drug targets of CYP1B1 and AKR1C3 are drug targets for detecting gene expression levels in bladder mucosal epithelium; the gene expression levels include RNA levels and protein levels.

3. The use according to claim 2, characterized in that: The gene expression level in the bladder mucosal epithelium is obtained by detecting the bladder mucosal epithelium inflammation model through PCR and flow cytometry; the bladder mucosal epithelium inflammation model is constructed by inducing inflammation in the normal bladder epithelial cell line SV-HUC-1 by lipopolysaccharide LPS.

4. A drug target for treating bladder inflammation, characterized in that: The method comprises CYP1B1 and AKR1C3 as described in any one of claims 1 to 3 as drug targets for treating bladder inflammation.

5. The drug target according to claim 4, characterized in that The specific siRNA of CYP1B1 and AKR1C3 can down-regulate the activity of SV-HUC-1 inflammatory cells in the bladder mucosal epithelium and reduce the expression of inflammatory factors in the SV-HUC-1 inflammatory cells.

6. The drug target according to claim 4, characterized in that The specific siRNA of CYP1B1 and AKR1C3 can downregulate the expression of CYP1B1 and AKR1C3 in inflammatory cells.

7. A kit, characterized in that: The invention comprises a reagent for detecting the gene expression of CYP1B1 and AKR1C3 as drug targets for detecting bladder inflammation according to any one of claims 1 to 3.

8. The kit according to claim 7, characterized in that Primers for amplification of CYP1B1 and AKR1C3 were also included.

9. The kit according to claim 8, characterized in that The primers used to amplify CYP1B1 were: 5'-TGAGTGCCGTGTGTTTCGG-3' and 5'-GTTGCTGAAGTTGCGGTTGAG-3'; the primers used to amplify AKR1C3 were: 5'-GTCATCCGTATTTCAACCGGAG-3' and 5'-CCACCCATCGTTTGTCTCGTT-3'.

10. The kit according to claim 7, characterized in that Also included are reagents for detecting the protein expression levels of IL6 and TNF-α, and antibodies for detecting IL6 and TNF-α.

Images