Application of d-borneol in preparation of medicine for treating renal fibrosis

Abstract

The invention relates to an application of d-borneol in preparation of medicines for treating renal fibrosis. The d-borneol can improve UUO mouse kidney interstitial collagen deposition, obviously reduce the expression level of fibrosis indexes COL-1, alpha-SMA, FN and TNC, can inhibit the fibrosis reaction of TGF-beta1 stimulated renal tubular epithelial cells, and has no obvious toxic or side effect. Therefore, the d-borneol serving as the renal fibrosis therapeutic agent has a wide clinical application prospect.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and specifically relates to the application of dextrorotatory borneol in the treatment of renal fibrosis. Background Art

[0002] Chronic kidney disease (CKD) has become an increasingly severe challenge in the field of global public health in recent years. Epidemiological statistics show that the prevalence of CKD globally is approximately 10.8%, which highlights the wide-ranging impact and heavy burden of this disease. CKD not only affects the quality of life of patients but may also gradually deteriorate to end-stage renal disease (ESRD), at which point patients will have to rely on dialysis or kidney transplantation to sustain life, which undoubtedly brings huge economic and psychological pressures to individuals, families, and even society.

[0003] Renal fibrosis is the core pathological mechanism in the progression of CKD and is a key pathogenic factor leading to end-stage renal disease, mainly manifested as the abnormal accumulation of extracellular matrix (ECM), which in turn causes disorder of renal tissue structure and decline of function. This process is regarded as the key driving factor for irreversible loss of renal function. Currently, the drugs used clinically for CKD patients mainly focus on controlling symptoms such as blood pressure and proteinuria, and drugs for reducing renal fibrosis have not been developed. Given the rapid growth of the prevalence of CKD and the urgent need for more effective treatment methods, it is particularly important to develop new, safe, and highly effective anti-renal fibrosis drugs. Therefore, exploring new renal protective drugs that can reduce renal fibrosis and delay the progression of CKD is of great significance for improving the prognosis of patients.

[0004] Dextrorotatory borneol, also known as "natural borneol", is recorded in "Newly Revised Materia Medica" that dextrorotatory borneol "maintains pathogenic factors in the heart and abdomen, rheumatism accumulation, and removes redness of the eyes and skin nebula". Dextrorotatory borneol is widely used in traditional Chinese medicine and has the effects of resuscitating the consciousness, clearing heat and detoxifying, relieving pain and itching, dispelling wind and cold, calming the mind, etc., but the application of dextrorotatory borneol in the prevention and treatment of renal fibrosis has not been reported. Summary of the Invention

[0005] The purpose of the present invention is to overcome the disadvantages and deficiencies existing in the prior art and provide a compound for the treatment of renal fibrosis.

[0006] Based on the above findings, the technical solutions for solving the above technical problems proposed by the present invention are as follows.

[0007] The first aspect of the present invention provides a compound for preventing or treating renal fibrosis, and the structure of the compound is shown in formula (I):

[0008]

[0009] The second aspect of the present invention provides a pharmaceutical composition for preventing or treating renal fibrosis, which comprises the above-mentioned compound of formula (I) and pharmaceutically acceptable excipients.

[0010] Preferably, the pharmaceutical composition further comprises other drugs for treating renal fibrosis.

[0011] Preferably, the other drugs for treating renal fibrosis are selected from one or more of pirfenidone, nintedanib, aliskiren, pentoxifylline, canagliflozin, and atrasentan.

[0012] Preferably, the pharmaceutically acceptable excipients are selected from one or more of fillers, disintegrants, binders, lubricants, solvents, emulsifiers, cosolvents, solubilizers, preservatives, pH regulators, osmotic pressure regulators, surfactants, coating materials, antioxidants, flavoring agents, and coloring agents.

[0013] Preferably, the dosage form of the pharmaceutical composition is tablets, granules, injections, pills or suspensions.

[0014] Preferably, the concentration of the compound of formula (I) in the pharmaceutical composition is 1-20 μM. More preferably, the concentration of the compound of formula (I) in the pharmaceutical composition is 10-20 μM. Further preferably, the concentration of the compound of formula (I) in the pharmaceutical composition is 15.6 μM.

[0015] The third aspect of the present invention provides the use of the above-mentioned compound of formula (I) or the above-mentioned pharmaceutical composition in the preparation of a drug for preventing or treating renal fibrosis.

[0016] Preferably, the renal fibrosis is renal fibrosis caused by unilateral ureteral ligation.

[0017] Preferably, the concentration of the compound of formula (I) in the drug is 1-20 μM. More preferably, the concentration of the compound of formula (I) in the pharmaceutical composition is 10-20 μM. Further preferably, the concentration of the compound of formula (I) in the pharmaceutical composition is 15.6 μM.

[0018] Preferably, the single-dose administration dosage of the compound of formula (I) is 10-60 mg / kg body weight. More preferably, the single-dose administration dosage of the compound of formula (I) is 12.5-50 mg / kg body weight. Further preferably, the single-dose administration dosage of the compound of formula (I) is 25-50 mg / kg body weight. Further preferably, the single-dose administration dosage of the compound of formula (I) is 50 mg / kg body weight.

[0019] The fourth aspect of the present invention provides the use of the compound of formula (I) or the above-mentioned pharmaceutical composition in the preparation of a medicament for treating chronic kidney disease.

[0020] Preferably, the chronic kidney disease is a chronic kidney disease characterized by renal fibrosis as a pathological feature.

[0021] Preferably, the concentration of the compound of formula (I) in the medicament is 1 - 20 μM. More preferably, the concentration of the compound of formula (I) in the pharmaceutical composition is 10 - 20 μM. Further preferably, the concentration of the compound of formula (I) in the pharmaceutical composition is 15.6 μM.

[0022] Preferably, the single-dose administration of the compound of formula (I) is 10 - 60 mg / kg body weight. More preferably, the single-dose administration of the compound of formula (I) is 12.5 - 50 mg / kg body weight. Further preferably, the single-dose administration of the compound of formula (I) is 25 - 50 mg / kg body weight. Further preferably, the single-dose administration of the compound of formula (I) is 50 mg / kg body weight.

[0023] The fifth aspect of the present invention provides the use of the compound of formula (I) or the above-mentioned pharmaceutical composition in the preparation of a medicament for treating renal tubular epithelial cell fibrosis.

[0024] Preferably, the renal tubular epithelial cells are human renal tubular epithelial cells HK2.

[0025] Preferably, the renal tubular epithelial cell fibrosis is TGF-β1-induced renal tubular epithelial cell fibrosis.

[0026] Preferably, the concentration of the compound of formula (I) in the medicament is 1 - 20 μM. More preferably, the concentration of the compound of formula (I) in the medicament is 10 - 20 μM. Further preferably, the concentration of the compound of formula (I) in the medicament is 15.6 μM.

[0027] Preferably, the single-dose administration of the compound of formula (I) is 10 - 60 mg / kg body weight. More preferably, the single-dose administration of the compound of formula (I) is 12.5 - 50 mg / kg body weight. Further preferably, the single-dose administration of the compound of formula (I) is 25 - 50 mg / kg body weight. Further preferably, the single-dose administration of the compound of formula (I) is 50 mg / kg body weight.

[0028] The sixth aspect of the present invention provides the use of the compound of formula (I) or the above-mentioned pharmaceutical composition in the preparation of a drug for reducing the expression level of fibrotic indexes in renal tubular epithelial cells, and the fibrotic indexes of renal tubular epithelial cells are selected from one or more of tenascin C (TNC), fibronectin (FN), type I collagen (COL-1), and alpha-smooth muscle actin (alpha-SMA).

[0029] Preferably, the reduction of the expression level of fibrotic indexes in renal tubular epithelial cells is to simultaneously reduce the expression levels of tenascin C (TNC), fibronectin (FN), type I collagen (COL-1), and alpha-smooth muscle actin (alpha-SMA).

[0030] Preferably, the concentration of the compound of formula (I) in the drug is 1-20 μM. More preferably, the concentration of the compound of formula (I) in the drug is 10-20 μM. Further preferably, the concentration of the compound of formula (I) in the drug is 15.6 μM.

[0031] Preferably, the single-dose administration dose of the compound of formula (I) is 10-60 mg / kg body weight. More preferably, the single-dose administration dose of the compound of formula (I) is 12.5-50 mg / kg body weight. Further preferably, the single-dose administration dose of the compound of formula (I) is 25-50 mg / kg body weight. Further preferably, the single-dose administration dose of the compound of formula (I) is 50 mg / kg body weight.

[0032] Advantages of the present invention:

[0033] The inventors unexpectedly found that borneol can reduce the expression levels of fibrotic indexes COL-1, alpha-SMA, FN, and TNC in an animal model of renal fibrosis, improve the renal tissue pathology of animals with renal fibrosis, inhibit renal interstitial collagen deposition, effectively reduce renal fibrosis, delay and / or reverse the course of chronic kidney disease, and has no obvious toxic and side effects. Therefore, borneol has broad clinical application prospects as a therapeutic agent for renal fibrosis. Description of the Drawings

[0034] Figure 1 Western Blot diagram of the protein expression of fibrotic indexes COL-1 and alpha-SMA in a mouse renal fibrosis model after administration of different concentrations of borneol;

[0035] Figure 2 Real-time PCR diagram of the mRNA expression of fibrotic indexes COL-1, alpha-SMA, TNC, and FN in a mouse renal fibrosis model after administration of different concentrations of borneol;

[0036] Figure 3This is the immunofluorescence image of α-SMA expression in the renal fibrosis model of mice after administration of different concentrations of borneol;

[0037] Figure 4 This is the immunofluorescence image of COL-1 expression in the renal fibrosis model of mice after administration of different concentrations of borneol;

[0038] Figure 5 Masson staining of renal interstitial collagen deposition in the renal fibrosis model of mice after administration of different concentrations of borneol;

[0039] Figure 6 The results of staining the heart, liver, spleen, and lung tissues of mice in the simple drug administration group using a hematoxylin and eosin (H&E) staining kit;

[0040] Figure 7 The figure shows the effect of borneol on the levels of blood creatinine and urea nitrogen in mouse serum. Figure 7 A is blood creatinine, Figure 7 B is urea nitrogen;

[0041] Figure 8 The figure shows the effect of borneol on the levels of alanine aminotransferase and aspartate aminotransferase in mouse serum. Figure 8 A is alanine aminotransferase, Figure 8 B is aspartate aminotransferase;

[0042] Figure 9 This is a comparison chart of the effects of different concentrations of borneol on the viability of renal tubular epithelial cells;

[0043] Figure 10 Real-time PCR images of COL-1 mRNA expression in renal tubular epithelial cells fibrosis induced by different concentrations of borneol;

[0044] Figure 11 The figure is the Real-time PCR image of borneol inhibiting the expression of FN and COL-1 mRNA in the fibrosis reaction of renal tubular epithelial cells induced by TGF-β1;

[0045] Figure 12 This is a Western Blot image showing that borneol inhibits the expression of α-SMA and COL-1 proteins in the TGF-β1-induced fibrosis of renal tubular epithelial cells;

[0046] Figure 13 This is the immunofluorescence image of α-SMA expression in the response of borneol to TGF-β1-induced fibrosis in renal tubular epithelial cells. DETAILED DESCRIPTION

[0047] The present invention will be further described in detail below in combination with test examples and the accompanying drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the definitions of the significance symbols of P values in the test examples and the accompanying drawings of this application are as follows: *P < 0.05, **P < 0.01, ***P < 0.001.

[0048] The drug of formula (I) used in the test examples of the present invention was provided by Jiangxi Linkelongnao Technology Co., Ltd., and is abbreviated as "d-borneol" in the test examples of the specification.

[0049] Test Example 1. Effects of d-borneol on renal tissue fibrosis in UUO model mice in vivo

[0050] 1. Test method

[0051] 1.1 Construction of UUO model

[0052] Thirty-six healthy male C57BL / 6 mice aged 6 - 8 weeks (body weight 22 - 24 g) were selected and randomly divided into a sham operation group (Sham), a group simply given d-borneol (d-borneol 50 mg / kg), a model group (UUO), a low-dose treatment group (UUO + d-borneol 12.5 mg / kg), a medium-dose treatment group (UUO + d-borneol 25 mg / kg), and a high-dose treatment group (UUO + d-borneol 50 mg / kg), with 6 mice in each group. The mice were fasted for 12 hours before the operation and allowed free access to water. After the mice were anesthetized, they were fixed in the prone position, the hair on the left back was shaved, and the surgical area was disinfected with 75% alcohol. A longitudinal incision was made 0.5 cm beside the left spine, the muscle layer was bluntly separated, and the left kidney and ureter were exposed. The ureter was doubly ligated with a suture 0.3 cm below the renal pelvis. The muscle and skin were sutured layer by layer, and disinfected with iodophor. In the sham operation group, only the ureter was separated without ligation. Administration started 24 hours after UUO surgery, and different concentrations of d-borneol were given by gavage once a day. The sham operation group was given an equal volume of normal saline.

[0053] 1.2 Western Blot method

[0054] Weigh the mouse kidney tissue, add protein lysate at a mass-to-volume ratio of 14 μL / mg, grind thoroughly, transfer it to an EP tube, and lyse it on a shaker at 4°C for 30 min, operating on ice throughout the process. Place the EP tube in a 4°C centrifuge and centrifuge at 12,000 rpm for 30 min, then take the supernatant. Add 1 / 4 volume of 5× protein loading buffer, mix well, heat at 100°C for 10 minutes, briefly centrifuge, and place on ice for later use. Prepare the stacking gel and separating gel, load 20 - 50 μg of protein into each well, and add prestained protein Marker at the same time. The initial voltage is 80 V (for the stacking gel), and after entering the separating gel, adjust it to 120 V until the bromophenol blue runs to the bottom of the gel. Use the semi-dry transfer method to transfer the membrane at a constant voltage of 128 V for 70 minutes. Place the membrane in a TBST solution containing 5% skim milk and block it on a shaker at room temperature for 1 h. Wash it three times with TBST, soak the membrane in the primary antibody, incubate overnight at 4°C, wash it three times with TBST, incubate the fluorescent secondary antibody in the dark for 1.5 h, wash it three times with TBST again, and develop and image. Detect the protein expression of fibrosis indicators COL-1 and α-SMA by Western Blot method and perform quantitative analysis. Repeat the experiment 6 times for each group.

[0055] 1.3, Real-time PCR method

[0056] Weigh the kidney tissue, add Trizol lysate at a mass-to-volume ratio of 600 μL / 15 mg, grind thoroughly, transfer it to an EP tube, lyse it at 4°C for 30 min, operate on ice throughout the process, extract RNA, reverse transcribe, and amplify. Repeat the experiment 6 times for each group.

[0057] 1.4, Immunofluorescence method

[0058] Embed the mouse kidney tissue in paraffin, and then section the paraffin. Place the sections in an oven at 65°C for 2 hours, and then successively place them in xylene and gradient ethanol for dewaxing. Repair them at high temperature with 1× EDTA repair solution, let them cool naturally at room temperature, wash them three times with PBS, drip 10% BSA to block for 0.5 h, wash them three times with PBS, drip the primary antibody, wash off the primary antibody after incubating for 24 h, drip the fluorescent secondary antibody, wash off the secondary antibody after incubating in the dark for 1.5 h, drip DAPI staining solution and incubate in the dark for 5 minutes, drip anti-fluorescence quenching agent, cover the slides, observe with a fluorescence inverted microscope, and take pictures. Repeat the experiment 6 times for each group.

[0059] 2. Test results

[0060] The results of Western Blot experiment showed that after 7 days of unilateral ureteral ligation, the protein levels of fibrosis indicators COL-1 and α-SMA increased significantly, while different concentrations of borneol decreased the expression of these proteins to varying degrees, and the high-dose group had the most significant reducing effect (see Figure 1 ).

[0061] The mRNA expression levels of fibrosis in kidney tissues were measured by real-time fluorescence quantitative PCR. From Figure 2 it can be seen that the mRNA expression levels of fibrosis indicators COL-1, α-SMA, TNC, and FN in the kidney tissues of mice in the UUO group were significantly increased, indicating that the mice had kidney fibrosis. Different concentrations of borneol inhibited the mRNA levels of fibrosis indicators COL-1, α-SMA, TNC, and FN in the UUO model to varying degrees, thereby inhibiting the progression of renal fibrosis in mice caused by unilateral ureteral ligation. The inhibitory effect was most significant in the high-dose group (50 mg / kg).

[0062] The results of immunofluorescence were as shown in Figure 3 and Figure 4 showed that borneol significantly reduced the expression of α-SMA and COL-1 in tissues caused by unilateral ureteral ligation, and the inhibitory effect was most significant at high doses.

[0063] It has been experimentally proven that borneol can play an important role in kidney fibrosis diseases. Therefore, this drug is expected to become a key drug for the prevention and treatment of chronic kidney diseases.

[0064] Experimental Example 2. Effects of Borneol on the Kidney Tissue Pathology of UUO Model Mice

[0065] 1. Experimental method (Masson method)

[0066] Modeling and drug administration were carried out according to the method of Experimental Example 1. The kidney tissues of mice were paraffin-embedded, and then the paraffin was sectioned. After the sections were placed in an oven at 65 °C for 2 hours, they were successively placed in xylene and gradient ethanol for dewaxing. After hematoxylin staining for 5 min, they were stained with ponceau acid fuchsin solution for 10 min, treated with phosphomolybdic acid aqueous solution for about 5 min, counterstained with aniline blue solution for 5 min, acidified with 1% glacial acetic acid for 1 min, dehydrated with gradient ethanol, and sealed.

[0067] 2. Experimental results

[0068] As shown in the Masson staining in Figure 5 after 7 days of unilateral ureteral ligation in mice, kidney fibrosis occurred. The main manifestation was a large amount of collagen deposition in the renal interstitium. The collagen deposition in the renal interstitium of mice in the UUO + borneol (50 mg / kg) group was significantly reduced, indicating that borneol had an obvious improvement effect on the kidney fibrosis pathology of UUO mice.

[0069] Experimental Example 3. Effects of Borneol on the Heart, Liver, Spleen, and Lung Tissues of Untreated Mice

[0070] 1. Experimental method

[0071] Healthy male C57BL / 6 mice (22 - 24 g) at 6 - 8 weeks of age were selected and raised in a pathogen - free environment (23 ± 2 °C and 55 ± 5% humidity). All mice had free access to food and water under a 12 - hour light / 12 - hour dark cycle.

[0072] A pure borneol mouse model was constructed: 6 mice were treated with intragastric administration of drugs every day, with a 24 - hour interval between each intragastric administration. The dosage of borneol was 50 mg / kg body weight. After 7 days, serum samples and kidney tissues were collected under anesthesia, and the contents of creatinine and urea nitrogen in the serum of the animal model were detected according to the instructions of the creatinine, urea nitrogen, alanine aminotransferase, and aspartate aminotransferase kits (purchased from Nanjing Jiancheng Bioengineering Institute). The heart, liver, spleen, lungs, and kidneys were embedded and subjected to HE staining.

[0073] (1) The method for creatinine determination is as follows:

[0074]

[0075] Creatinine content (μmol / l) = [(Measured A2 - K * Measured A1) - (Blank A2 - K * Blank A1)] / [(Standard A2 - K * Standard A1) - (Blank A2 - K * Blank)] * Standard concentration (442 μmol / l)

[0076] Note: Dilution factor K = (Sample volume + Volume of Enzyme Solution A) / (Sample volume + Volume of Enzyme Solution A + Volume of Enzyme Solution B) = 186 / 246

[0077] (2) The method for urea nitrogen test is as follows:

[0078]

[0079] Urea nitrogen content (mmol / l) = (Measured OD value - Blank measured value) / (Standard OD value - Blank OD value) * Standard concentration (10 mmol / l) * Dilution factor before sample testing

[0080] (3) The method for alanine aminotransferase test is as follows:

[0081]

[0082] Calculation method for liquid samples such as serum (plasma):

[0083] ALT activity (U / l) = ALT activity obtained by substituting into the standard curve (Karmen unit) * 0.482 * N

[0084] Among them, 0.482 is the conversion factor from Karmen unit to U / l; N is the dilution factor before sample testing.

[0085] (4) The method for aspartate aminotransferase test is as follows:

[0086]

[0087] Calculation method for liquid samples such as serum (plasma):

[0088] AST activity (U / l) = AST activity obtained by substituting into the standard curve (Karmen unit) * 0.482 * N

[0089] Among them, 0.482 is the conversion from Karmen unit to U / l; N is the dilution factor before sample testing.

[0090] (5) The HE staining method is as follows:

[0091] Embed the mouse heart, liver, spleen, and lung tissues in paraffin, and then section the paraffin. Place the sections in an oven at 65 °C for 2 hours, and then sequentially place them in xylene and gradient ethanol for dewaxing. After dewaxing, stain the cell nuclei with hematoxylin and wash away the floating color with distilled water. Differentiate in 1% hydrochloric acid alcohol differentiation solution for 30 s. Stain the cytoplasm with eosin, then wash away the floating color with distilled water, dehydrate with gradient ethanol, and mount the sections after clearing with xylene.

[0092] 2. Test results

[0093] As shown by HE staining Figure 6 There is no abnormal pathological structure in the mouse heart, liver, spleen, and lung tissues in the pure borneol group, and there is no significant difference in the pathological state of the mouse heart, liver, spleen, and lung tissues between the pure borneol group and the normal control group, indicating that borneol at this dose has no obvious toxicity to other organs.

[0094] The results of blood urea nitrogen and serum creatinine, the renal function indicators, are as Figure 7 shown. It can be seen from the figure that there is no significant difference in blood urea nitrogen and creatinine in the serum between the pure borneol group and the normal control group, indicating that borneol has no toxicity to the kidneys.

[0095] The results of alanine aminotransferase and aspartate aminotransferase in the blood, the liver function indicators, are as Figure 8 shown. It can be seen from the figure that there is no significant difference in alanine aminotransferase and aspartate aminotransferase in the serum between the pure borneol group and the normal control group, indicating that borneol has no toxicity to the liver.

[0096] Test Example 4. Inhibitory effect of borneol on TGF-β1-induced renal tubular epithelial cell fibrosis reaction in vitro

[0097] 1. Test method

[0098] 1.1 CCK8 method

[0099] Human renal tubular epithelial cells (HK2) were seeded in 96-well plates at a seeding density of approximately 4,000 cells / well. After the cells adhered to the plate, the culture medium was aspirated and replaced with culture medium containing different concentrations of borneol. The culture medium without borneol was used as the normal control group (NC). After continued culture for 24 hours, 10 μl of CCK8 solution was added to each well. After the CCK8 solution acted for 2 hours, the OD values of each well were measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and the results were recorded. A graph of cell viability against dose was plotted. Cell viability = (OD value of experimental group cells - OD value of blank group cells) / (OD value of control group cells - OD value of blank group cells) × 100%.

[0100] 1.2, Real-time PCR

[0101] HK2 cells were seeded in 12-well plates at a seeding density of approximately 0.5×10 5 cells / well and divided into a normal group (NC), a group with only borneol added (borneol 15.6 μM), a TGF-β1-induced model group (TGF-β1), a low-dose borneol group (TGF-β1 + borneol 3.9 μM), a medium-dose borneol group (TGF-β1 + borneol 7.8 μM), and a high-dose borneol group (TGF-β1 + borneol 15.6 μM). The selected concentration of TGF-β1 was 20 ng / ml. After stimulation for 12 hours, the cells were washed three times with PBS, collected, and total RNA was extracted, reverse-transcribed, and amplified. Each group was repeated three times.

[0102] 1.3, Western Blot

[0103] HK2 cells in the logarithmic growth phase were seeded in a 6-well plate at a seeding density of approximately 1.0×10 5 cells / well and divided into a normal group (NC), a group with only borneol added (borneol 15.6 μM), a TGF-β1-induced model group (TGF-β1), a low-dose borneol group (TGF-β1 + borneol 3.9 μM), a medium-dose borneol group (TGF-β1 + borneol 7.8 μM), and a high-dose borneol group (TGF-β1 + borneol 15.6 μM). The selected concentration of TGF-β1 was 20 ng / ml. After stimulation for 24 hours, the cells were washed three times with PBS, collected, and total protein was extracted. The expression of fibrosis markers COL-1 and α-SMA proteins was detected by Western Blot and quantitatively analyzed. Each group was repeated three times.

[0104] 1.4, Immunofluorescence

[0105] HK2 cells in the logarithmic growth phase were seeded in a 6-well plate containing glass slides at a seeding density of approximately 1.0×105 cells / ml, and they were divided into a normal group (NC), a group with only (+)-borneol added (+)-borneol 15.6 μM), a TGF-β1-induced model group (TGF-β1), a low-dose (+)-borneol group (TGF-β1 + (+)-borneol 3.9 μM), a medium-dose (+)-borneol group (TGF-β1 + (+)-borneol 7.8 μM), and a high-dose (+)-borneol group (TGF-β1 + (+)-borneol 15.6 μM). The selected concentration of TGF-β1 was 20 ng / ml. After stimulation for 24 hours, they were washed three times with PBS, fixed with paraformaldehyde for 10 minutes, washed three times with PBS, blocked with 10% BSA for 0.5 hour, washed three times with PBS, primary antibody was added, after incubation for 24 hours, the primary antibody was washed off, fluorescent secondary antibody was added, after incubation in the dark for 1.5 hours, the secondary antibody was washed off, DAPI staining solution was added and incubated in the dark for 5 minutes, anti-fluorescence quenching agent was added, mounted, observed under a fluorescence inverted microscope, photographed, and each group repeated the experiment 3 times.

[0106] 2. Test results

[0107] The results of the CCK-8 experiment were as Figure 9 shown, where Figure 9 A was the effect of (+)-borneol at a concentration of 0.8 - 100 μM on the survival rate of HK2 cells, Figure 9 B was the effect of (+)-borneol at a concentration of 7.8 - 1000 μM on the survival rate of HK2 cells. It could be seen from the figure that at a concentration of 0.8 - 125 μM, there was no significant difference in the cell survival rate between the (+)-borneol group and the normal control group (NC), indicating that (+)-borneol had no obvious toxicity.

[0108] The results of the Real-time PCR experiment showed that when TGF-β1-induced renal tubular epithelial cells were treated with (+)-borneol at a concentration of 3.9 - 125 μM, 15.6 μM of (+)-borneol most significantly reduced the mRNA level of COL-1 in the fibrotic response, indicating that the drug effect of (+)-borneol was the best at this concentration ( Figure 10 ). In addition, the mRNA levels of FN and COL-1 in the model group were significantly up-regulated after treatment with TGF-β1, and this phenomenon was improved after treatment with (+)-borneol, and the high-dose group had the most obvious effect ( Figure 11 ). The above test results suggested that (+)-borneol could inhibit the cell fibrotic response caused by TGF-β1.

[0109] The results of Western Blot were as Figure 12 shown (using β-actin as an internal reference). After HK2 cells treated with TGF-β1 were treated with (+)-borneol, the protein expression levels of the fibrotic indexes COL-1 and α-SMA were significantly inhibited, and the high-dose group had the most obvious effect, suggesting that (+)-borneol could inhibit the cell fibrotic response caused by TGF-β1.

[0110] The immunofluorescence results were as follows Figure 13 shown. (+)-borneol significantly reduced the expression of α-SMA in TGF-β1-induced HK2 cells in a concentration-dependent manner, suggesting that (+)-borneol can effectively inhibit the cellular fibrotic response caused by TGF-β1.

[0111] The above experimental examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above experimental examples. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A compound for preventing or treating renal fibrosis, characterized in that, The structure of the said compound is shown in formula (I):

2. A pharmaceutical composition for preventing or treating renal fibrosis, characterized in that, The said pharmaceutical composition comprises the compound of formula (I) as claimed in claim 1 and a pharmaceutically acceptable excipient.

3. The pharmaceutical composition according to claim 2, wherein The said pharmaceutical composition further comprises other drugs for treating renal fibrosis.

4. The pharmaceutical composition according to claim 3, wherein The said other drugs for treating renal fibrosis are selected from one or more of pirfenidone, nintedanib, aliskiren, pentoxifylline, canagliflozin, and atrasentan.

5. The pharmaceutical composition according to claim 2, wherein The dosage form of the said pharmaceutical composition is tablet, granule, injection, pill or suspension.

6. Use of the compound of formula (I) as claimed in claim 1 or the pharmaceutical composition as claimed in any one of claims 2 - 5 in the preparation of a drug for preventing or treating renal fibrosis.

7. The application according to claim 6, wherein The said renal fibrosis is renal fibrosis caused by unilateral ureteral ligation.

8. Use of the compound of formula (I) as claimed in claim 1 or the pharmaceutical composition as claimed in any one of claims 2 - 5 in the preparation of a drug for treating chronic kidney disease.

9. Use of the compound of formula (I) as claimed in claim 1 or the pharmaceutical composition as claimed in any one of claims 2 - 5 in the preparation of a drug for treating renal tubular epithelial cell fibrosis.

10. Use of the compound of formula (I) according to claim 1 or the pharmaceutical composition according to any one of claims 2-5 in the preparation of a drug for reducing the expression of renal fibrosis indexes, characterized in that, The said renal fibrosis index is selected from one or more of tenascin C, fibronectin, type I collagen, and α - smooth muscle actin.

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