Application of recombinant ITLN1 protein in preparation of products for treating renal interstitial fibrosis

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses application of recombinant ITLN1 protein in preparation of a product for treating renal interstitial fibrosis. The invention discloses application of ITLN1 in treatment of renal interstitial fibrosis, ITLN1 can activate AMPK so as to increase fatty acid oxidation and relieve renal interstitial fibrosis, and a new theoretical basis and strategy are provided for clinical treatment of chronic kidney diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the application of recombinant ITLN1 protein in the preparation of a product for treating renal interstitial fibrosis. Background Art

[0002] Renal interstitial fibrosis is a common pathway for chronic kidney disease caused by various reasons to progress to end-stage renal disease and is the main factor affecting the progression of chronic kidney disease. Therefore, alleviating the development of renal interstitial fibrosis may be an effective measure to delay the progression of chronic kidney disease. Currently, there is no effective treatment for renal interstitial fibrosis.

[0003] Intrinsic lectin-1 (ITLN1) is a novel adipokine in the same family as leptin and adiponectin, with a full length of 313 amino acids and a molecular weight of 34Kda. ITLN1 can regulate inflammation, vasomotion and endothelial function, cell proliferation, apoptosis and cell differentiation, and has been reported to be an effective protective factor in many diseases. However, the role of ITLN1 in renal interstitial fibrosis has not been reported. Summary of the invention

[0004] The purpose of the present invention is to provide the use of recombinant ITLN1 protein in the preparation of a product for treating renal interstitial fibrosis, so as to solve the above technical problems.

[0005] The present invention provides use of a recombinant ITLN1 protein in treating renal interstitial fibrosis. The amino acid sequence of the ITLN1 protein is SEQ ID NO: 2; and the nucleotide sequence is SEQ ID NO: 1.

[0006] Preferably, the recombinant ITLN1 protein is injected at a concentration of 0.15 μg / g body weight.

[0007] Preferably, the product contains recombinant ITLN1 protein as the only active ingredient.

[0008] Preferably, the recombinant ITLN1 protein can improve pathological changes such as renal tubular damage and interstitial fibrosis deposition caused by unilateral ureteral obstruction and folic acid nephropathy.

[0009] Preferably, the recombinant ITLN1 protein can reduce the expression of fibrosis markers Vimentin and Collagen I in unilateral ureteral obstruction and folate nephropathy group.

[0010] Preferably, the recombinant ITLN1 protein has a protective effect on cell fibrosis phenotype.

[0011] The present invention also relates to the use of the recombinant ITLN1 protein in treating a renal interstitial fibrosis model caused by unilateral ureteral obstruction (UUO) and folic acid nephropathy (FAN).

[0012] The present invention discloses the application of ITLN1 in the preparation and treatment of renal interstitial fibrosis. ITLN1 can relieve renal interstitial fibrosis by activating AMPK and thereby increasing fatty acid oxidation, thereby providing a new theoretical basis and strategy for the clinical treatment of chronic kidney disease and a new direction for the clinical treatment of chronic kidney disease.

[0013] In the mouse models of renal interstitial fibrosis caused by unilateral ureteral obstruction (UUO) and folic acid nephropathy (FAN), the degree of renal interstitial fibrosis in mice was improved by tail vein injection of recombinant ITLN1 protein, providing a potential therapeutic option and new ideas for the prevention and treatment of renal interstitial fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0015] Figure 1 HE staining, PAS staining, Masson staining, and Sirius red staining of renal tissues of mice in each group of the UUO model and FAN model of the present invention.

[0016] Figure 2 The immunohistochemical images of vimentin and Coa I staining in the renal tissues of mice in each group of the UUO model and the FAN model of the present invention are shown.

[0017] Figure 3 The expression levels of fibrosis-related proteins such as FN, vimentin, and α-SMA detected by Western blot in the renal tissues of mice in each group of the UUO model and the FAN model in the present invention.

[0018] Figure 4 The expression levels of AMPK, p-AMPK, ACC1, p-ACC1, CPT1a and other related pathway proteins detected by Western blot in the renal tissues of each group of mice in the UUO model and FAN model of the present invention.

[0019] Figure 5 After HK2 cells were given recombinant ITLN1 protein, the expression of various fibrosis proteins and pathway proteins was detected by Western blot.

[0020] Figure 6 After the HK2 cells of the present invention overexpressed ITLN1, the expression levels of various fibrosis proteins and various pathway proteins were detected by Western blot.

[0021] Figure 7 After the AMPK inhibitor BML-275 and the recombinant ITLN1 protein were administered to the HK2 cells of the present invention, the expression levels of the proteins were detected by Western blot.

[0022] Figure 8 According to the present invention, BML-275 and recombinant ITLN1 protein were added to the HK2 cell line overexpressing ITLN1, and the expression level of each protein was detected by Western blot. DETAILED DESCRIPTION

[0023] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only 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 creative work are within the scope of protection of the present invention.

[0024] 1. Experimental Animals

[0025] The experimental animals were healthy C57BL / 6J male mice of 6-8 weeks old and weighing 20-24 g, purchased from Zhuhai Baishitong Biotechnology Co., Ltd., SPF grade, and raised in the SPF Grade Animal Experiment Center of Jinan University.

[0026] Animals were kept in an environment with a temperature of 20-25°C, a humidity of 45-60%, and a 12-hour day-night cycle, with free access to water and food. The bedding was changed regularly every week to keep the living environment clean.

[0027] 2. Solution Preparation

[0028] Preparation of recombinant human ITLN1 protein solution: 100 μg of recombinant ITLN1 protein was added to 10 ml PBS to prepare an ITLN1 solution with a final concentration of 10 μg / ml. After mixing, the solution was dispensed into 1.5 ml EP tubes and stored at -20°C for later use.

[0029] Preparation of folic acid solution: Dissolve folic acid in 0.3 mol / L sodium bicarbonate solution. The solvent of the sodium bicarbonate solution is PBS.

[0030] 3. Establishment of unilateral ureteral obstruction (UUO) model in mice

[0031] (1) Mice were anesthetized by airway with an initial concentration of 2% isoflurane, maintained by inhalation of a concentration of 0.5-1%. The mice were fixed on their backs on a small animal heating pad, the skin of the left lower abdomen was disinfected, and the left ureter was found by incision layer by layer. The ureteropelvic transition and the middle section of the ureter were ligated respectively, and finally the peritoneum and skin were sutured in sequence.

[0032] The modeling method is as follows: before surgery, the surgical instruments are sterilized and dried under high pressure for standby use. The gas anesthesia machine adjusts the isoflurane to an initial concentration of 2% to anesthetize the mice. The operation is maintained by mask inhalation at a concentration of 0.5-1%. During the operation, the changes in the respiratory rhythm of the mice are closely monitored to adjust the inhalation concentration of isoflurane in time. After the anesthesia takes effect, the mice are placed on a small animal constant temperature heating pad covered with sterile surgical towels, the skin is prepared, the left lower abdomen is disinfected with 75% alcohol, the skin and subcutaneous tissue are cut layer by layer in a relatively sterile environment, and the left intestine is turned to the opposite side. At this time, a white, thin, linear structure can be seen, which is the left ureter. The renal pelvis is found along the ureter. The ureter is ligated with a 5-0 surgical thread at a position 0.5 cm away from the transition between the renal pelvis and ureter, and the ureter is ligated again at 0.5-1 cm distal to the ligation point, and the ureter is cut between the two ligation points. In the sham operation group, only the left ureter is separated without ligation. Finally, restore the position of the intestine, close the abdominal cavity layer by layer, suture the subcutaneous tissue and skin, cover the surgical site with disinfected and sterile gauze, and place the mouse in a clean mouse cage to wait for it to wake up.

[0033] (2) Grouping

[0034] Sham operation group (Sham): only the ureter was separated without ligation, and the time from model establishment to sacrifice was 2 weeks in total.

[0035] Model group (UUO): One week after surgical ligation of the left ureter, 100 μl of normal saline was injected into the tail vein for 4 consecutive days, and the time from the beginning of modeling to sacrifice was 2 weeks in total.

[0036] Treatment group (UUO+ITLN1): One week after surgical ligation of the left ureter, the recombinant human ITLN1 protein was injected into the tail vein at a rate of 0.15 μg / g mouse body weight for 4 consecutive days. The time from the beginning of modeling to sacrifice was 2 weeks in total.

[0037] There were 4 mice in each group.

[0038] After 2 weeks, the mice were killed and the left kidney tissue was recovered. The kidney tissue capsule was removed and separated into four parts. One quarter was stored in 4% paraformaldehyde for fixation and used for embedding and sectioning. H&E, Masson, PAS and Sirius red staining and immunohistochemical staining were performed, and the remaining three quarters were frozen in a -80℃ refrigerator for future use. It was determined whether ITLN1 has a protective effect on renal interstitial fibrosis.

[0039] 4. Construction of Folic acid nephropathy (FAN) model

[0040] (1) A single intraperitoneal injection of folic acid at a dose of 250 μg / g mouse body weight.

[0041] (2) The folic acid dose was calculated based on 250 μg / g of mouse body weight. A single intraperitoneal injection of folic acid solution was performed. The total modeling time was calculated from the first day of injection to the end of the third week.

[0042] (3) Grouping

[0043] The control group (Control) did not receive any treatment, and the time of sacrifice from the beginning of modeling was 3 weeks in total.

[0044] Model group (FAN): Two weeks after a single intraperitoneal injection of folic acid, 100 μl of normal saline was injected into the tail vein for 4 consecutive days. The time from the beginning of modeling to sacrifice was 3 weeks in total.

[0045] Treatment group (FAN+ITLN1); 2 weeks after a single intraperitoneal injection of folic acid, human recombinant ITLN1 protein was injected into the tail vein for 4 consecutive days for a total of 3 weeks.

[0046] There were 4 mice in each group.

[0047] After 3 weeks, the mice were killed and bilateral renal tissues were recovered. The renal tissue capsule was removed and separated into four parts. One quarter was stored in 4% paraformaldehyde for fixation and used for embedding and sectioning. H&E, Masson, PAS and Sirius red staining and immunohistochemical staining were performed, and the remaining three quarters were frozen in a -80℃ refrigerator for future use. It was determined whether ITLN1 has a protective effect on renal interstitial fibrosis.

[0048] 5. Preparation of Kidney Pathological Sections

[0049] Mouse kidney tissue was fixed in 4% paraformaldehyde solution for 48 hours, and then the kidney tissue blocks were placed in 75%, 95%, and 100% ethanol solutions in turn, and each solution was placed twice, each time for 15 minutes. The obtained kidney tissue blocks were then placed in anhydrous ethanol:xylene solution of 1:1 for 10 minutes, xylene I for 10 minutes, and xylene II for 7 minutes, and then paraffinized and embedded.

[0050] The renal tissue blocks obtained in the above steps were placed in paraffin I (60°C) for 1 hour, paraffin II (60°C) for 1 hour, and paraffin III (60°C) for 1 hour. The paraffin blocks were placed on a freezing table for 5-10 minutes, cut into slices of about 3-4 μm using a microtome, and finally the obtained slices were placed in a warm water bath at about 44°C, and then baked in a 60°C oven for 2 hours.

[0051] 6. Staining

[0052] (1) Hematoxylin-Eosin (H&E) staining

[0053] Place the paraffin sections in xylene I for 20 minutes, xylene II for 20 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 95% alcohol, 90% alcohol, 80% alcohol and 70% alcohol for 5 minutes each, and rinse with tap water to dewax. Then put the sections in hematoxylin stain for 3-5 minutes, wash with tap water for 1-2 minutes until the color of the sections gradually fades, differentiate with 1% hydrochloric acid alcohol for 10 seconds, then quickly wash with tap water for 1-2 minutes, 1% ammonia water for blueing, and rinse with tap water again for 1 minute. Then put the sections in eosin stain for 5 minutes, and rinse with tap water for 1 minute. Finally, put the sections in 95% alcohol I for 5 minutes, 95% alcohol II for 5 minutes, anhydrous ethanol I, anhydrous ethanol II and anhydrous ethanol III for 5 minutes each, and then put them in xylene I and xylene II for 5 minutes each to make them transparent, and seal them with neutral gum.

[0054] (2) Periodic Acid-Schiff staining (PAS)

[0055] The paraffin sections were dewaxed and hydrated. The specific steps were as follows: the paraffin sections were placed in xylene I for 20 minutes, xylene II for 20 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 95% alcohol, 90% alcohol, 80% alcohol and 70% alcohol for 5 minutes each, and then rinsed with tap water for dewaxing. The sections were then placed in hematoxylin stain for 3-5 minutes, washed with tap water for 1-2 minutes until the color of the sections gradually faded, differentiated with 1% hydrochloric acid alcohol for 10 seconds, then quickly washed with tap water for 1-2 minutes, 1% ammonia water for blueing, and rinsed with tap water again for 1 minute. The sections were then placed in eosin stain for 5 minutes and rinsed with tap water for 1 minute. Finally, the sections were placed in 95% alcohol I for 5 minutes, 95% alcohol II for 5 minutes, anhydrous ethanol I, anhydrous ethanol II and anhydrous ethanol III for 5 minutes each, and then placed in xylene I and xylene II for 5 minutes each to make them transparent, and sealed with neutral gum.

[0056] Then, the paraffin sections were placed in the Chevron stain for 30 minutes, protected from light, and rinsed with running water for 5 minutes; the sections were placed in the hematoxylin stain for 3-5 minutes, washed with tap water, differentiated with differentiation solution, washed with tap water, blued with blue solution, and rinsed with running water. The sections were sequentially placed in anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, anhydrous ethanol III for 5 minutes, xylene for 5 minutes, and xylene II for 5 minutes to make them transparent, and sealed with neutral gum.

[0057] (3) Masson staining

[0058] Dewax and hydrate the paraffin sections. Specifically: Place the paraffin sections in xylene I for 20 minutes, xylene II for 20 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 95% alcohol, 90% alcohol, 80% alcohol and 70% alcohol for 5 minutes each, and rinse with tap water to dewax. Then put the sections in hematoxylin stain for 3-5 minutes, wash with tap water for 1-2 minutes until the color of the sections gradually fades, differentiate with 1% hydrochloric acid alcohol for 10 seconds, then quickly wash with tap water for 1-2 minutes, 1% ammonia water for blueing, and rinse with tap water again for 1 minute. Then put the sections in eosin stain for 5 minutes and rinse with tap water for 1 minute. Finally, put the sections in 95% alcohol I for 5 minutes, 95% alcohol II for 5 minutes, anhydrous ethanol I, anhydrous ethanol II and anhydrous ethanol III for 5 minutes each, then put them in xylene I and xylene II for 5 minutes each to make them transparent, and seal them with neutral gum.

[0059] Subsequently, the paraffin sections were soaked in potassium dichromate solution overnight and rinsed with tap water. Then, iron hematoxylin A solution and B solution were mixed in equal proportions to form iron hematoxylin dye solution, and the sections were placed in the iron hematoxylin dye solution for 3 minutes, rinsed with tap water, differentiated with differentiation solution, rinsed with tap water, and then blued with blue-returning solution, and rinsed with running tap water. Subsequently, the sections were immersed in Ponceau acid fuchsin for 5-10 minutes and rinsed with tap water. Then, the sections were immersed in phosphomolybdic acid aqueous solution for 1-3 minutes. After phosphomolybdic acid, no water was needed to wash, but they were directly placed in aniline blue dye solution for 3-6 minutes. The sections were differentiated with 1% glacial acetic acid and dehydrated with two cylinders of anhydrous ethanol. Finally, the sections were placed in a third cylinder of anhydrous ethanol for 5 minutes, transparent in xylene for 5 minutes, and sealed with neutral gum.

[0060] (4) Sirius Red staining

[0061] Paraffin sections are dewaxed and hydrated. Specifically, paraffin sections are placed in xylene I for 20 minutes, xylene II for 20 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 95% alcohol, 90% alcohol, 80% alcohol and 70% alcohol for 5 minutes each, and dewaxed with tap water. Then the sections are placed in hematoxylin stain for 3-5 minutes, washed with tap water for 1-2 minutes until the color of the sections gradually fades, differentiated with 1% hydrochloric acid alcohol for 10 seconds, then quickly washed with tap water for 1-2 minutes, 1% ammonia water for blueing, and rinsed with tap water again for 1 minute. Then the sections are placed in eosin stain for 5 minutes and rinsed with tap water for 1 minute. Finally, the sections are placed in 95% alcohol I for 5 minutes, 95% alcohol II for 5 minutes, anhydrous ethanol I, anhydrous ethanol II and anhydrous ethanol III for 5 minutes each, and then placed in xylene I and xylene II for 5 minutes each to make them transparent, and sealed with neutral gum.

[0062] Then, the slices were stained in Sirius red dye for 8-10 minutes, dehydrated in two or three cylinders of anhydrous ethanol, and then placed in clean xylene for 5 minutes to make it transparent, and sealed with neutral gum.

[0063] (5) Staining results

[0064] H&E, PAS, Masson staining and Sirius red staining Figure 1 The results showed that the infiltration of inflammatory cells in the kidney tissue of the mice in the UUO group increased, and the tubular epithelial cells showed shrinkage, vacuolar degeneration, and tubular dilation, and other changes of tubular injury (H&E staining, PAS staining), and the accumulation of blue / red collagen fiber components in the renal interstitium (Masson staining, Sirius red staining). Compared with the Shan group (control group), the infiltration of inflammatory cells in the kidney tissue of the mice in the UUO group increased, and the tubular epithelial cells showed shrinkage, vacuolar degeneration, and tubular dilation, and other changes of tubular injury (H&E staining, PAS staining), Masson staining showed increased blue fiber deposition in the renal interstitium, and Sirius red staining showed increased accumulation of red collagen fibers. In the UUO+ITTL1 group with the addition of ITLN1 protein, the infiltration of inflammatory cells in the kidney tissue and the shrinkage, vacuolar degeneration, and tubular dilation of the tubular epithelial cells in the mice were alleviated compared with the UUO group, and the deposition of fibrin in the renal interstitium was also reduced compared with the UUO group.

[0065] It can be concluded that ITLN1 intervention can improve pathological changes such as renal tubular damage and interstitial fibrosis deposition caused by UUO.

[0066] 7. Immunohistochemistry (IHC)

[0067] Paraffin sections are routinely dewaxed and hydrated. Specifically, the paraffin sections are placed in xylene I for 20 minutes, xylene II for 20 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 95% alcohol, 90% alcohol, 80% alcohol and 70% alcohol for 5 minutes each, and dewaxed with tap water. Then the sections are placed in hematoxylin stain for 3-5 minutes, washed with tap water for 1-2 minutes until the color of the sections gradually fades, differentiated with 1% hydrochloric acid alcohol for 10 seconds, then quickly washed with tap water for 1-2 minutes, 1% ammonia water for blueing, and rinsed with tap water again for 1 minute. Then the sections are placed in eosin stain for 5 minutes and rinsed with tap water for 1 minute. Finally, the sections are placed in 95% alcohol I for 5 minutes, 95% alcohol II for 5 minutes, anhydrous ethanol I, anhydrous ethanol II and anhydrous ethanol III for 5 minutes each, and then placed in xylene I and xylene II for 5 minutes each to make them transparent, and sealed with neutral gum.

[0068] Subsequently, the tissue sections were placed in a repair box filled with citric acid antigen repair buffer (pH 6.0) in a microwave oven for antigen repair. The sections were heated for 8 minutes at medium heat, stopped for 8 minutes, and then heated for 7 minutes at medium-low heat. During this process, the buffer should be prevented from evaporating excessively and the sections should not be dried. After natural cooling, the slides were placed in PBS (pH 7.4) and washed on a decolorizing shaker for 3 times, 5 minutes each time. After the sections were slightly dried, a circle was drawn around the tissue with a tissue pen (to prevent the antibody from flowing away). Then 3% BSA was added to the circle and incubated at room temperature for 30 minutes to block. After blocking, the blocking solution was gently shaken off, and the primary antibodies Vimentin (Shenyang Wanlei Biology) and Collagen I (Proteintech) prepared in a certain ratio were added to the sections. The sections were placed flat in a humidified box and incubated overnight at 4°C (a small amount of water was added to the humidified box to prevent the antibody from evaporating). After the primary antibody incubation was completed, the slides were placed in PBS (pH 7.4) and washed on a decolorizing shaker for 3 times, 5 minutes each time. After the slices are slightly dried, add the secondary antibody of the corresponding species to the primary antibody in the circle and cover it, then add HRP-labeled goat anti-rabbit secondary antibody (Proteintech) and incubate at room temperature in the dark for 50 minutes. After incubation with the secondary antibody, place the slide in PBS (pH7.4) and wash it 3 times on a decolorizing shaker, shaking for 5 minutes each time. After the slices are slightly dried, add DAB color developer in the circle and control the color development under a microscope. After the color development is complete, rinse with distilled water or tap water, counterstain with hematoxylin, differentiate with 1% hydrochloric acid alcohol (about 1s), rinse with tap water, turn blue with ammonia water, and rinse with running water. Finally, put the slices in 75% alcohol for 5 minutes-85% alcohol for 5 minutes--anhydrous ethanol I for 5 minutes-anhydrous ethanol II for 5 minutes-xylene I for 5 minutes to dehydrate and make them transparent, then take them out of xylene to dry slightly and seal them with neutral gum.

[0069] Results Figure 2 The results showed that ITLN1 reduced the expression of fibrotic proteins Vimentin and CollagenI in the renal tissue of UUO and FAN mice. In the UUO model, compared with the Sham group, the expression of Vimentin and CollagenI in the UUO group increased significantly, while the expression of Vimentin and CollagenI in the UUO+ITLN1 group decreased compared with that in the UUO group; in the FAN group, compared with the Control group, the expression of Vimentin and CollagenI in the FAN group increased significantly compared with that in the FAN group, while the expression of Vimentin and CollagenI in the FAN+ITLN1 group decreased compared with that in the FAN group. It can be seen that ITLN1 reduced the expression of fibrotic markers Vimentin and CollagenI in the UUO and FAN groups.

[0070] 8. Western blot

[0071] Prepare gel according to the required gel concentration. Place the two prepared gel plates in a vertical electrophoresis tank, add freshly prepared 1×SDS-PAGE electrophoresis buffer between the gels, pull out the electrophoresis comb, add protein markers and protein samples to each well in a certain sample loading order, and perform electrophoresis on the concentrated gel at a constant voltage of 80V, then change the voltage to 120V and continue electrophoresis. Place the cut PVDF membrane in anhydrous ethanol to activate it thoroughly. After the electrophoresis is completed, take out the glass plate, soak the PVDF membrane and glass plate in the pre-cooled 1× transfer buffer for 15 minutes, place the "sandwich" structure made of sponge pad, filter paper, gel, PVDF membrane, filter paper, and sponge pad, and transfer the membrane at a constant current of 400mA for 30 minutes. Finally, incubate the membrane in primary antibodies of different concentrations at 4°C overnight.

[0072] Western blot antibody information: primary antibodies were purchased from Proteintech; Fibronectin, CollagenI, α-SMA, AMPK, p-AMPK, p-ACC1, ACC1, CPT1a, ITLN1, β-actin (mouse), and Vimentin were purchased from Wanle Biotechnology.

[0073] Western blot results Figure 3 :ITLN1 can reduce the expression of fibrotic proteins Fibronectin, Vimentin, and α-SMA in the renal tissues of mice in the UUO and FAN groups, and alleviate renal interstitial fibrosis in mice. Figure 4 As shown, ITLN1 can also increase the expression of p-AMPK and p-ACC1 in the renal tissue of UUO model and FAN model mice, increase the expression of CPT1a, and increase fatty acid oxidation.

[0074] 9. Detection of fibrosis indicators and pathway-related indicators

[0075] (1) HK2 cells (Fenghui Bio) in the logarithmic growth phase were collected. The cells were starved for 12 h the night before the addition of TGFβ1 recombinant protein. The recombinant human ITLN1 protein was added 2 h after the addition of TGFβ1 the next day. Cell proteins were collected 24 h later for Western blot detection of fibrosis indicators and pathway-related indicators. 200 μl RIPA lysis buffer and 20 μl protease phosphatase inhibitors were added to the collected cells. The collected cells were centrifuged at 4°C and 14000 rpm / min for 5 minutes. The cell supernatant was collected for BCA protein concentration determination and Western blot detection.

[0076] The results are as follows Figure 5As shown in the results, after adding recombinant ITLN1 protein, the expressions of Fibronectin, Vimentin and α-SMA in HK2 cells under TGFβ1 stimulation were significantly lower than those in the TGFβ1 stimulation group, while the expressions of p-AMPK, p-ACC1 and CPT1a were also significantly higher than those in the TGFβ1 stimulation group. ITLN1 can reduce the expression of fibrosis indicators in fibrotic cells, and increase the expressions of p-AMPK and p-ACC1.

[0077] The results are as follows Figure 6 As shown in the figure, after adding TGFβ1, the expression of cell fibrosis proteins in HK2 cells overexpressing ITLN1 (ITLN1 plasmid was purchased from Saiguo Bio) was significantly reduced compared with the parental and PLV.NC groups, while the expression of p-AMPK, p-ACC1 and CPT1a increased significantly. This shows that endogenous increase in ITLN1 expression can also reduce the expression of fibrosis-related proteins in HK2 cells stimulated by TGFβ1, while increasing the expression of fatty acid oxidation rate-limiting enzymes.

[0078] (3) HK2 cells were cultured in the same manner as in Example 5. After adding the recombinant human ITLN1 protein, the AMPK inhibitor BML-275 was added. After 24 hours, cell proteins were collected for Western blot detection of fibrosis indicators and pathway-related indicators. The Western blot method and antibody information were the same as in the animal experiment section.

[0079] The results are as follows Figure 7 As shown in the data, in the BML-275 group, the expressions of fibrosis proteins such as Fibronectin, Vimentin, and α-SMA were significantly lower than those in the TGFβ1+ITLN1 group. At the same time, the expressions of p-AMPK, p-ACC1, and CPT1a were also significantly lower than those in the TGFβ1+ITLN1 group, indicating that BML-275 weakened the protective effect of ITLN1 on cell fibrosis and reduced the ability of ITLN1 to activate AMPK and ACC1 phosphorylation.

[0080] (4) After HK2 cells overexpressed ITLN1, BML-275 was added 2 hours after the addition of TGFβ1 recombinant protein. Cell samples were collected 24 hours later to extract cell proteins for Western blot detection of fibrosis indicators and pathway-related indicators. The Western blot method and antibody information were the same as those in the animal experiment section.

[0081] The results are as follows Figure 8As shown in the figure, after adding TGFβ1 and BML-275, the expression of cell fibrosis proteins in HK2 cells overexpressing ITLN1 did not change significantly compared with the parental+TGFβ1 and PLV.NC+TGFβ1 groups, and the expression of p-AMPK and p-ACC1 did not increase significantly, and the expression of CPT1a did not change significantly, indicating that BML-275 reduced the protective effect of the increase of endogenous ITLN1 on the cell fibrosis phenotype.

[0082] The preferred specific embodiments of the present invention are described in detail above. It should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, any technical solution that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. Use of recombinant ITLN1 protein in the preparation of a product for treating renal interstitial fibrosis, characterized in that: The amino acid sequence of the ITLN1 protein is SEQ ID NO:

2.

2. Use of the recombinant ITLN1 protein according to claim 1 in preparing a product for treating renal interstitial fibrosis, characterized in that: The recombinant ITLN1 protein was injected at a concentration of 0.15 μg / g body weight.

3. Use of the recombinant ITLN1 protein according to claim 1 in preparing a product for treating renal interstitial fibrosis, characterized in that: The product contains recombinant ITLN1 protein as the only active ingredient.

4. Use of the recombinant ITLN1 protein according to claim 1 in preparing a product for treating renal interstitial fibrosis, characterized in that: The recombinant ITLN1 protein can improve pathological changes such as renal tubular damage and interstitial fibrosis deposition caused by unilateral ureteral obstruction and folic acid nephropathy.

5. Use of the recombinant ITLN1 protein according to claim 1 in preparing a product for treating renal interstitial fibrosis, characterized in that: The recombinant ITLN1 protein can reduce the expression of fibrosis markers Vimentin and Collagen I in unilateral ureteral obstruction and folic acid nephropathy groups.

6. Use of the recombinant ITLN1 protein according to claim 1 in preparing a product for treating renal interstitial fibrosis, characterized in that: The recombinant ITLN1 protein has a protective effect on cell fibrosis phenotype.

7. Use of the recombinant ITLN1 protein according to claim 1 in preparing a product for treating renal interstitial fibrosis, characterized in that: The recombinant ITLN1 protein can reduce the expression of fibrosis indicators Fibronectin, Vimentin, and α-SMA in fibrotic cells, and can also increase the expression of p-AMPK, p-ACC1, and CPT1a, thereby increasing fatty acid oxidation.

8. Use of recombinant ITLN1 protein in treating renal interstitial fibrosis in a renal interstitial fibrosis model, characterized in that: The model is caused by unilateral ureteral obstruction and folate nephropathy.

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