A method for preparing an anti-peritoneal fibrosis dialysis fluid containing intestinal alkaline phosphatase

Abstract

The present invention discloses an application of alkaline phosphatase in the preparation of a peritoneal fibrosis-preventing dialysis fluid. The present invention first proves that the application of alkaline phosphatase to prepare a peritoneal dialysis fluid with anti-peritoneal fibrosis can inhibit the infiltration of peritoneal macrophages and inhibit the macrophage-coordinated EMT process of peritoneal mesothelial cells, thereby playing a role in preventing and treating peritoneal fibrosis, and is expected to be applied clinically to improve the technical survival rate of peritoneal dialysis patients.

Description

Technical Field

[0001] The present invention relates to the technical field of peritoneal dialysis, and more particularly to a method for preparing an anti-peritoneal fibrosis dialysis fluid containing intestinal alkaline phosphatase. Background Art

[0002] Peritoneal dialysis is an effective renal replacement therapy for end-stage renal disease. Currently, approximately 11% of renal replacement therapy patients worldwide choose peritoneal dialysis. Over the past decade, the number of patients receiving peritoneal dialysis in my country has increased fourfold, making it one of the most important dialysis methods for patients with end-stage renal disease in my country.

[0003] Peritoneal dialysis relies on intact peritoneal structure and function. However, long-term peritoneal dialysis often causes changes in peritoneal morphology and function. Once it progresses to severe peritoneal fibrosis, patients will experience insufficient dialysis or ultrafiltration failure, leading to failure of peritoneal dialysis technology. The use of sugar-containing peritoneal dialysis fluid is an important factor promoting peritoneal fibrosis. At present, commercialized peritoneal dialysis fluids at home and abroad are all sugar-containing dialysis fluids. Therefore, blocking the progression of peritoneal dialysis-related peritoneal fibrosis through drug intervention is of great clinical significance for improving patients' long-term prognosis and improving the quality of peritoneal dialysis.

[0004] Inflammatory responses and epithelial-mesenchymal transition (EMT) of peritoneal mesothelial cells are two of the most important mechanisms in the pathogenesis of peritoneal fibrosis. EMT is primarily characterized by mesenchymal-like morphological changes in peritoneal mesothelial cells, enhanced migration ability, disrupted intercellular junctions, and a significant increase in extracellular matrix. Transforming growth factor β (TGF-β) is a key factor regulating the EMT process in peritoneal fibrosis. The inflammatory response is primarily mediated by activated peritoneal macrophages. Peritoneal macrophages primarily differentiate into M1 and M2 macrophages. M2 macrophages can secrete TGF-β1 and other factors to induce peritoneal fibrosis, while M1 macrophages can directly induce the transformation of peritoneal mesothelial cells into fibroblasts by activating the TLR4 signaling pathway. Sugar-containing dialysate during peritoneal dialysis can induce these inflammatory responses and EMT, leading to the development of peritoneal fibrosis.

[0005] Therefore, how to develop an ideal drug regimen to prevent and treat peritoneal fibrosis after peritoneal dialysis is a technical problem that urgently needs to be solved by people in this field. Summary of the Invention

[0006] In view of this, the object of the present invention is to provide an application of alkaline phosphatase in the preparation of dialysis fluid for preventing and treating peritoneal fibrosis after peritoneal dialysis, so as to address the deficiencies in the prior art.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The invention discloses an application of alkaline phosphatase in preparing dialysis fluid for preventing and treating peritoneal fibrosis after peritoneal dialysis.

[0009] Furthermore, the alkaline phosphatase is a tissue-specific alkaline phosphatase.

[0010] Furthermore, the tissue-specific alkaline phosphatase is intestinal alkaline phosphatase.

[0011] Alkaline phosphatase (ALP) is a monophospholipid hydrolase widely distributed in human tissues, including the liver, bone, intestine, kidney, and placenta. It consists of two subunits and contains four isoenzymes, which are primarily divided into tissue-nonspecific and tissue-specific ALPs. Tissue-nonspecific ALP is primarily expressed in the liver, kidney, and bone; tissue-specific ALPs include intestinal alkaline phosphatase (IAP), placental alkaline phosphatase, and germ cell alkaline phosphatase. ALP catalyzes the removal of 5'-phosphate groups from nucleic acids, converting the 5'-P termini of DNA or RNA fragments to 5'-OH termini. ALP exerts an anti-inflammatory effect by dephosphorylating pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide, thereby blocking macrophage activation via the TLR4 pathway. Furthermore, under conditions of hypoxia and inflammation, ALP converts ATP into the end product adenosine, which binds to A2A adenosine receptors on inflammatory cells such as macrophages, thereby suppressing inflammation and promoting tissue protection.

[0012] Clinical studies have found that intravenous administration of tissue-nonspecific ALP in the treatment of sepsis-related acute kidney injury improves renal function and significantly reduces inflammation. Intravenous ALP administration in patients undergoing coronary artery bypass grafting resulted in significantly lower levels of inflammatory cytokines (IL-6, IL-8, and TNF-α) compared to controls. Furthermore, recent research has shown that in transgenic rat models overexpressing IAP, intestinal absorption of LPS and lipids is reduced, thereby improving atherosclerosis. Another study found that IAP can repair aging-related intestinal barrier dysfunction, suppress serum levels of the inflammatory factors IL-6, IL-1β, and TNFα, improve metabolic status, and enhance survival in aged mice.

[0013] Therefore, based on the role of alkaline phosphatase in inhibiting the inflammatory response of macrophages, the application of alkaline phosphatase in the preparation of dialysis fluid for the prevention and treatment of peritoneal fibrosis may bring new hope for the prevention and treatment of peritoneal fibrosis after peritoneal dialysis.

[0014] ALP is currently in Phase II clinical trials for the treatment of acute kidney injury caused by severe sepsis. ALP has a molecular weight of 56 kDa and consists of two monomers, each composed of 449 amino acids. Each monomer has an active center, consisting of a triplet of aspartic acid (Asp) 101, serine (Ser) 102, and alanine (Ala) 103, arginine (Arg) 166, a water molecule, three metal ions, and its ligand amino acids. The complete ALP molecule exhibits an α / β topology. ALP is mostly excreted in the bile via the liver.

[0015] This invention innovatively adds alkaline phosphatase to conventional peritoneal dialysis fluid to create a dialysate with anti-peritoneal fibrosis properties. Animal experiments have confirmed that this novel dialysate significantly reduces peritoneal macrophage infiltration and peritoneal fibrosis in mice induced by high-glucose peritoneal dialysis fluid. These findings suggest that peritoneal dialysis fluid prepared with alkaline phosphatase can effectively prevent and treat peritoneal dialysis fibrosis.

[0016] It can be seen from the above technical solution that compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] 1. The present invention demonstrates for the first time that alkaline phosphatase can be used to prepare peritoneal dialysis fluid with anti-peritoneal fibrosis properties. This can inhibit the infiltration of peritoneal macrophages and the EMT process of macrophages in conjunction with peritoneal mesothelial cells, thereby playing a role in preventing and treating peritoneal fibrosis. This is expected to be used clinically to improve the technical survival rate of peritoneal dialysis patients.

[0018] 2. Alkaline phosphatase is an endogenous, synthesizable substance with good compatibility with finished peritoneal dialysis fluid and the human body, and has minimal side effects. When added to peritoneal dialysis fluid for local administration, only a small amount is absorbed into the bloodstream, with minimal systemic impact.

[0019] 3. The peritoneal dialysis solution containing alkaline phosphatase of the present invention is relatively simple to prepare and easy to use. Before use, the prepared alkaline phosphatase is added to the finished peritoneal dialysis solution and left in the peritoneal dialysis solution during peritoneal dialysis treatment without the need for additional oral administration or injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The curves show the time-dependent changes in alkaline phosphatase activity in peritoneal dialysis fluid containing alkaline phosphatase (A) and serum alkaline phosphatase activity in mice treated with peritoneal dialysis fluid containing alkaline phosphatase (B).

[0021] Figure 2 Comparison of serum alkaline phosphatase activity in mice treated with different drugs for 6 hours. Control: negative control group; Model: high glucose model group; IAP ip: alkaline phosphatase intraperitoneal infusion group; IAP ig: alkaline phosphatase gavage group.

[0022] Figure 3 Comparison of peritoneal pathological staining and fibrosis markers between mice treated with intraperitoneal perfusion of dialysate containing alkaline phosphatase and other groups. Control, negative control; Model, high glucose model; IAP ip, alkaline phosphatase intraperitoneal perfusion; IAP ig, alkaline phosphatase gavage.

[0023] Figure 4 Comparison of peritoneal macrophage immunofluorescence between mice treated with alkaline phosphatase-containing peritoneal dialysis solution and other groups. Control, negative control; Model, high glucose model; IAP ip, alkaline phosphatase intraperitoneal perfusion; IAP ig, alkaline phosphatase gavage. F4 / 80+CCR7+ cells are labeled as M1 macrophages; F4 / 80+CD206+ cells are labeled as M2 macrophages.

[0024] Figure 5 This study was conducted to investigate the effects of alkaline phosphatase on mesothelial cell morphology and fibrosis markers following 24-hour co-culture of M1 macrophages and mesothelial cells. LPS, lipopolysaccharide; IAP, intestinal alkaline phosphatase.

[0025] Figure 6 Figure 3 shows the inhibitory effect of alkaline phosphatase on the secretion of inflammatory cytokines IL-6 (A) and TNF-α (B) by macrophages stimulated by LPS. DETAILED DESCRIPTION

[0026] The following is a clear and complete description of 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 making any creative efforts are within the scope of protection of the present invention.

[0027] Example 1 The anti-peritoneal fibrosis effect of alkaline phosphatase was confirmed by animal experiments and its anti-fibrosis mechanism was confirmed by in vitro experiments

[0028] 1. Materials and Methods

[0029] 1) Sources of experimental materials

[0030] Tissue and blood alkaline phosphatase (AKP / ALP) activity detection kit Solebao Intestinal alkaline phosphatase Sigma α-tubμLin antibody, F4 / 80 antibody, CD206 antibody Cell Signaling Technology TGF-β1 antibody Abcam Collagen-I Antibody Southern Biotech α-SMA antibody Sigma Fibronectin antibody, E-Cadherin antibody BD Biosciences IL-6 and TNF-α ELISA kits R&D system company 4.25% peritoneal dialysis solution Baxter

[0031] 2) Preparation of anti-peritoneal fibrosis dialysate containing alkaline phosphatase

[0032] Intestinal alkaline phosphatase was dissolved in glycerol aqueous solution and added to 4.25% peritoneal dialysis fluid to prepare a peritoneal dialysis fluid containing alkaline phosphatase at a concentration of 150 U / mL. In animal experiments, it was freshly prepared before treatment.

[0033] 3) Animal Experiment: Mouse Peritoneal Fibrosis Model and Experimental Grouping

[0034] A peritoneal fibrosis model was established by intraperitoneal injection of 4.25% high-glucose peritoneal dialysis solution in SPF-grade C57BL / 6 mice. All animal experiments complied with the animal ethics requirements of Sun Yat-sen University.

[0035] ① High glucose model group (n=6): intraperitoneal injection of 100 mL / kg of 4.25% high glucose peritoneal dialysis solution, once a day for 28 days.

[0036] ② High glucose + IAP intraperitoneal perfusion group (n=6): intraperitoneal injection of 100 mL / kg of 4.25% high glucose peritoneal dialysis solution + 300 IUIAP, once a day for 28 days.

[0037] ③Negative control group (n=6): intraperitoneal injection of equal dose of normal saline once a day for 28 days.

[0038] 4) In vitro cell culture and treatment

[0039] ① Induction of human macrophage differentiation: THP-1 human monocytes were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS), 10 U / mL penicillin and 10 μg / mL streptomycin. 6 The cells were seeded at a density of 100 μg / mL in a six-well plate and cultured in serum-free RPMI1640 medium containing 200 ng / mL phorbol methyl ester (PMA) and 0.3% BSA for 72 h to induce differentiation into macrophages (M0). Then, 20 ng / mL IFN-γ + 100 ng / mL LPS were added to the M0 macrophages and cultured for 24 h to obtain M1 macrophages for the next experiment.

[0040] ② Identification of human macrophages:

[0041] I. Observation under a phase contrast microscope: After THP-1 cells differentiated into M0 macrophages, the cells adhered to the wall, increased in size, and extended their tentacles. The cell size of M1 macrophages increased further than that of M0 macrophages, and their tentacles extended.

[0042] II. Surface marker detection: Flow cytometry was used to detect M0 macrophage markers CD11b and CD14; qPCR was used to detect M1 macrophage markers iNOS, CXCL10, IL-6, and TNFα.

[0043] ③ Co-culture of human M1 macrophages and peritoneal mesothelial cell line (HMrSV5): M1 macrophages were seeded in six-well plates, and HMrSV5 cells were washed once with serum-free DMEM medium. 6The cells were directly added to the macrophage layer for co-culture; serum-free culture was performed for 24 h and the cells were divided into the following groups: LPS pretreatment group: 1 μg / mL LPS was added for 1 h, followed by glycerol aqueous solution; LPS+IAP intervention group: 1 μg / mL LPS was added for 1 h, followed by 50 IU / mL IAP (dissolved in glycerol aqueous solution); serum-free control group: serum-free DMEM medium was added, followed by glycerol aqueous solution.

[0044] The morphological changes of peritoneal mesothelial cells at different time points were observed under a microscope, and the cell supernatant and cells were collected for detection.

[0045] 5) Qualitative observation of peritoneal morphology and fibrosis

[0046] Masson staining: The peritoneal tissue was fixed in PLP solution for 4 h, dehydrated and paraffin-embedded as usual, the sections were dewaxed to water, immersed in Bouin's fixative at room temperature overnight, rinsed with running water to remove the yellow stain on the slides, stained with Weigert's ironwood solution for 5 min, rinsed with running water for 5 min, stained with Biebrich Scarlet acid solution for 5 min, washed with distilled water, stained with phosphomolybdic acid for 5 min, stained with aniline blue solution for 20 min, treated with 1% acetic acid solution for 2 min, dehydrated with gradient alcohol, transparentized with xylene, and mounted with neutral gum.

[0047] HE staining: fix, dehydrate, embed and slice, dewax to water and stain with Masson stain, stain with hematoxylin for 5 minutes, 1% hydrochloric acid alcohol for 20 seconds, rinse with tap water until the nucleus turns blue and the cytoplasm is not blue, stain with eosin for 1-2 minutes until the cytoplasm turns pink, rinse with tap water to remove excess red, rinse with distilled water, dehydrate in gradients, make transparent with xylene, and seal with neutral resin.

[0048] 6) Western Blot

[0049] Peritoneal tissue, about the size of a mung bean, was removed from a -80°C freezer and added to 200 μL of protein lysis buffer. The cells were ground in a grinder at 12,000 rpm for 15 minutes at 4°C. The supernatant was collected and the protein concentration was determined using a BCA assay. 2× loading buffer was added and the cells were boiled at 100°C for 5–10 minutes to denature the proteins. An 8%–16% gradient gel was loaded and electrophoresed. The cells were then wet-transferred to a PVDF membrane at 100V for 90 minutes. After transfer, the membrane was removed and washed twice in TBST for 10 minutes. Blocked with 5% skim milk for 1 hour at room temperature, washed twice in TBST for 10 minutes, and then incubated overnight at 4°C with the primary antibody dilution (1:1000). The cells were then washed twice in TBST for 10 minutes. The cells were then incubated with the secondary antibody dilution (1:5000) for 1 hour at room temperature, and then three times for 10 minutes. The membranes were exposed and analyzed using a ChemiScope 6200 Touch luminescence imaging system.

[0050] 7) Immunofluorescence detection

[0051] The sections were fixed, dehydrated, embedded, and deparaffinized until water was available for Masson staining. Antigen retrieval was performed in an autoclave using antigen retrieval solution for 8 hours. Blocking was performed with 3% BSA solution for 1 hour at room temperature. Antibodies were prepared in 1% BSA and diluted with primary antibody (1:50) in the solution. The sections were incubated in a humidified chamber at 4°C overnight. The sections were washed twice with PBS and diluted with fluorescent secondary antibody (1:500) in the solution for 1 hour at room temperature. The sections were washed three times with PBS and stained with DAPI (1:500) for 5 minutes. The sections were then washed twice with PBS. The sections were mounted with anti-quencher, air-dried in a fume hood, and stored at -80°C. Images were taken using a laser confocal microscope.

[0052] 8) Enzyme-linked immunosorbent assay

[0053] Enzyme-linked immunosorbent assay (ELSIA) was performed according to the manufacturer's recommended procedures and conditions.

[0054] 9) Statistical analysis

[0055] GraphPad Prism 7 and SPSS 25 were used for statistical analysis. One-way analysis of variance was used for comparison between groups, and P < 0.05 was considered statistically significant.

[0056] 2. Results

[0057] 1) Changes in alkaline phosphatase activity in the anti-peritoneal fibrosis dialysate prepared by the present invention and in vivo metabolism of alkaline phosphatase activity during treatment

[0058] The alkaline phosphatase-containing dialysate prepared by the present invention maintains stable enzyme activity, which is a prerequisite for its anti-peritoneal fibrosis effect. Before the experiment, the enzyme activity of the anti-peritoneal fibrosis dialysate prepared by the present invention was monitored, and the results were as follows: Figure 1 As shown in A. Figure 1 As shown in Figure 1, the alkaline phosphatase activity in the anti-peritoneal fibrosis peritoneal dialysis fluid prepared by the present invention can remain stable for up to 6-8 hours, and even at 24 hours, its activity can still be maintained at over 60%. In clinical treatment, patients using peritoneal dialysis fluid have a residence time of 4-6 hours per exchange of peritoneal dialysis fluid. Therefore, the stable alkaline phosphatase activity in the anti-peritoneal fibrosis dialysate prepared by the present invention can meet the clinical treatment time requirements.

[0059] The time-dependent changes in serum alkaline phosphatase activity during the peritoneal perfusion treatment of mice with the anti-peritoneal fibrosis peritoneal dialysis solution prepared by the present invention are as follows: Figure 1 As shown in B. Figure 1 As shown in Figure B, compared to the changes in enzyme activity in peritoneal dialysis fluid, serum alkaline phosphatase activity peaked at 2 hours and then rapidly declined, reaching only 2% of the enzyme activity in peritoneal dialysis fluid at 6 hours. Even at its peak at 2 hours, serum enzyme activity was only 15% of the enzyme activity in peritoneal dialysis fluid.

[0060] The serum alkaline phosphatase activity of the anti-peritoneal fibrosis peritoneal dialysis solution prepared by the present invention after 6 hours of peritoneal treatment was compared with that of other control groups. Figure 2 As shown. Figure 2 It can be seen that the alkaline phosphatase in the peritoneal dialysis solution prepared by the present invention is absorbed into the blood in small amounts and is rapidly metabolized. It can be inferred that its main effect is localized in the abdominal cavity, with little impact on the whole body, making it an ideal local abdominal treatment mode.

[0061] 2) The anti-peritoneal fibrosis dialysate prepared by the present invention inhibits the progression of peritoneal fibrosis in mice induced by high-glucose peritoneal dialysis fluid

[0062] The peritoneal fibrosis model of C57BL / 6 mice was successfully established after 4 weeks of intraperitoneal injection of 2 mL of 4.25% high glucose peritoneal dialysis solution into mice. Figure 3 As shown. Figure 3 HE and Masson staining revealed significant thickening of the parietal subperitoneal region, increased fibrous tissue, and extensive inflammatory cell infiltration in the high-glucose model group. Compared with the high-glucose model group, mice treated with the anti-peritoneal fibrosis dialysate prepared by the present invention (IAP intraperitoneal infusion group) showed significantly less peritoneal thickness, fibrosis, and inflammatory infiltration. The expression of TGF-β1 and Collagen-I, indicators of peritoneal fibrosis, was lower than in the high-glucose model group, while E-cadherin, a marker of epithelial integrity, was significantly higher in the high-glucose model group. These results demonstrate that intraperitoneal infusion of the alkaline phosphatase-containing peritoneal dialysis fluid prepared by the present invention effectively inhibits the EMT process of peritoneal mesothelial cells in mice induced by high-glucose peritoneal dialysis fluid, exerting an anti-peritoneal fibrosis effect.

[0063] 3) Intraperitoneal infusion of IAP inhibits high-glucose peritoneal dialysis solution-induced peritoneal macrophage infiltration in mice

[0064] Peritoneal macrophage infiltration and the inflammatory response it mediates are important steps in the process of peritoneal fibrosis. In the above animal model, immunofluorescence was used to detect the infiltration of macrophages in the visceral peritoneum of each group of mice. Figure 4 As shown. Figure 4 It can be seen that the M1 type (F4 / 80+ CCR7+) macrophages in the peritoneal tissue of mice in the intraperitoneal treatment group (IAP intraperitoneal perfusion group) using the anti-peritoneal fibrosis dialysate prepared by the present invention ( Figure 4 A) and M2 (F4 / 80+ CD206+) macrophages ( Figure 4 The infiltration of B cells in the high-glucose model group was significantly reduced. These results demonstrate that intraperitoneal perfusion of IAP can inhibit peritoneal macrophage infiltration in mice. Therefore, the inhibitory effect of the dialysate prepared in this invention on peritoneal macrophage infiltration can mechanistically demonstrate the important pharmacological effect of this product in preventing peritoneal fibrosis.

[0065] 4) In vitro experiments show that alkaline phosphatase inhibits the EMT process of peritoneal mesothelial cells induced by M1 macrophages

[0066] Previous studies have found that when M1 macrophages are co-cultured with peritoneal mesothelial cells, they can induce EMT changes in peritoneal mesothelial cells and upregulate the expression of peritoneal fibrosis-related markers. Therefore, the present invention further observed the effect of intestinal alkaline phosphatase on the co-culture of human M1 macrophages and human peritoneal mesothelial cells in an in vitro cell experiment. The results are as follows Figure 5 As shown. Figure 5 As shown, after 24 hours of co-culture, most peritoneal mesothelial cells in the negative control group and LPS stimulation group showed obvious fibroblast-like morphological changes, while the peritoneal mesothelial cells in the alkaline phosphatase treatment group showed no obvious changes, and peritoneal fibrosis markers (TGF-β1 and Fibronectin) were significantly downregulated. These results indicate that alkaline phosphatase can inhibit the EMT process of peritoneal mesothelial cells assisted by M1 macrophages.

[0067] 5) Alkaline phosphatase inhibits the secretion of inflammatory cytokines by LPS-stimulated macrophages

[0068] Studies have shown that inflammatory cytokines, especially IL-6, play an important role in the progression of peritoneal fibrosis. In vitro cell experiments of the present invention found that the secretion of IL-6 and TNF-α inflammatory cytokines by macrophages in the alkaline phosphatase treatment group was significantly lower than that in the LPS stimulation group ( Figure 6 A and Figure 6 This result indicates that alkaline phosphatase can inhibit the secretion of inflammatory cytokines by macrophages, and may further exert its anti-peritoneal fibrosis effect through this mechanism.

[0069] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing an anti-peritoneal fibrosis dialysis fluid containing intestinal alkaline phosphatase, characterized in that: The specific steps include: Intestinal alkaline phosphatase was dissolved in glycerol aqueous solution and then added to 4.25% peritoneal dialysis fluid to prepare an anti-peritoneal fibrosis dialysis fluid containing intestinal alkaline phosphatase at a concentration of 150 U / mL.

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