PD aggravated AKI pathological process verification method

Abstract

The invention relates to the field of periodontitis pathology exploration, and discloses a PD aggravated AKI pathology process verification method. Directly verifying that accumulation of the cells in the kidney aggravates the pathological process of the initial stage of AKI during PD through a macrophage clearing or refilling means; by means of means such as a transgenic mouse of which the CSF-1 is specifically knocked out in the renal tubular epithelial cells, receptor closing and the like, kidney macrophage accumulation during PD mediation of the CSF-1 from the renal tubular epithelial cells is further proved; mMP8 plays a bridge role in connecting'mouth-kidney ', so that it is emphasized to verify that increase of MMP-8 in serum during PD is a key medium for promoting renal tubular epithelial cells to secrete CSF-1, and specific mechanisms such as an action mode, a receptor and an intracellular signal channel are disclosed by means of an IP-MS technology, transcriptome sequencing and the like; the invention solves the problems that the pathological process is significantly aggravated when the existing PD mouse induces AKI, and there is no experimental evidence for the pathological process in the initial stage of AKI aggravated by kidney macrophage accumulation and macrophage activation.

Description

Technical Field

[0001] The present invention relates to the field of pathological exploration of periodontitis, and in particular to a method for verifying the process of PD aggravating the pathological process of AKI. Background Art

[0002] The results of the Fourth National Oral Epidemiological Survey show that the periodontal health rate of adults in China is only 9.1%, and the incidence rate of periodontitis (PD) is as high as 85%; worldwide, about 70 million people suffer from severe PD. PD is a chronic infectious disease caused by oral flora dysregulation, mainly manifested as periodontal pocket formation, suppuration in the pocket, and alveolar bone resorption, etc. It was previously believed that PD only caused local pathological changes and rarely caused death, but subsequent evidence confirmed that it is closely related to the occurrence of many major diseases, including digestive system cancers, cardiovascular diseases, Alzheimer's disease, diabetes, pregnancy miscarriages, etc. Recently, clinical cohort studies have shown that PD is related to chronic kidney injury, but there is a lack of direct experimental evidence and the mechanism of action is unclear; moreover, whether PD affects the pathological process of acute kidney injury (AKI) has not been reported. AKI is a difficult clinical problem, with multiple pathogenic factors (infection, trauma, drug side effects, etc.), a high incidence rate (10%-15% hospital incidence rate), poor prognosis (more than 50% of AKI stage 2-3 patients' renal function cannot recover within 30 days), and a high mortality rate. The condition of AKI is complex and there is no specific therapeutic drug or means. Therefore, predicting its early occurrence risk and identifying the key factors promoting the pathological process are crucial for early detection and early intervention of high-risk patients. Given the high incidence rate of PD, clarifying the correlation with the occurrence and development of AKI is of great significance for raising the vigilance of susceptible populations, reducing the incidence risk of AKI, and improving the prognosis of patients.

[0003] In previous work, we observed that the pathological process was significantly aggravated when PD mice were induced to develop AKI, and there was accumulation of renal macrophages; literature research showed that macrophage activation aggravated the pathological process in the initial stage of AKI. However, there is a lack of direct experimental evidence to link the two. Summary of the Invention

[0004] The present invention aims to provide a method for verifying the process of PD aggravating the pathological process of AKI, which solves the problem that there is no experimental evidence for the significant aggravation of the pathological process and the accumulation of renal macrophages when PD mice are induced to develop AKI, and the aggravation of the pathological process of AKI in the initial stage by macrophage activation.

[0005] To achieve the above object, the present invention provides the following methods and systems: A method for verifying the process of PD aggravating the pathological process of AKI provided by the present invention is: S1: Induce AKI in PD mice by depleting or adoptively transferring additional macrophages in vivo, observe the changes in pathological indicators, and verify that the accumulation of renal macrophages during PD is a key factor leading to the exacerbation of the pathological process during AKI onset. S2: Cultivate renal tubular epithelial cells and mouse bone marrow monocytes using the Transwell cell co-culture method, and use flow cytometry to detect the periodic changes in macrophage content, prove the effect on the self-renewal ability of in-situ renal macrophages, and confirm that the increased secretion of CSF-1 by renal tubular epithelial cells during PD leads to the accumulation of renal macrophages. S3: Detect the levels of MMP-8 in the sera of normal and PD mice, the expression levels of CSF-1 in the kidneys, and the levels of macrophage infiltration in the control group and the artemisinin-treated group of mice by ELISA. After cisplatin-induced AKI, detect the changes in the relevant pathological indicators of the above mice to clarify that the elevated MMP-8 in the serum during PD is a key mediator promoting the secretion of CSF-1 by renal tubular epithelial cells. S4: Elucidate the specific mechanism by which MMP-8 regulates the secretion of CSF-1 by renal tubular epithelial cells by exploring the enzymatic / non-enzymatic regulatory pathways, substrates / receptors, and major signaling pathways.

[0006] Preferably, the step of inducing AKI in PD mice by depleting or adoptively transferring additional macrophages in vivo, observing the changes in pathological indicators, and verifying that the accumulation of renal macrophages during PD is a key factor leading to the exacerbation of the pathological process during AKI onset includes: preparing a PD mouse model from experimental mice to obtain PD mice, treating the PD mice by ligating the cervical teeth and inoculating Porphyromonas gingivalis, and stably forming PD after 6 weeks; injecting 200 μl of liposome suspension of clodronate disodium into the tail vein of the mice to deplete macrophages; after 48 hours, injecting 2 mg / ml of cisplatin saline solution into the tail vein of the mice, and detecting the serum creatinine value and urea nitrogen content of the mice and observing the changes in renal pathological sections after 5 days; extracting bone marrow monocytes from normal mice, inducing their differentiation into a large number of macrophages in vitro, collecting the macrophages, inoculating them into normal or PD mice via the tail vein at a dose of 5×10^6 cells / mouse, inoculating normal or the above PD mice in the control group with the same volume of normal saline, waiting for 24 hours, and then inducing AKI in all mice with cisplatin solution and observing the index changes after 5 days.

[0007] Preferably, a PD mouse model is prepared using experimental mice. The steps for obtaining PD mice include: ligating the gingival sulci of the bilateral mandibular premolars and the first and second molars of the mice with 3.0 silk threads, and simultaneously applying Porphyromonas gingivalis to the ligated teeth, once every week; after about 6 weeks, periodontal pockets can be seen to form, with a large amount of plaque accumulation on the tooth surface, in the pocket, and on the root surface. Histopathological observation shows erosion of the sulcular epithelium, and X-ray examination indicates obvious alveolar bone loss and significantly reduced bone density, then the PD mouse model is successfully established.

[0008] Preferably, normal or the PD mice in the control group are inoculated with the same volume of normal saline. After waiting for 24 hours, all mice are induced with cisplatin solution to cause AKI. The steps for observing the changes in indicators after 5 days include: sacrificing the mice to obtain fresh kidneys, fixing them with 4% paraformaldehyde for about 24 hours, dehydrating them, embedding them in paraffin, and cutting them into slices about 4 μm thick; dewaxing the slices to water according to the following steps: xylene I for 10 min - xylene II for 10 min - absolute ethanol for 5 min - absolute ethanol for 5 min - 95% alcohol for 5 min - 90% alcohol for 5 min - 80% alcohol for 5 min - 70% alcohol for 5 min - distilled water wash; immersing the slices in hematoxylin staining solution for 5 min, rinsing with tap water, differentiating with 1% hydrochloric acid alcohol for several seconds, rinsing with tap water again, blueing with 0.6% ammonia water, and rinsing with running water; immersing the slices in eosin staining solution for 2 min; dehydrating and clearing the slices in 95% alcohol for 5 min - 95% alcohol for 5 min - absolute ethanol for 5 min - absolute ethanol for 5 min - xylene for 5 min - xylene for 5 min in sequence; taking out the slices from xylene, air-drying them, and mounting them; examining under a microscope and collecting and analyzing pictures.

[0009] Preferably, the Transwell cell co-culture method is used to culture renal tubular epithelial cells and mouse bone marrow monocytes. Flow cytometry is used to detect the periodic changes in the content of macrophages, to prove the effect on the self-renewal ability of renal in-situ macrophages, and to confirm the steps that the increased secretion of CSF-1 by renal tubular epithelial cells during PD leads to the accumulation of renal macrophages, including: continuously co-culturing the primary renal tubular epithelial cells stimulated by 20% PD / normal mouse serum with the sorted bone marrow monocytes for 7 days. With or without adding a CSF-1 receptor blocking antibody, the content of macrophages differentiated from the bone marrow monocytes is detected by flow cytometry every 24 hours to prove the effect on the differentiation efficiency of macrophages; after co-culturing the primary renal tubular epithelial cells with the isolated renal infiltrating macrophages for 3 days, counting the macrophages and detecting the cell cycle changes by flow cytometry to prove the effect on the self-renewal ability of renal in-situ macrophages; constructing transgenic mice with specific knockout of CSF-1 in renal tubular epithelial cells, inducing a PD model, and detecting the content of renal infiltrating macrophages by flow cytometry; and further observing the changes in pathological indicators after inducing AKI with cisplatin.

[0010] Preferably, the method for extracting and separating the kidney-infiltrating macrophages and the renal tubular epithelial cells is as follows: Take fresh mouse kidneys, fully cut the renal tissues into pieces after removing the renal capsules, add 20 ml of 0.2% collagenase IV digestive solution, and incubate in an incubator at 37°C for 1 h; Remove undigested impurities with a 40-μm filter to obtain a cell suspension; After cell centrifugation, erythrocyte lysis and washing, resuspend with the complete medium for mouse renal tubular epithelial cells and spread on a 6-well plate; Place in a cell incubator for 1 h, and the adherent cells are kidney-infiltrating macrophages; Aspirate the suspended cells, change the adherent cells to macrophage medium, and inoculate the suspended cells in a new 6-well plate. Change the medium for the first time after three days, and then change the medium every other day to obtain the purified primary renal tubular epithelial cells.

[0011] Preferably, the steps for clarifying that the increase in MMP-8 in serum promotes the secretion of CSF-1 by renal tubular epithelial cells when PD occurs by detecting the levels of MMP-8 in the sera of normal and PD mice, the expression level of CSF-1 in the kidneys, and the macrophage infiltration level in the control group and the artemisinin-injected group by ELISA, and detecting the changes in the relevant pathological indexes of the above mice after cisplatin-induced AKI include: After the PD mice are treated with ligature of the neck of the tooth and inoculation with Porphyromonas gingivalis, intragastrically administer 40 mg / kg artemisinin every 3 days until PD is formed at 6 weeks; Use ELISA to detect the levels of MMP-8 in the sera of normal and PD mice, the expression level of CSF-1 in the kidneys, and the macrophage infiltration level in the control group and the intragastrically administered group. After cisplatin-induced AKI, detect the changes in the pathological indexes of the above mice; Breed MMP-8 gene knockout mice, detect the changes in the pathological indexes of the mice, and after constructing the PD model, detect the changes in the pathological indexes of the mice again.

[0012] Preferably, the steps for clarifying the specific mechanism by which MMP-8 regulates the secretion of CSF-1 by renal tubular epithelial cells by exploring the enzyme activity / non-enzyme activity regulation pathway, the action substrate / receptor, and the main signaling pathway include: In the last week of constructing the PD mouse model, the control and PD mice are subcutaneously injected with 20 mg / kg Ilomastat every 48 hours, and the levels of MMP-8 in the sera of the mice, the expression level of CSF-1 in the kidneys, and the macrophage infiltration level are detected. After cisplatin-induced AKI, detect the changes in the pathological indexes of the mouse kidneys; In vitro, treat primary renal tubular epithelial cells with 100 ng / ml mouse MMP-8 recombinant protein combined with 10 μM Ilomastat for 3, 6, and 12 hours, and detect the mRNA expression level of CSF-1 in the cells and the protein secretion level in the culture supernatant; If the use of an enzyme activity inhibitor does not interfere with the function of MMP8 in promoting the secretion of CSF-1 by primary renal tubular epithelial cells, it is judged that the function is exerted through a non-enzyme activity pathway.

[0013] Preferably, the steps for elucidating the specific mechanism by which MMP-8 regulates the secretion of CSF-1 by renal tubular epithelial cells by exploring the enzymatic / non-enzymatic regulatory pathway, the action substrate / receptor, and the main signaling pathway further include: after incubating the primary renal tubular epithelial cells with MMP-8 recombinant protein for 6 hours, collecting the cells, performing immunoprecipitation-mass spectrometry proteomic identification and detection, and screening for target proteins with high binding activity to MMP-8. For the screened target proteins, cell lines with knockout of the protein in TCMK-1 were constructed respectively. After stimulation with 100 ng / ml MMP-8 recombinant protein in vitro, the intracellular mRNA expression level of CSF-1 and the protein secretion level in the culture supernatant were detected; after determining the final candidate binding protein according to the detection results, it was knocked out in the primary mouse renal tubular epithelial cells cultured in vitro using adenovirus.

[0014] Preferably, the steps for elucidating the specific mechanism by which MMP-8 regulates the secretion of CSF-1 by renal tubular epithelial cells by exploring the enzymatic / non-enzymatic regulatory pathway, the action substrate / receptor, and the main signaling pathway further include: stimulating the primary mouse renal tubular epithelial cells with 100 ng / ml MMP-8 recombinant protein in vitro for 6 hours, collecting the cells for transcriptome sequencing, enriching and displaying highly enriched signaling pathways through bioinformatics analysis, and verifying by WB detection in vitro experiments. For the verified signaling pathway, a specific inhibitor targeting its activity was selected to intervene in the primary mouse renal tubular epithelial cells stimulated with MMP-8 recombinant protein in vitro, and the changes in the intracellular mRNA expression level of CSF-1 and the protein secretion level in the culture supernatant were detected; after intervening with the specific inhibitor in the PD mice, the changes in the expression level of CSF-1 and the macrophage infiltration level in the kidneys were detected, and after cisplatin-induced AKI, the changes in the pathological indexes of the mice were detected.

[0015] The beneficial effects of the present invention are reflected in that: According to recent clinical research reports, PD is related to the occurrence of chronic kidney injury, but whether it affects the pathological process of AKI and the specific regulatory pathways have not been studied. The present invention intends to systematically prove that although PD is not the direct cause of AKI, it is an important risk factor exacerbating the pathological process of the latter. Clarifying this phenomenon can, on the one hand, enhance the risk awareness of PD patients regarding the occurrence of kidney diseases, and on the other hand, it is of great significance for early detection and early intervention in high-risk AKI patients; therefore, the research object of the present invention has distinct characteristics; as a local disease, PD can systematically affect kidney health, but the specific regulatory pathways have not been clarified. Based on the phenomenon that periodontitis exacerbates the pathological process of AKI, the present invention first proposes and verifies the regulatory axis of "PD→MMP8→tubular epithelial cells→CSF-1→macrophages→AKI". Therefore, the present invention has theoretical innovation; the present invention intends to focus on solving the mechanism of how the PD biomarker MMP8 regulates tubular epithelial cells, which is the key link connecting PD with the distal kidney, and no such technology has been seen before. Therefore, this application has obvious innovation in the research mechanism. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts do not necessarily draw according to the actual scale.

[0017] Figure 1 It is a schematic flow chart of a method for verifying the exacerbation of AKI pathological process by PD provided by an embodiment of the present invention; Figure 2 It is a schematic technical route diagram of a method for verifying the exacerbation of AKI pathological process by PD provided by an embodiment of the present invention; Figure 3 In it, A is a comparison chart of blood urea nitrogen content of AKI patients with a history of PD and AKI patients without a history of PD, B is a comparison chart of serum creatinine content of AKI patients with a history of PD and AKI patients without a history of PD; C is a comparison chart of the proportion of stage 2 / 3 patients of AKI patients with a history of PD and AKI patients without a history of PD; D is a comparison chart of kidney pathological section detection of AKI patients with a history of PD and AKI patients without a history of PD; E is a comparison chart of blood urea nitrogen detection of mice; F is a comparison chart of blood creatinine detection of mice; Figure 4In it, A is a comparison chart of the proportion of macrophages in the immune cells infiltrating the kidneys of PD mice and normal mice; B is a comparison chart of the number of macrophages in the immune cells infiltrating the kidneys of PD mice and normal mice; C is a comparison chart of the ROS level of effector-related molecule expression in macrophages; D is a comparison chart of the changes in the expression levels of IL-1β, TNF-α, and iNOS in cells detected by qRT-PCR; E is a comparison chart of macrophage apoptosis; F is the proliferation marker Ki-67 + Cell proportion comparison chart; Figure 5 In it, A is a chart of the expression of various factors that may mediate macrophage accumulation in the kidney tissues of PD mice detected by qRT-PCR; B is a chart of the change in the expression level of CSF-1 in infiltrating macrophages in the kidney detected by flow cytometry; C is a chart of the change in the mRNA level of intracellular CSF-1 detected by stimulating renal tubular epithelial cells with the serum of PD mice in vitro; D is a chart of the change in the protein level of CSF-1 in the culture supernatant detected by ELISA; E is a chart of the relative mRNA expression level of CSF-1 in macrophages; F is a chart of the expression level of CSF-1 in the culture supernatant of macrophages.

[0018] Figure 6 In it, A is a chart of the change in the protein level of MMP-8 in the serum of PD mice; B is a chart of the relative change in the mRNA level of CSF-1 detected in cells by directly stimulating renal tubular epithelial cells with recombinant MMP-8 protein in vitro for 48 hours; C is a chart of the change in the expression level of CSF-1 in the culture supernatant of cells detected by directly stimulating renal tubular epithelial cells with recombinant MMP-8 protein in vitro for 48 hours. Detailed implementation mode

[0019] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0020] The terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish different objects, rather than to describe a specific order. In addition, the terms "comprising" and "having" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, device, product or terminal that includes a series of steps or units is not limited to the listed steps or units, but optionally further includes steps or units not listed, or optionally further includes other steps or units inherent to these processes, methods, products or terminals.

[0021] As used herein, the mention of "embodiments" means that the specific features, structures, or characteristics described in connection with the embodiments can be included in at least one embodiment of the present invention. The phrase appears in various places in the specification and does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment mutually exclusive with other embodiments. Those skilled in the art will explicitly and implicitly understand that the embodiments described herein can be combined with other embodiments.

[0022] Recently, clinical cohort studies have shown that PD is associated with chronic kidney injury, but there is a lack of direct experimental evidence and the mechanism of action is unclear; moreover, whether PD affects the pathological process of acute kidney injury (AKI) has not been reported. AKI is a difficult clinical problem, characterized by multiple pathogenic factors (infection, trauma, drug side effects, etc.), a high incidence rate (10%-15% in-hospital incidence rate), poor prognosis (more than 50% of patients with AKI stage 2-3 cannot recover renal function within 30 days), and a high mortality rate. The condition of AKI is complex and there is no specific therapeutic drug or means. Therefore, predicting the risk of its early occurrence and identifying the key factors promoting the pathological process are crucial for early detection and early intervention in high-risk patients. Given the high incidence rate of PD, clarifying its correlation with the occurrence and development of AKI is of great significance for raising the vigilance of susceptible populations, reducing the incidence risk of AKI, and improving the prognosis of patients; in previous work, we observed that the pathological process was significantly aggravated when PD mice were induced to have AKI, and there was an accumulation of renal macrophages; literature research shows that macrophage activation exacerbates the pathological process in the initial stage of AKI. However, there is a lack of direct experimental evidence to link the two.

[0023] The present invention aims to provide a method for verifying that PD exacerbates the pathological process of AKI, which solves the problem that there is no experimental evidence that the pathological process is significantly aggravated when PD mice are induced to have AKI, and there is an accumulation of renal macrophages, and macrophage activation exacerbates the pathological process in the initial stage of AKI.

[0024] As Figure 1 shown, the specific implementation manner of the present invention provides a method for verifying that PD exacerbates the pathological process of AKI, including the following steps: S1: After depleting or adoptively transferring additional macrophages in PD mice, induce AKI in the mice, observe the changes in pathological indicators, and verify that the accumulation of renal macrophages during PD is the key factor leading to the exacerbation of the pathological process during the occurrence of AKI.

[0025] In the embodiments of the present invention, a PD mouse model is prepared from experimental mice to obtain PD mice. The steps of preparing a PD mouse model from experimental mice to obtain PD mice include: ligating the gingival sulci of the bilateral mandibular premolars and the first and second molars of the mice with 3.0 silk threads, and simultaneously applying Porphyromonas gingivalis to the ligated teeth, applying it once every week; after about 6 weeks, the formation of periodontal pockets can be seen, with a large amount of plaque accumulation on the tooth surface, in the pockets and on the root surface. Histopathological observation shows erosion of the sulcular epithelium, and X-ray examination indicates obvious alveolar bone loss and significant reduction in bone density, then the PD mouse model is successfully established; the PD mice are treated by the method of ligating the cervical part of the teeth combined with inoculating Porphyromonas gingivalis, and PD is stably formed after 6 weeks; 200 μl of liposome suspension of disodium clodronate is injected into the tail vein of the mice to remove macrophages; after 48 hours, 2 mg / ml of cisplatin saline solution is injected into the mice via the tail vein, and the serum creatinine value and urea nitrogen content of the mice are detected 5 days later, and the changes in the pathological sections of the kidneys are observed; bone marrow monocytes are extracted from normal mice and induced to differentiate into a large number of macrophages in vitro, the macrophages are collected, and inoculated into normal or PD mice via the tail vein at a dose of 5×10^6 cells / mouse. The normal or PD mice in the control group are inoculated with the same volume of normal saline. After waiting for 24 hours, all the mice are induced with AKI using cisplatin solution, and the changes in the observed indexes are measured 5 days later. The steps of inoculating the normal or PD mice in the control group with the same volume of normal saline, waiting for 24 hours, and then inducing AKI in all the mice with cisplatin solution and measuring the changes in the observed indexes 5 days later include: sacrificing the mice to obtain fresh kidneys, fixing them with 4% paraformaldehyde for about 24 hours, embedding them in paraffin after dehydration, and cutting them into slices about 4 μm thick; dewaxing the slices to water according to the following steps: xylene I for 10 min - xylene II for 10 min - absolute ethanol for 5 min - absolute ethanol for 5 min - 95% alcohol for 5 min - 90% alcohol for 5 min - 80% alcohol for 5 min - 70% alcohol for 5 min - distilled water wash; immersing the slices in hematoxylin staining solution for 5 min, rinsing with tap water, differentiating with 1% hydrochloric acid alcohol for several seconds, rinsing with tap water again, bluing with 0.6% ammonia water, and rinsing with running water; immersing the slices in eosin staining solution for 2 min; dehydrating and clearing the slices in 95% alcohol for 5 min - 95% alcohol for 5 min - absolute ethanol for 5 min - absolute ethanol for 5 min - xylene for 5 min - xylene for 5 min in sequence; taking them out of the xylene, air-drying, and mounting; microscopic examination and image acquisition and analysis.

[0026] S2: The renal tubular epithelial cells and mouse bone marrow monocytes are cultured by the Transwell cell co-culture method, and flow cytometry is used to detect the periodic changes in the macrophage content, so as to prove the influence on the self-renewal ability of the in-situ macrophages in the kidneys, and confirm that the increase in the secretion of CSF-1 by renal tubular epithelial cells during PD leads to the accumulation of renal macrophages.

[0027] In the embodiments of the present invention, primary renal tubular epithelial cells stimulated with 20% PD / normal mouse serum were continuously co-cultured with sorted bone marrow monocytes for 7 days. With or without adding CSF-1 receptor blocking antibody, the content of macrophages differentiated from bone marrow monocytes was detected by flow cytometry every 24 hours to prove the effect on the differentiation efficiency of macrophages. After co-culturing primary renal tubular epithelial cells with isolated kidney-infiltrating macrophages for 3 days, the macrophages were counted, and the cell cycle changes were detected by flow cytometry to prove the effect on the self-renewal ability of in-situ kidney macrophages. Transgenic mice with specific knockout of CSF-1 in renal tubular epithelial cells were constructed. After inducing the PD model, the content of kidney-infiltrating macrophages was detected by flow cytometry; and after further inducing AKI with cisplatin, the changes in pathological indexes were observed. The methods for extracting and separating kidney-infiltrating macrophages and renal tubular epithelial cells were as follows: Fresh mouse kidneys were taken, and after removing the renal capsule, the renal tissue was fully minced. After adding 20 ml of 0.2% collagenase IV digestive solution, it was incubated in an incubator at 37°C for 1 h; the cell suspension was obtained after removing undigested impurities with a 40-μm filter; after cell centrifugation, erythrocyte lysis and washing, it was resuspended with complete medium for mouse renal tubular epithelial cells and plated in a 6-well plate; after being placed in a cell culture incubator for 1 h, the adherent cells were kidney-infiltrating macrophages; the suspended cells were aspirated, the adherent cells were changed to macrophage medium, and the suspended cells were inoculated in a new 6-well plate. The medium was changed for the first time after 3 days, and then every other day, and purified primary renal tubular epithelial cells could be obtained.

[0028] S3: Detect the MMP-8 levels, CSF-1 expression levels and macrophage infiltration levels in the normal and PD mouse sera and kidneys of the control group and the group intragastrically administered with abrotanine by ELISA. After cisplatin-induced AKI, detect the changes in the relevant pathological indexes of the above mice to clarify that the increase in serum MMP-8 during PD is the key mediator for promoting the secretion of CSF-1 by renal tubular epithelial cells.

[0029] In the embodiments of the present invention, after PD mice were treated with ligature at the cervical margin of the tooth combined with inoculation with Porphyromonas gingivalis, 40 mg / kg abrotanine was intragastrically administered every 3 days until PD was formed at 6 weeks; ELISA was used to detect the MMP-8 levels, CSF-1 expression levels and macrophage infiltration levels in the normal and PD mouse sera and kidneys of the control group and the intragastrically administered group; after cisplatin-induced AKI, the pathological index changes of the above mice were detected; MMP-8 gene knockout mice were bred, the pathological index changes of the mice were detected, and after constructing the PD model, the pathological index changes of the mice were detected again.

[0030] S4: Elucidate the specific mechanism of MMP-8 regulating the secretion of CSF-1 by renal tubular epithelial cells by exploring the enzyme activity / non-enzyme activity regulation pathway, action substrates / receptors, and main signal pathways.

[0031] In the embodiment of the present invention, in the last week of constructing the PD mouse model, the control and PD mice were subcutaneously injected with 20 mg / kg Ilomastat every 48 hours to detect the MMP-8 level in the mouse serum, the CSF-1 expression level and macrophage infiltration level in the kidney, and the changes in the pathological indexes of the mouse kidney after cisplatin-induced AKI; in vitro, primary renal tubular epithelial cells were treated with 100 ng / ml mouse MMP-8 recombinant protein combined with 10 μM Ilomastat for 3, 6, and 12 hours to detect the intracellular mRNA expression level of CSF-1 and the protein secretion level in the culture supernatant; if the enzyme activity inhibitor did not interfere with the effect of MMP8 in promoting the secretion of CSF-1 by primary renal tubular epithelial cells, it was judged to function through a non-enzymatic pathway; it also included: after primary renal tubular epithelial cells were incubated with MMP-8 recombinant protein for 6 hours, the cells were collected for immunoprecipitation-mass spectrometry proteomic identification and detection to screen for target proteins with high binding activity to MMP-8. For the screened target proteins, cell lines with knockout of the protein in TCMK-1 were constructed respectively, and after being stimulated with 100 ng / ml MMP-8 recombinant protein in vitro, the intracellular mRNA expression level of CSF-1 and the protein secretion level in the culture supernatant were detected; after determining the final candidate binding protein according to the detection results, it was knocked out in the primary renal tubular epithelial cells of mice cultured in vitro using adenovirus; it also included: primary renal tubular epithelial cells of mice were stimulated with 100 ng / ml MMP-8 recombinant protein in vitro for 6 hours, the cells were collected for transcriptome sequencing, the signal pathways with high enrichment display were analyzed by bioinformatics, and verified by WB detection in vitro experiments. For the verified signal pathways, specific inhibitors targeting their activities were selected to intervene in the primary renal tubular epithelial cells of mice stimulated with MMP-8 recombinant protein in vitro, and the changes in the intracellular mRNA expression level of CSF-1 and the protein secretion level in the culture supernatant were detected; after the specific inhibitor was used to intervene in the PD mice in vivo, the changes in the CSF-1 expression level and macrophage infiltration level in the kidney were detected, and after cisplatin-induced AKI, the changes in the pathological indexes of the mice were detected.

[0032] A total of 351 case records of AKI patients hospitalized in Chongqing People's Hospital from 2018 to 2021 were collected. Through follow-up visits, medical history tracing and other means, it was determined that 94 of them had experienced PD within 60 days before the onset of the disease, and 247 had no PD history. Through statistical analysis, we found that AKI patients with a history of PD had higher serum creatinine and urea nitrogen levels compared to the group without a history (Figure 3 A, B), and a higher proportion of patients were in the AKI 2 / 3 stage (Figure 3C). In further research, we constructed a PD mouse model and induced the occurrence of AKI with cisplatin. Through kidney pathological section detection (Figure 3D), mouse serum creatinine and urea nitrogen detection (Figure 3E, F), it was also found that the AKI kidney injury in PD mice was more severe.

[0033] In the pre-experiment, flow cytometry was used for detection, and it was found that the proportion and number of macrophages in the infiltrating immune cells of the kidneys of PD mice were significantly increased compared to normal mice (Figure 4A, B); the expression of effector-related molecules in these macrophages was detected, and no significant difference in ROS levels was found (Figure 4C); macrophages were isolated and the expression levels of IL-1β, TNF-α and iNOS in the cells were detected by qRT-PCR, and no obvious changes were found (Figure 4D); further detection of macrophage homeostasis found that there was no obvious change in its apoptosis level, but the proportion of cells of the proliferation marker Ki-67 + was significantly increased, indicating a significant enhancement of the proliferation ability (Figure 4 E, F).

[0034] The expression levels of various factors that may mediate macrophage accumulation in the kidney tissues of PD mice were detected by qRT-PCR, including chemokines MCP-1, OPN, CCL2, CCL5, CXCL16, CX3CR1, complement C3b, C3a, C5a, adhesion molecules ICAM-1, VCAM-1, migration inhibitory protein MIF and cytokine CSF-1. It was found that although the expressions of MCP-1, CCL2 and CCL5 also increased, the increase in CSF-1 was the most significant, more than 10 times higher (Figure 5A). Further, we used flow cytometry to detect the expression level of CSF-1 in the infiltrating macrophages of the kidney and found no significant change (Figure 5B). However, when tubular epithelial cells were stimulated with the serum of PD mice in vitro, an increase in the intracellular CSF-1 mRNA level by more than 10 times was detected (Figure 5 C), and the protein level of CSF-1 in the culture supernatant was also significantly increased by ELISA detection (Figure 5D); no significant change was observed when macrophages were stimulated in the same way in vitro (Figure 5E, F) It was detected that the protein level of MMP-8 in the serum of PD mice was significantly increased (Figure 6A); when renal tubular epithelial cells were directly stimulated with recombinant MMP-8 protein in vitro for 48 hours, it was detected that both the mRNA level of CSF-1 in the cells and the protein level in the culture supernatant were significantly increased (Figure 6B, C).

[0035] The beneficial effects of the present invention are reflected in that: through recent clinical research reports, it is known that PD is related to the occurrence of chronic kidney injury, but whether it affects the pathological process of AKI and the specific regulatory pathway have not been studied. The present invention intends to systematically prove that although PD is not the direct cause of AKI, it is an important risk factor exacerbating the pathological process of the latter. Clarifying this phenomenon can, on the one hand, enhance the risk awareness of PD patients regarding the occurrence of kidney diseases, and on the other hand, it is of great significance for early detection and early intervention in high-risk AKI patients; therefore, the research object of the present invention has distinct characteristics; as a local disease, PD can systematically affect kidney health, but the specific regulatory pathway has not been clarified. Based on the phenomenon that periodontitis exacerbates the pathological process of AKI, the present invention first proposes and verifies the regulatory axis of "PD→MMP8→renal tubular epithelial cells→CSF-1→macrophages→AKI". Therefore, the present invention has theoretical innovation; the present invention intends to focus on solving the mechanism by which the PD biomarker MMP8 regulates renal tubular epithelial cells, which is the key link connecting PD with the distal kidney, and no such technology has been seen before. Therefore, this application has obvious innovation in the research mechanism.

[0036] The above are only embodiments of the present invention, and common general technical solutions or characteristics in the solutions are not described in detail here; it should be pointed out that for those skilled in the art, without departing from the solution of the present invention, several modifications and improvements can still be made, and these should also be regarded as the protection scope of the present invention, and these will not affect the effects of the present invention and the practicality of the patent. The protection scope required by this application should be subject to the content of its claims, and the specific implementation manners and the like recorded in the specification can be used to interpret the content of the claims.

Claims

1. A method for verifying the pathological process of PD exacerbating AKI, characterized in that: The method comprises: S1: By removing or adoptively transferring additional macrophages from PD mice to induce AKI in mice, and observing the changes in pathological indicators, we verified that the accumulation of renal macrophages during PD is a key factor that leads to the aggravation of the pathological process when AKI occurs; S2: Transwell cell co-culture method was used to culture renal tubular epithelial cells and mouse bone marrow mononuclear cells, and flow cytometry was used to detect the cyclic changes in macrophage content to prove the effect on the self-renewal ability of in situ macrophages in the kidneys, confirming that the increased secretion of CSF-1 by renal tubular epithelial cells during PD led to the accumulation of renal macrophages; S3: The serum MMP-8 level, kidney CSF-1 expression level and macrophage infiltration level of normal and PD mice in the control group and the oral absinthe group were detected by ELISA. After cisplatin-induced AKI, the changes of relevant pathological indicators of the above mice were detected to clarify that the increase of serum MMP-8 during PD is the key mediator to promote the secretion of CSF-1 by renal tubular epithelial cells; S4: The specific mechanism by which MMP-8 regulates the secretion of CSF-1 by renal tubular epithelial cells was elucidated by exploring the enzyme activity / non-enzyme activity regulation pathways, substrates / receptors, and major signaling pathways.

2. The method for verifying the pathological process of PD exacerbating AKI according to claim 1, characterized in that: The steps of inducing AKI in mice by removing or adoptively transferring additional macrophages in PD mice, observing changes in pathological indicators, and verifying that the accumulation of macrophages in the kidneys during PD is a key factor leading to the aggravation of the pathological process when AKI occurs include: A PD mouse model was prepared by using experimental mice to obtain PD mice, and the PD mice were treated by a method of ligating the cervical ligament combined with inoculation of Porphyromonas gingivalis, and PD was stably formed after 6 weeks; Inject 200ul of disodium clodophosphate liposome suspension into the tail vein of the mouse to remove macrophages; After 48 hours, the mice were injected with 2 mg / ml cisplatin saline solution via the tail vein. After 5 days, the serum creatinine and urea nitrogen content of the mice were detected, and the changes in kidney pathological sections were observed. Bone marrow mononuclear cells were extracted from normal mice and induced to differentiate into a large number of macrophages in vitro. The macrophages were collected and inoculated into normal or PD mice via the tail vein at a dose of 5×10^6 cells / mouse. The normal or PD mice in the control group were inoculated with the same volume of normal saline. After waiting for 24 hours, AKI was induced in all mice with cisplatin solution, and the changes in indicators were observed after 5 days.

3. A method for verifying the pathological process of PD exacerbating AKI according to claim 2, characterized in that: The step of preparing a PD mouse model by using experimental mice to obtain PD mice comprises: The gingival sulcus of the bilateral mandibular premolars and the first and second molars of the mice was ligated with 3.0 silk thread, and Porphyromonas gingivalis was applied to the ligated teeth at intervals of one week. After about 6 weeks, periodontal pockets were formed, and a large amount of plaque accumulated on the tooth surface, pockets and root surfaces. Histopathological observations showed erosion of the epithelium in the sulcus. X-ray examinations showed obvious alveolar bone loss and a significant decrease in bone density. The PD mouse model was successfully prepared and established.

4. The method for verifying the pathological process of PD exacerbating AKI according to claim 2, characterized in that: The steps of inoculating the normal mice or the PD mice in the control group with the same volume of physiological saline, waiting for 24 hours, inducing AKI in all mice with cisplatin solution, and observing the changes of indicators after 5 days, include: Mice were killed and fresh kidneys were taken out. The kidneys were fixed with 4% paraformaldehyde for about 24 hours, embedded in paraffin after dehydration, and cut into slices of about 4 μm. The slices were dewaxed to water according to the following steps: xylene I for 10 minutes, xylene II for 10 minutes, anhydrous ethanol, 5 minutes, anhydrous ethanol, 5 minutes, 95% alcohol, 5 minutes, 90% alcohol, 5 minutes, 80% alcohol, 5 minutes, 70% alcohol, and 5 minutes of distilled water. The slices were immersed in hematoxylin stain for 5 minutes, rinsed with tap water, differentiated with 1% hydrochloric acid alcohol for a few seconds, washed with tap water again, and then blued with 0.6% ammonia water, and rinsed with running water. The slices were immersed in eosin stain for 2 minutes. The slices were placed in 95% alcohol, 5 minutes, 95% alcohol, 5 minutes, anhydrous ethanol, 5 minutes, anhydrous ethanol, 5 minutes, xylene, 5 minutes, and 5 minutes of dehydration and transparency in turn. The slices were taken out of xylene to dry and sealed. The slices were examined under a microscope, and the images were collected and analyzed.

5. The method for verifying the pathological process of PD exacerbating AKI according to claim 1, characterized in that: The steps of using the Transwell cell co-culture method to culture renal tubular epithelial cells and mouse bone marrow mononuclear cells, using flow cytometry to detect the periodic changes in macrophage content, proving the effect on the self-renewal ability of renal in situ macrophages, and confirming that the increased secretion of CSF-1 by renal tubular epithelial cells during PD leads to the accumulation of renal macrophages include: Primary renal tubular epithelial cells stimulated with 20% PD / normal mouse serum were co-cultured with the sorted bone marrow mononuclear cells for 7 days, and the content of macrophages differentiated from the bone marrow mononuclear cells was detected by flow cytometry every 24 hours with or without the addition of CSF-1 receptor blocking antibody to demonstrate the effect on the efficiency of macrophage differentiation; After the primary renal tubular epithelial cells were co-cultured with the isolated kidney-infiltrating macrophages for 3 days, the macrophages were counted, and the cell cycle changes were detected by flow cytometry to demonstrate the effect on the self-renewal ability of the macrophages in situ of the kidney; Transgenic mice with specific knockout of CSF-1 in renal tubular epithelial cells were constructed. After inducing the PD model, the content of kidney-infiltrating macrophages was detected by flow cytometry. The changes in pathological indicators were further observed after AKI was induced by cisplatin.

6. A method for verifying the pathological process of PD exacerbating AKI according to claim 5, characterized in that: The extraction and separation method of the kidney-infiltrating macrophages and the renal tubular epithelial cells is as follows: take a fresh mouse kidney, remove the renal capsule and fully chop the kidney tissue, add 20 ml of 0.2% collagenase IV digestion solution, and place it in a 37°C incubator for incubation for 1 hour; use a 40μm filter to remove undigested impurities to obtain a cell suspension; after centrifugation, red lysis and washing of the cells, resuspend them in mouse renal tubular epithelial cell complete culture medium and spread them in a 6-well plate; place them in a cell culture incubator for 1 hour, and the adherent cells are kidney-infiltrating macrophages; aspirate the suspended cells, replace the adherent cells with macrophage culture medium, and inoculate the suspended cells in a new 6-well plate. Change the medium for the first time after three days, and then change the medium every other day to obtain the purified primary renal tubular epithelial cells.

7. The method for verifying the pathological process of PD exacerbating AKI according to claim 1, characterized in that: The steps of detecting the serum MMP-8 level, the CSF-1 expression level and the macrophage infiltration level in the normal and PD mice of the control group and the mice gavage with absinthe by ELISA, detecting the changes of the relevant pathological indicators of the above mice after cisplatin-induced AKI, and clarifying that the increase of serum MMP-8 during PD is the key medium for promoting the secretion of CSF-1 by renal tubular epithelial cells, include: The PD mice were treated with cervical ligation combined with Porphyromonas gingivalis inoculation, and then intragastrically administered with 40 mg / kg absinthe every 3 days until PD was formed at 6 weeks; ELISA was used to detect the serum MMP-8 level, kidney CSF-1 expression level and macrophage infiltration level of normal and PD mice in the control group and gavage group. After cisplatin-induced AKI, the changes of pathological indicators of the above mice were detected. MMP-8 gene knockout mice were bred and the changes in pathological indicators of mice were detected. After the PD model was established, the changes in pathological indicators of mice were detected again.

8. The method for verifying the pathological process of PD exacerbating AKI according to claim 1, characterized in that: The steps of clarifying the specific mechanism of MMP-8 regulating the secretion of CSF-1 by renal tubular epithelial cells by exploring the enzyme activity / non-enzyme activity regulation pathway, substrate / receptor, and main signaling pathway include: In the last week of establishing the PD mouse model, control and PD mice were subcutaneously injected with 20 mg / kg Ilomastat every 48 hours to detect the level of MMP-8 in mouse serum, the expression level of CSF-1 and the level of macrophage infiltration in the kidney, and the changes in the renal pathological indicators of mice after cisplatin-induced AKI; Primary renal tubular epithelial cells were treated with 100 ng / ml mouse MMP-8 recombinant protein combined with 10 μM Ilomastat in vitro for 3, 6, and 12 hours, and the intracellular mRNA expression level of CSF-1 and the protein secretion level in the culture supernatant were detected; if the use of enzyme activity inhibitors did not interfere with the effect of MMP8 in promoting the secretion of CSF-1 by primary renal tubular epithelial cells, it was judged that the function was exerted through a non-enzymatic pathway.

9. The method for verifying the pathological process of PD exacerbating AKI according to claim 1, characterized in that: The step of clarifying the specific mechanism of MMP-8 regulating the secretion of CSF-1 by renal tubular epithelial cells by exploring the enzyme activity / non-enzyme activity regulation pathway, substrate / receptor, and main signaling pathway also includes: After the primary renal tubular epithelial cells were incubated with MMP-8 recombinant protein for 6 hours, the cells were collected and subjected to immunoprecipitation-mass spectrometry proteomic identification and detection to screen the target protein with high binding activity with MMP-8. For the target protein obtained by screening, cell lines with the protein knocked out in TCMK-1 were constructed respectively, and after in vitro stimulation with 100ng / ml MMP-8 recombinant protein, the intracellular mRNA expression level of CSF-1 and the protein secretion level in the culture supernatant were detected; After the final candidate binding protein was determined based on the test results, it was knocked out in primary mouse tubular epithelial cells cultured in vitro using adenovirus.

10. The method for verifying the pathological process of PD exacerbating AKI according to claim 1, characterized in that: The step of clarifying the specific mechanism of MMP-8 regulating the secretion of CSF-1 by renal tubular epithelial cells by exploring the enzyme activity / non-enzyme activity regulation pathway, substrate / receptor, and main signaling pathway also includes: The primary renal tubular epithelial cells of the mice were stimulated with 100ng / ml MMP-8 recombinant protein in vitro for 6 hours, and the cells were collected for transcriptome sequencing. The signal pathways with high enrichment were analyzed by bioinformatics, and verified by WB detection in vitro. For the verified signal pathways, specific inhibitors targeting their activity were selected to intervene in the primary renal tubular epithelial cells of mice stimulated by MMP-8 recombinant protein in vitro, and the changes in the mRNA expression level of CSF-1 in the cells and the protein secretion level in the culture supernatant were detected; after the intervention of the specific inhibitor in the PD mice, the changes in the CSF-1 expression level and the macrophage infiltration level in the kidney were detected, and after cisplatin-induced AKI, the changes in the pathological indicators of the mice were detected.

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