Method for detecting effect of NHH in sepsis encephalopathy by applying CLP model

Abstract

The invention relates to the field of biology, and particularly provides a method for detecting the effect of NHH in sepsis encephalopathy by using a CLP model, which comprises the following steps: in a CLP mouse model, after skin disinfection under isoflurane anesthesia, making an abdominal midline incision to expose cecum; the cecum is ligated in the middle between the distal electrode and the cecum base to induce moderate sepsis. The invention provides a method for detecting the effect of NHH in sepsis encephalopathy by applying a CLP model, and aims to research whether the occurrence of NHH in a CLP mouse model is caused by the increase of ammonia-producing bacteria in intestinal tracts or not by applying 16S rDNA and metabonomics technologies. The pathophysiology change and potential mechanism of NHH-induced brain injury in the CLP mouse model are illuminated on animal and cell levels through coprophilous fungus transplantation, morphology, functionality, molecular biology and neuroimaging methods. And a new insight is provided for the pathological mechanism of SAE treatment and a potential new drug target.

Description

Technical Field

[0001] The present invention relates to the field of biology, and in particular, to a method for detecting the role of NHH in septic encephalopathy by using a CLP model. Background Art

[0002] Sepsis-associated encephalopathy (SAE) is a major cause of short-term and long-term death in septic patients. The global incidence and mortality are on the rise, posing a serious threat to public health and urgently needing to be solved. Although inflammation, mitochondrial dysfunction, and microglial activation have been reported as potential mechanisms of SAE, the exact pathogenesis is still unclear, and there is still a lack of effective preventive and intervention measures. Ammonia is an important neurotoxin and plays an important role in hepatic encephalopathy. Hyperammonemia (NHH) caused by non-liver diseases has been reported more and more frequently in critically ill patients, especially in septic patients. Our previous study found that NHH is related to SAE, and the etiology of NHH in septic patients may be attributed to factors such as increased intestinal ammonia production and enhanced amino acid catabolism. The gastrointestinal tract is an immune system that is easily damaged after sepsis, especially in septic shock, and intestinal flora dysbiosis is likely to occur after intestinal injury. Amino acids respond to intestinal bacteria to produce ammonia. Therefore, we hypothesize that the ecological dysregulation of intestinal microbiota and increased amino acid metabolism after sepsis may be the cause of NHH. Astrocyte edema is one of the important mechanisms leading to the occurrence of SAE accompanied by neuronal damage, and it is also an important cell in the occurrence of hepatic encephalopathy caused by ammonia. Aquaporin 4 (AQP4) is a selective membrane-bound water channel with a high expression level in astrocytes. AQP4 is not only an important molecule controlling the influx and efflux of water in astrocytes, but also activates astrocytes by increasing the expression of AQP4. AQP4 plays an important role in astrocyte injury. Therefore, we hypothesize that the entry of NHH into the brain and the upregulation of astrocyte AQP4 expression may be the mechanism of SAE. Summary of the Invention

[0003] The main object of the present invention is to provide a method for detecting the role of NHH in septic encephalopathy by using a CLP model, aiming to study the occurrence of NHH in a CLP mouse model by using 16S rDNA and metabolomics techniques, and whether it is caused by an increase in ammonia-producing bacteria in the intestine. Through fecal transplantation, morphology, function, molecular biology, and neuroimaging methods, the pathophysiological changes and potential mechanisms of NHH-induced brain injury in a CLP mouse model were clarified at the animal and cell levels.

[0004] To achieve the above object, according to one aspect of the present invention, there is provided a method for detecting the role of NHH in septic encephalopathy by using a CLP model, including the following steps:

[0005] In the CLP mouse model, under isoflurane anesthesia, after skin disinfection, a midline abdominal incision was made to expose the cecum; the cecum was ligated in the middle between the distal pole and the bottom of the cecum to induce moderate sepsis.

[0006] Further, the anesthesia concentration of the isoflurane is 1-3%.

[0007] Further, the midline abdominal incision is 0.8-1.2 cm.

[0008] Further, the midline abdominal incision was sutured with sterile 6-0 silk thread.

[0009] Further, after the establishment of the CLP model, feces of mice in each group were collected, and 16S rDNA sequencing technology was used to observe the changes in the types and abundances of microorganisms in each group.

[0010] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for detecting the role of NHH in sepsis encephalopathy using the CLP model, aiming to study whether the occurrence of NHH in the CLP mouse model is caused by an increase in ammonia-producing bacteria in the intestine. Through fecal transplantation, morphology, function, molecular biology, and neuroimaging methods, the pathophysiological changes and potential mechanisms of NHH-induced brain injury in the CLP mouse model were elucidated at the animal and cell levels. It provides new insights into the pathological mechanism of SAE treatment and potential new drug targets. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0012] The structures, ratios, sizes, etc. shown in the drawings of this specification are only used to cooperate with the content disclosed in the specification for those skilled in this technology to understand and read, and are not used to limit the limited conditions for the implementation of the present invention. Therefore, they do not have a substantial technical meaning. Any modification of the structure, change of the proportional relationship, or adjustment of the size should still fall within the scope that can be covered by the technical content disclosed in the present invention without affecting the effects that the present invention can produce and the purposes that can be achieved.

[0013] Figure 1 It is a graph of the liver function-related data of three control groups at 24h, 48h, and 72h after CLP in the preferred embodiment of the present invention to exclude the increase in ammonia caused by severe liver injury.

[0014] Figure 2It is the state diagram related to the changes of intestinal flora after the establishment of the CLP model in the preferred embodiment of the present invention;

[0015] Figure 3 It is the metabolomics analysis diagram of LC-MS / MS in each group in the preferred embodiment of the present invention;

[0016] Figure 4 It is the research result diagram of the changes of fMRI, behavior, hippocampus and blood-brain barrier permeability in each group of mice in the preferred embodiment of the present invention;

[0017] Figure 5 It is the research result diagram of proving that NHH causes brain damage at the animal level in the preferred embodiment of the present invention;

[0018] Figure 6 It is the result diagram of proving the mechanism of NHH causing brain damage at the cell level in the preferred embodiment of the present invention;

[0019] Figure 7 It is the overall experimental schematic diagram in the preferred embodiment of the present invention. Detailed implementation manners

[0020] To make the invention purpose, features and advantages of the present invention more obvious and understandable, 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 embodiments described below are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0021] The technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and through specific implementation manners.

[0022] Previous studies by the inventors found that the ammonia level increased in sepsis patients with normal liver function, and the serum ammonia level was related to the degree of consciousness disorder. The inventors speculated that non-hepatic hyperammonemia (NHH) might be a potential mechanism of SAE. To verify the role of NHH in SAE, the inventors used the CLP surgery to evaluate the response of mice to SAE. To avoid liver injury caused by severe CLP, the inventors used a moderate CLP mouse model. In addition, the inventors evaluated the HE staining and the levels of alanine aminotransferase, aspartate aminotransferase and total bilirubin in three groups at 24 h, 48 h and 72 h after CLP ( Figure 1 a and Figure 1 b). HE staining showed that the hepatocytes of the three groups of mice were arranged neatly and the hepatic lobule structure was complete, and there were no significant abnormalities in the liver function indexes of the three groups (Figure 1 b). However, the inventors found that the serum of the CLP group (469.91 ± 34.14 vs

[0023] 375.93 ± 52.26 vs 375.12 ± 18.56) and the hippocampal ammonia level (52.57 ± 6.27 vs 38.46 ± 6.29 vs

[0024] 39.17 ± 2.26) were significantly higher than those of the control group and the sham operation group ( Figure 1 c).

[0025] After the establishment of the CLP model, the ileal villus length and crypt depth after CLP were lower than those of the control group and the sham operation group, and the pH value increased ( Figure 2 a, Figure 2 b). CLP damaged the intestinal structure of mice and changed the intestinal pH value level. After fecal microbiota transplantation (FMT), the 16S rDNA sequencing technology was used to observe the changes in the species and abundances of microorganisms in each group. After CLP, at the class level, the abundance of Bacteroidetes was reduced compared with the control group and the sham operation group, while the abundance of Bacilli increased. After fecal microbiota transplantation, the abundance of Bacteroidetes increased and the abundance of Bacilli decreased in the community Barplot (P < 0.05) ( Figure 2 c). According to the Chao1 index (P < 0.05), it was determined that the microbial diversity of the CLP group was significantly lower than that of the control group and the sham operation group, but there were no significant differences in the Shannon index and Simpson index ( Figure 2 d). In addition, the β-diversity of the microbiome shown by unweighted Unifrac and principal coordinate analysis (PCoA) indicated that there were significant differences in the community compositions between the CLP group and the other three groups, suggesting that CLP significantly affected the composition of the intestinal flora ( Figure 2 e and Figure 2 f). Linear discriminant analysis effect size (LEfSe) analysis showed that the key bacteria in the sham operation group were Bacteroidales, Muribaculaceae, Erysipelotrichaceae, Allobaculum, Prevotellaceae, Alloprevotella, etc. In CLP mice, Bacilli, Clostridia, Firmicutes, Lactobacillales, Enterobacterales, Streptococcus, and Enterococcus were dominant ( Figure 2 g), and after fecal microbiota transplantation, the relative abundances of Bacilli and Clostridia decreased, while the relative abundances of Spirochaetaceae, Moraxellaceae, Clostridia_vadinBB60_group, and Gastranaerophilales increased ( Figure 2h). The above results indicate that CLP significantly alters the types and abundances of the gut microbiota. The inventors performed functional annotation and differential analysis to further examine the effects of the altered gut microbiota. The results of the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) functional annotations showed that the relative abundance value of amino acid metabolism in the CLP group was higher than that in other groups. The functional differential analysis also showed that "carbohydrate metabolism", "amino acid metabolism", "other amino acid metabolism", and "infectious diseases: bacteria" in the CLP group were stimulated ( Figure 2 j) (P < 0.05) and were inhibited after fecal bacteria transplantation ( Figure 2 k) (P < 0.05). The PCA analysis of the microbiota functions indicated that there were significant differences in the microbiota functions between the CLP group and the sham operation group, and the microbiota functions were similar after fecal microbiota transplantation ( Figure 2 l). Therefore, it is speculated that the amino acid level may be related to the changes in the gut microbiota.

[0026] The metabolomics analysis by LC-MS / MS showed that the amino acid count accounted for 55% of all identified metabolites ( Figure 3 a). The PCA of metabolomics showed differences among the four groups of metabolites ( Figure 3 b). The amino acid level in the CLP group was significantly higher than that in the sham operation group, and the Log2FC values of the volcano plot and the heat map of the cluster analysis were between 1.15 - 60.87 (P < 0.05). After fecal microbiota transplantation, the amino acid substances in the CLP group decreased significantly, and the Log2FC values were between 0.10 - 0.49 (P < 0.05) ( Figure 3 d). In addition, the chord diagrams of the CLP group and the sham operation group, as well as the CLP+FMT and CLP groups, once again confirmed the significant differences in the amino acid levels (P < 0.05), and there was a correlation between the amino acids (r > 0.8) ( Figure 3 e). The KEGG pathway analysis of the metabolites showed that amino acid metabolism increased after CLP, and the differential abundance was higher than that in the sham operation group ( Figure 3 f). In addition, after fecal microbiota transplantation in CLP mice, the ammonia levels in the blood and hippocampal tissues decreased ( Figure 3 d). The above results indicate that fecal microbiota transplantation can reduce the amino acid metabolism and ammonia levels in the CLP group. Previous studies have found that amino acid metabolism is the main source of ammonia.

[0027] This example analyzed the associations between the gut microbiota and metabolites in each group. The bacilli in the CLP and sham operation groups were positively correlated with the metabolism of amino acids ( Figure 3 g). Therefore, the inventors speculated that the increase in bacilli after CLP may be related to the occurrence of NHH. Previous studies have shown that bacilli are one of the ammonia-producing bacteria.

[0028] The results of T2-weighted tests showed that the water absorption intensities of the sham operation group and the control group were significantly higher than those of the control group, indicating an increase in the water content in the brain tissues of mice after CLP, a decrease in ammonia levels after fecal microbiota transplantation, and a decrease in the water content of the brain tissues of CLP mice (T2-weighted siginal intensity: control group: 9197.28 ± 572.62; sham operation group: 9105.12 ± 365.82; CLP group: 10777.75 ± 471.50; CLP+FMT: 9675.30 ± 726.61). The results of ASL examinations showed that the blood flow in the hippocampal region of CLP mice decreased significantly, and the cerebral blood flow improved after fecal microbiota transplantation in CLP mice (Hippocampal rCBF: control group: 83.71 ± 7.83; sham operation group: 85.26 ± 8.45; CLP group: 48.54 ± 8.00; CLP+FMT: 69.16 ± 8.83). The results of DTI tests showed that the FA and AD of the hippocampal tissues of CLP mice were significantly higher than those of the sham operation group and the control group [(Hippocampal FA: control group: 0.303 ± 0.032; sham operation group: 0.30 ± 0.02; CLP group: 0.244 ± 0.028; CLP+FMT: 0.289 ± 0.024),

[0029] (Hippocampal AD: control group: 0.78 ± 0.04; sham operation group: 0.78 ± 0.05; CLP group: 0.65 ± 0.07; CLP+FMT: 0.74 ± 0.04)]. The RD and MD of the hippocampal tissues of CLP mice were significantly lower than those of the sham operation group and the control group [(Hippocampal MD: control group: 0.54 ± 0.04; sham operation group: 0.54 ± 0.03; CLP group: 0.62 ± 0.04; CLP+FMT: 0.55 ± 0.03), (Hippocampal RD: control group: 0.44 ± 0.05; sham operation group: 0.44 ± 0.04; CLP group: 0.58 ± 0.06; CLP+FMT: 0.49 ± 0.04)]. The results of DTI studies showed that CLP could lead to damage to the integrity of nerve fibers in mice, and improving the integrity of nerve fibers after reducing ammonia levels expression Figure 4 a). The results of imaging studies showed that CLP could lead to brain edema, reduced cerebral blood flow, damage to the integrity of nerve fiber bundles, fecal microbiota transplantation, and reduced ammonia levels expression in mice, and the above-mentioned injuries were improved. Nissl staining of hippocampal tissues was completed 72 hours after CLP surgery. The research results showed that CLP could lead to a decrease in hippocampal neuron cells, and reducing ammonia levels could reduce the damage of CLP to neuron cells Figure 4b). Evans blue dye extravasation was used to evaluate the change in vascular permeability 72 hours after CLP. Evans blue extravasation into the whole brain was measured in the CLP group. A significant increase in Evans blue extravasation was observed in CLP mice, which was significantly reduced in CLP+FTM mice (Evans Blue: Control group: 5.00±0.36; Sham group: 4.82±0.20; CLP group: 9.29±0.89; CLP+FMT: 6.64±0.69)( Figure 4 c). CLP not only damages neuronal cells but also disrupts the blood-brain barrier. Reducing ammonia levels can alleviate the brain injury caused by CLP. On the 7th and 8th days after CLP, the cognitive memory function of novel object recognition was evaluated in each group of mice. The results showed that the discrimination index of mice decreased after CLP and increased after fecal microbiota transplantation (Discrimination index: Control group: 0.57±0.09; Sham group: 0.50±0.08; CLP group: 0.36±0.05; CLP+FMT: 0.47±0.06)( Figure 4 e). On the 9th - 13th days after CLP, the Morris water maze was used to evaluate the spatial learning and memory function of each group of mice. Compared with the sham group and the control group, the number of platform crossings and the time spent in the target quadrant of the CLP group were significantly reduced, and the escape latency was prolonged( Figure 4 f)[(Number of platform crossings: Control group: 5.50±1.60; Sham group: 5.13±1.46; CLP group: 1.75±1.04; CLP+FMT: 3.75±1.28),(Time spent in target quadrant: Control group: 34.16±8.27; Sham group: 31.22±7.38; CLP group: 12.30±5.31; CLP+FMT: 23.06±8.00)]. The results of behavioral studies showed that the cognitive function of mice was impaired after CLP, and reducing ammonia levels could improve the cognitive function of CLP mice. In addition, the success of the SAE model was further confirmed.

[0030] The results of Western blot, QT-PCR, and immunofluorescence staining showed that after 72 hours of CLP, AQP4 [(WB: Control group: 0.994±0.152; Sham group: 1.112±0.092; CLP

[0031] Group: 1.79 ± 0.30; CLP + FMT: 1.40 ± 0.22); (QT-PCR: Control group: 1.00045 ± 0.000327; Sham operation group: 1.048817 ± 0.874434; CLP group: 1.971100 ± 0.3162803; CLP + FMT: 1.49277 ± 0.180085] and GFAP [(WB: Control group: 0.97471 ± 0.253466; Sham operation group: 1.025539 ± 0.1616037; CLP group: 1.371542 ± 0.1455747; CLP + FMT: 1.23378 ± 0.141342); (QT-PCR: Control group: 1.00028 ± 0.000445; Sham operation group: 1.069033 ± 0.1339411; CLP group: 2.643267 ± 0.5571180; CLP + FMT: 1.81998 ± 0.274833] protein and gene expression, the number of GFAP positive cells (Control group: 42.17 ± 3.71; Sham operation group: 45.33 ± 4.76; CLP group: 61.00 ±

[0032] 3.41; CLP + FMT: 52.00 ± 4.24), the positive rate of the number of AQP4 cells in the hippocampal tissue accounting for the number of GFAP cells increased significantly (Control group: 23.26 ± 4.68; Sham operation group: 26.03 ± 3.68; CLP group: 34.52 ± 4.44; CLP + FMT: 26.35 ± 4.10)( Figure 5 a, Figure 5 b, Figure 5 c), after fecal microbiota transplantation, ammonia levels decreased (CLP group: 52.57 ± 6.27; CLP + FMT: 45.36 ± 2.16). In addition, 1H-MRS studies found that after CLP, the levels of Glu / Cr and NAA / C in the mouse brain decreased, indicating neuronal damage, while the level of Glx / Cr increased. After fecal bacteria transplantation, blood ammonia levels decreased, neuronal damage improved, and the level of Glx / Cr decreased ( Figure 5 d). At the same time, the use of the Elisa method to detect the glutamine levels in the blood and hippocampal tissue of mice in each group was consistent with the 1H-MRS results ( Figure 5 e). Previous studies have found that ammonia enters the brain, combines with glutamate to produce glutamine, and further activates the expression of astrocytic AQP4, leading to brain damage. To further verify the mechanism of action of NHH in the brains of CLP mice, the following cell experiments were conducted.

[0033] To test whether ammonia affects astrocytes, astrocytes were treated with NH4Cl for 72 h. Immunofluorescence results showed that astrocytes were activated, and the number of GFAP-positive cells increased significantly compared with the control group (109.33 ± 3.51 vs 185.67 ± 12.34)( Figure 6 a). Subsequently, the co-expression of AQP4 and GFAP also increased( Figure 6 a). WB and QT-PCR results showed that the expression levels of AQP4 protein (1.00 ± 0.07 vs 1.25 ± 0.11) and gene (1.00 ± 0.00 vs 1.30 ± 0.03) increased after NH4CL stimulation( Figure 6 b and Figure 6 c). To further demonstrate the effect of AQP4 on astrocytes, the inventors treated astrocytes with the AQP4 inhibitor TGN20. Immunofluorescence showed that the activation level of astrocytes decreased, the number of GFAP-positive cells decreased (172.00 ± 12.77), and the co-expression level with AQP4 decreased( Figure 6 a). The expression levels of AQP4 protein (0.90 ± 0.040) and gene (0.41 ± 0.05) decreased. Therefore, cell experiments confirmed that ammonia can induce an increase in AQP4 expression in astrocytes and activate astrocytes.

[0034] In summary, after sepsis, the intestinal flora is disordered, the ureaplasma is significantly increased, amino acid metabolism and ammonia production are increased, ammonia enters the brain and upregulates the AQP4 level of astrocytes, resulting in brain edema, cerebral ischemia, impaired integrity of nerve fiber structure, neuronal cell damage and cognitive dysfunction in CLP mice, which may be the potential mechanism of SAE. This example provides new insights into the pathological mechanism of SAE treatment and potential new drug targets.

[0035] For ease of description, spatial relative terms, such as "above", "over", "on the upper surface", "above", etc., can be used here to describe the spatial positional relationship of a device or feature shown in the figure with other devices or features. It should be understood that spatial relative terms are intended to include different orientations in use or operation in addition to the orientation described in the figure of the device. For example, if the device in the figure is inverted, the device described as "above" or "over" other devices or structures will be positioned "below" or "beneath" other devices or structures after inversion. Thus, the exemplary term "above" can include both the orientations of "above" and "below". The device can also be positioned in other different ways (rotated 90 degrees or in other orientations), and corresponding interpretations are made for the spatial relative descriptions used here.

[0036] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular forms are also intended to include the plural forms. In addition, it should also be understood that when the terms "comprise" and / or "include" are used in this specification, they specify the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0037] It should be noted that the terms "first", "second", etc. in the description, claims and above-mentioned drawings of the present application are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged under appropriate circumstances so that the embodiments of the present application described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "comprise" and "have" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, system, product or device that comprises a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units not clearly listed or inherent to these processes, methods, products or devices.

[0038] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for detecting the effect of NHH in septic encephalopathy using a CLP model, characterized in that: The steps include: In the CLP mouse model, under isoflurane anesthesia, after skin disinfection, a midline abdominal incision was made to expose the cecum; the cecum was ligated midway between the distal pole and the base of the cecum to induce moderate sepsis.

2. The method for detecting the effect of NHH in septic encephalopathy using the CLP model according to claim 1, characterized in that: The anesthesia concentration of isoflurane is 1-3%.

3. The method for detecting the effect of NHH in septic encephalopathy using the CLP model according to claim 1, characterized in that: The abdominal midline incision was 0.8-1.2 cm.

4. The method for detecting the effect of NHH in septic encephalopathy using the CLP model according to claim 1, characterized in that: The abdominal midline incision was sutured with sterile 6-0 silk sutures.

5. The method for detecting the effect of NHH in septic encephalopathy using the CLP model according to claim 1, characterized in that: After the CLP model was established, the feces of mice in each group were collected, and 16S rDNA sequencing technology was used to observe the changes in the types and abundance of microorganisms in each group.

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