Culture device for intestinal microbial metabolite detection and application in depression diagnosis

Abstract

The invention relates to a culture device for intestinal microbial metabolite detection and application in depression diagnos.The culture device comprises a shell and a flexible container which is arranged in the shell and used for containing excrement, a lantern ring is arranged outside the flexible container, and the lantern ring is formed by spirally surrounding an elastic sheet structure and provided with two end points; the two end points extend reversely, and telescopic supporting parts are connected between the two end points and the shell. A pushing mechanism is arranged on the side, away from the two end points, of the lantern ring and used for extruding and pushing the lantern ring, and the two end points can slide along the telescopic supporting part when the lantern ring is extruded and pushed. According to the technical scheme, by simulating the peristalsis of the intestinal tract and periodically extruding the detection sample in the container, a growth condition close to the in-vivo growth condition is provided for the intestinal flora in the detection sample, so that the accuracy of the intestinal flora machine metabolite during final detection is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of intestinal microbial metabolite detection, and in particular to a culture device for intestinal microbial metabolite detection and its application in diagnosing depression. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic, nonspecific intestinal inflammatory disease that affects the ileum, colon, and rectum and is more common in young and middle-aged people.

[0003] The onset of IBD is closely related to genetic susceptibility, environmental factors, intestinal microecology and immunity. Due to the nature of chronic diseases, IBD requires lifelong treatment, which affects the quality of life of patients. The connection between IBD and the nervous system has been widely studied. Literature reports that the incidence of emotional problems such as depression and anxiety in IBD patients is significantly higher than that in the general population, but most patients do not receive the mental health treatment they need. A systematic review found that the incidence of depression in patients with active IBD was as high as 34.7%, and the incidence of depression in patients with inactive IBD was about 19.9%. IBD patients with depressive symptoms have an increased risk of outbreaks, treatment escalation, hospitalization, emergency department visits and surgery; similarly, patients with depression are often diagnosed with IBD, and psychiatric symptoms are associated with adverse disease activity in IBD. Therefore, comorbidity between mental illness and gastrointestinal diseases is common.

[0004] The concept of "brain-gut axis" proposed in recent years explains to a certain extent the correlation between the above-mentioned comorbidities, that is, there is a complex bidirectional regulation between the brain and the gastrointestinal tract, and the mechanisms involved include inflammatory immune response, autonomic nervous system, enteric nervous system, and the role of intestinal microbiota and its metabolites. In other words, depression can be predicted based on the intestinal flora and its metabolites.

[0005] Before testing the intestinal flora and its metabolites, the test samples need to be cultured to expand the base of the flora and its metabolites. Currently, when culturing intestinal flora, a stable and suitable growth environment needs to be provided, including constant temperature, humidity and the necessary supply of mixed gases required for growth.

[0006] However, some existing intestinal flora culture devices are relatively simple in structure, and the culture dishes are often fixed inside the devices and cannot be moved. They cannot simulate the bending and expansion and contraction of the intestines, and thus cannot establish an intestinal flora ecosystem in vitro and cultivate most intestinal flora. Summary of the invention

[0007] In view of this, the purpose of the present invention is to provide a culture device for detecting intestinal microbial metabolites to solve the problem that the culture dishes in the prior art are often fixedly connected inside the device and cannot move, and cannot simulate the bending and expansion and contraction of the intestine, thereby failing to establish an intestinal bacterial ecosystem in vitro and cultivate the majority of intestinal flora.

[0008] The present invention is achieved through the following technical solutions:

[0009] A culture device for detecting intestinal microbial metabolites, comprising a shell and a flexible container arranged in the shell for accommodating excrement, wherein a collar is arranged outside the flexible container, the collar is composed of an elastic sheet structure spirally wound around, and has two end points, the two end points extend in opposite directions, and are both connected to the shell with a telescopic support part;

[0010] A pushing mechanism is arranged on the side of the collar away from the two end points, and the pushing mechanism is used to squeeze and push the collar, and the two end points can slide along the telescopic support part when the collar is squeezed and pushed.

[0011] In this embodiment, the pushing mechanism includes a push rod and a driving part for pushing the push rod. One end of the push rod extends outward through the shell and is connected to the driving part, and the other end is connected to a push plate that rests on the outer wall of the ring. The push rod slides with the shell.

[0012] In this embodiment, the driving part includes a sleeve extending parallel to the push rod, the sleeve is arranged outside the push rod, one end of the sleeve is fixedly connected to the housing, and the other end is installed with a sealing cover, and a vent hole is opened on the sealing cover;

[0013] A piston is slidably and sealingly fitted in the sleeve, and the piston is connected to the end of the push rod.

[0014] In this embodiment, an air supply mechanism is also included, the air supply mechanism includes a mounting frame and a rotating plate rotatably mounted in the mounting frame, a gas injection hole penetrating along the thickness direction is opened at the center of the top surface of the rotating plate, a gas guide groove is opened at the center of the bottom surface of the rotating plate, the gas guide groove is connected to the gas injection hole, an exhaust hole penetrating along the thickness direction is opened at the bottom of the gas guide groove, an exhaust groove is opened on the top surface of the rotating plate, the exhaust groove extends out of the side of the rotating plate, and the exhaust groove and the exhaust hole are symmetrically arranged on opposite sides of the center of the rotating plate;

[0015] The mounting frame includes a mounting ring and a cover plate, the mounting ring is sleeved outside the bottom surface of the rotating plate and fixedly mounted on the side wall of the housing, the cover plate covers the top surface of the rotating plate, a plurality of connecting rods are arranged between the edge of the cover plate and the mounting ring, and the two ends of the connecting rods are fixedly connected to the cover plate and the mounting ring respectively;

[0016] The cover plate is connected with an air inlet joint and an air outlet joint, the air inlet joint is opposite to and connected with the air injection hole, the air outlet joint is located on the rotation track of the air outlet hole and the air outlet groove, and the air outlet joint is connected with the air vent through a pipe;

[0017] A driving motor for driving the rotating plate to rotate is installed on the shell.

[0018] In this embodiment, the driving motor is installed in the housing, an annular support platform is provided at the center of the bottom surface of the air guide groove, and a plurality of limiting teeth are provided on the outer circumference of the annular support platform;

[0019] A transmission plate is arranged in the annular support platform, the center of the transmission plate is connected to the output shaft of the driving motor, and a plurality of limiting grooves are opened on the outer edge, and the limiting grooves are engaged with the limiting teeth;

[0020] The transmission plate is provided with a plurality of air guide holes communicating with the air guide grooves and the air injection holes.

[0021] In this embodiment, at least two of the collars are provided, and the two collars are longitudinally overlapped to form a tubular structure sleeved outside the flexible container.

[0022] In this embodiment, the two telescopic support parts both include a support rod and a support tube connected to the joint, one end of the two support rods are respectively connected to the two joints, and the other end extends backwards, one end of the two support tubes are respectively sleeved on the end of the two support rods away from the joint, and the other end is connected to the shell.

[0023] In this embodiment, a support spring is arranged in the support tube, and two ends of the support spring abut against the end of the support rod and the housing respectively.

[0024] In this embodiment, the shell is provided with through holes respectively communicating with the interiors of the two support tubes.

[0025] In this embodiment, the ring is wrapped with a flexible protective layer.

[0026] The beneficial effects of the present invention are:

[0027] The intestinal microbial metabolite detection culture device simulates the peristalsis of the intestine to periodically squeeze the detection sample in the container, thereby providing the intestinal flora in the detection sample with growth conditions close to those in the body, thereby improving the accuracy of the intestinal flora machine metabolites in the final detection.

[0028] Other advantages, objectives and features of the present invention will be described in the following description to some extent, and to some extent, will be obvious to those skilled in the art based on the following examination and study, or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The three-dimensional Figure 1 ;

[0030] Figure 2 The three-dimensional Figure 2 ;

[0031] Figure 3 It is a schematic diagram of the internal structure of the present invention;

[0032] Figure 4 is a schematic diagram of the structure of the driving mechanism;

[0033] Figure 5 It is a structural schematic diagram of the telescopic support part;

[0034] Figure 6 is a schematic diagram of the top surface structure of the rotating plate;

[0035] Figure 7 is a schematic diagram of the bottom structure of the rotating plate;

[0036] Figure 8 It is a schematic diagram of the connection structure between the rotating plate and the driving motor;

[0037] Fig. 9 is a schematic diagram of the structure of the ring;

[0038] Fig.10 Schematic diagram of the integration and differential expression analysis of I BD datasets;

[0039] Fig.11 Illustration of the important module genes in the MDD dataset screened by WGCNA;

[0040] Fig.12 Schematic diagram of PPI analysis between IBD-related secretory proteins and important MDD genes;

[0041] Fig.13 Illustration of potential diagnostic biomarkers for IBD-related MDD identified by machine learning methods;

[0042] Fig.14 Provide illustrations for the development of diagnostic nomogram models and efficacy evaluation;

[0043] Fig.15Schematic representation of single gene enrichment analysis and immune infiltration analysis of candidate biomarkers;

[0044] Fig.16 Schematic representation of the expression patterns of four candidate diagnostic markers in serum samples of I BD and I BD-MDD validated for cohort data;

[0045] In the figure: 1. shell; 2. flexible container; 3. sleeve; 4. end point; 5. push rod; 6. push plate; 7. sleeve; 8. sealing cover; 9. vent; 10. piston; 11. rotating plate; 12. air injection hole; 13. air guide groove; 14. exhaust hole; 15. exhaust groove; 16. mounting ring; 17. cover plate; 18. connecting rod; 19. driving motor; 20. limiting tooth; 21. transmission plate; 22. limiting tooth; 23. air guide hole; 24. support rod; 25. support tube; 26. support spring; 27. through hole; 28. flexible protective layer. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Generally, the components of the embodiments of the present invention described and shown in the drawings here can be arranged and designed in various different configurations.

[0047] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, further definition and explanation thereof is not required in subsequent drawings.

[0049] In the above description of the present invention, it should be noted that the terms "one side", "the other side", etc. indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, or the orientation or positional relationship in which the product of the invention is usually placed when in use, which is only for the convenience of describing the present invention and simplifying the description, and does not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", etc. are only used to distinguish the description, and cannot be understood as indicating or implying relative importance.

[0050] In addition, the term "same" does not mean that the parts must be absolutely the same, but slight differences are allowed. The term "vertical" only means that the positional relationship between the parts is more vertical than "parallel", and does not mean that the structure must be completely vertical, but can be slightly tilted.

[0051] See also Figure 1-9 The present invention provides a technical solution: a culture device for detecting intestinal microbial metabolites, comprising a shell 1 and a flexible container 2 arranged in the shell 1 for accommodating excrement, wherein a collar 3 is arranged outside the flexible container 2, wherein the collar 3 is composed of an elastic sheet structure spirally wound around and has two end points 4, wherein the two end points 4 extend in opposite directions and are connected to the shell 1 with a telescopic support portion;

[0052] The collar 3 is provided with a pushing mechanism on one side away from the two end points 4, and the pushing mechanism is used to squeeze and push the collar 3, and the two end points 4 can be squeezed and pushed by the collar 3 to slide along the telescopic support portion.

[0053] The shell 1 can be a sealed space, and its interior can maintain stable temperature and humidity by connecting other external devices to provide stable growth conditions for intestinal flora. The flexible container 2 is installed in the shell 1 for placing the test sample, and a collar 3 is arranged on the outer shell. The inner wall of the collar 3 can be connected and fixed to the outer wall of the flexible container 2 by adhesion. The collar 3 is made of a sheet structure with elasticity, and can be made of plastic or thin steel sheet. The collar 3 is spiral, and the collar 3 has two end points 4. When the side away from the end point 4 is pressurized, the two end points 4 move to both sides respectively.

[0054] In the present solution, the pushing mechanism is used to squeeze the side of the ring 3 away from the outlet, and the telescopic support parts respectively constitute the sliding track limiting structures of the two end points 4. When the two end points 4 of the ring 3 are respectively moved to both sides under the push of the pushing mechanism, the ring 3 will shrink inward as a whole to squeeze the outer wall of the flexible container 2. When the ring 3 shrinks inward, it can store energy elastically, and the pushing mechanism can push back and forth. When the thrust of the pushing mechanism is cancelled, the ring 3 can expand through its own elastic reset, causing the flexible container 2 to expand, which can simulate the peristalsis of the intestine and periodically squeeze the test sample in the container, providing the intestinal flora in the test sample with growth conditions close to those in the body, thereby improving the accuracy of the intestinal flora machine metabolites in the final test.

[0055] In this embodiment, the pushing mechanism includes a push rod 5 and a driving part for pushing the push rod 5. One end of the push rod 5 extends outward through the shell 1 and is connected to the driving part, and the other end is connected to a push plate 6 that rests on the outer wall of the ring 3. The push rod 5 is slidably matched with the shell 1.

[0056] The push rod 5 can be moved under the action of the driving part to push the side of the ring 3 away from the opening. The push plate 6 is connected to the end of the push rod 5 close to the ring 3, which can increase the contact area with the outer wall of the ring 3, so that it can push and squeeze the ring 3 more stably. The push plate 6 can be a V-shaped structure that is engaged with the outside of the ring 3, and its upper and lower sides both extend toward one side of the ring 3, so that the side facing the ring 3 is a groove-shaped structure, and the ring 3 is clamped in the groove-shaped structure, which can further improve the stability of the connection structure with the ring 3.

[0057] In this embodiment, the driving part includes a sleeve 7 extending parallel to the push rod 5, and the sleeve is arranged outside the push rod 5. One end of the sleeve 7 is fixedly connected to the housing 1, and the other end is installed with a sealing cover 8, and a vent hole 9 is opened on the sealing cover 8;

[0058] A piston 10 is slidably and sealingly fitted in the sleeve 7 , and the piston 10 is connected to the end of the push rod 5 .

[0059] One end of the sleeve 7 is fixedly connected to the housing 1, and the end is a closed structure. The other end is sealed by setting a sealing cover 8. A vent hole 9 is provided on the sealing cover 8, and air or liquid can be pressed into the sleeve 7 through the vent hole 9. When the air or liquid is pressed into the sleeve 7, the piston 10 located in the sleeve 7 will be pushed and moved by the pressure of the air or liquid, that is, the push rod 5 is driven to move and squeeze the ring 3. When the air or liquid stops being introduced into the sleeve 7, the push rod 5 will move in the reverse direction under the elastic reset action of the ring 3, and the air or liquid in the sleeve 7 will be discharged from the vent hole 9 in the reverse direction through the piston 10.

[0060] In this embodiment, an air supply mechanism is also included, and the air supply mechanism includes a mounting frame and a rotating plate 11 rotatably mounted in the mounting frame, a gas injection hole 12 penetrating along the thickness direction is opened at the center of the top surface of the rotating plate 11, a gas guide groove 13 is opened at the center of the bottom surface of the rotating plate 11, the gas guide groove 13 is connected to the gas injection hole 12, an exhaust hole 14 penetrating along the thickness direction is opened at the bottom of the gas guide groove 13, an exhaust groove 15 is opened on the top surface of the rotating plate 11, the exhaust groove 15 extends out of the side of the rotating plate 11, and the exhaust groove 15 and the exhaust hole 14 are symmetrically arranged on opposite sides of the center of the rotating plate 11;

[0061] The mounting frame includes a mounting ring 16 and a cover plate 17. The mounting ring 16 is sleeved outside the bottom surface of the rotating plate 11 and fixedly mounted on the side wall of the housing 1. The cover plate 17 covers the top surface of the rotating plate 11. A plurality of connecting rods 18 are arranged between the edge of the cover plate 17 and the mounting ring 16. The two ends of the connecting rods 18 are fixedly connected to the cover plate 17 and the mounting ring 16 respectively.

[0062] The cover plate 17 is connected with an air inlet joint and an air outlet joint, the air inlet joint is opposite to and connected with the air injection hole 12, the air outlet joint is located on the rotation track of the air outlet 14 and the air outlet groove 15, and the air outlet joint is connected with the air vent 9 through a pipeline;

[0063] The housing 1 is provided with a driving motor 19 for driving the rotating plate 11 to rotate.

[0064] The mounting frame is composed of a cover plate 17, a connecting rod 18 and a mounting ring 16. The connecting rod 18 serves as a connecting structure between the cover plate 17 and the mounting ring 16, so that the cover plate 17 and the mounting ring 16 can maintain structural stability, and the cover plate 17 and the mounting ring 16 are hollowed out. The rotating plate 11 is located in the mounting frame, and the top and bottom surfaces of the rotating plate 11 are respectively sealed with the bottom surface of the cover plate 17 and the outer wall of the shell 1, and the air inlet joint on the cover plate 17 and the air injection hole 12 on the rotating plate 11 are opposite and connected, and air or liquid can be introduced into the air inlet joint through an external air source, and the introduced air or liquid (hereinafter referred to as "air or liquid" as "fluid") will enter the air injection hole 12;

[0065] In addition, since the bottom surface of the rotating plate 11 is provided with an air guide groove 13, the bottom of the air guide groove 13 is provided with an exhaust hole 14, and the top surface of the rotating plate 11 is provided with an exhaust groove 15, the exhaust interface is located on the rotation track of the exhaust hole 14 and the exhaust groove 15, during the rotation process of the rotating plate 11, the exhaust groove 15 and the exhaust hole 14 will rotate synchronously with the rotating plate 11;

[0066] When the exhaust hole 14 rotates to be connected with the exhaust joint, the fluid entering the air injection hole 12 will pass through the exhaust hole 14 and be connected with the exhaust joint, and finally enter the sleeve 7 through the pipeline and the vent hole 9 to push the piston 10 to move;

[0067] When the exhaust groove 15 rotates to be relatively connected with the exhaust joint, the fluid in the sleeve 7 will be elastically reset at the collar 3 to push the piston 10 to move and be discharged from the vent hole 9 and then discharged from the exhaust groove 15 through the pipeline;

[0068] As described above, it is only necessary to continuously introduce fluid into the air inlet connector, and the fluid can be intermittently introduced into the sleeve 7 under the rotation of the rotating plate 11, and the fluid in the cylinder can be discharged through the elastic reset of the ring 3 in the gap of the introduced fluid.

[0069] In this embodiment, the driving motor 19 is installed in the housing 1, and an annular support platform is provided at the center of the bottom surface of the air guide groove 13, and a plurality of limiting teeth 20 are provided on the outer circumference of the annular support platform;

[0070] A transmission plate 21 is arranged in the annular support platform, the center of the transmission plate 21 is connected to the output shaft of the driving motor 19, and a plurality of limiting grooves 22 are opened on the outer edge, and the limiting grooves 22 are engaged with the limiting teeth 20;

[0071] The transmission plate 21 is provided with a plurality of air guide holes 13 communicating with the air guide grooves 13 and the air injection holes 12 .

[0072] The driving motor 19 serves as a power source for driving the rotating plate 11 to rotate, and its output shaft is connected to the transmission plate 21. The transmission plate 21 cooperates with a number of limit teeth 20 in the annular sink to realize the transmission connection between the driving motor 19 and the rotating plate 11. The air guide hole 13 opened on the transmission plate 21 is used to connect the air guide groove 13 and the air injection hole 12 to provide a channel for fluid flow.

[0073] In this embodiment, at least two of the collars 3 are provided, and the two collars 3 are longitudinally overlapped to form a tubular structure sleeved outside the flexible container 2 .

[0074] If two sleeve rings 3 are provided, the exhaust joints on the cover plate 17 and the exhaust holes 14 provided on the rotating plate 11 are provided with two opposite ones, and the sleeves 7 etc. are provided with the same number, then the two sleeve rings 3 will work alternately during the rotation of the rotating plate 11, and intermittently squeeze different parts of the flexible container 2.

[0075] In this embodiment, the two telescopic support parts each include a support rod 24 and a support tube 25 connected to the joint, one end of the two support rods 24 is respectively connected to the two joints, and the other end extends backwards, one end of the two support tubes 25 is respectively sleeved on the end of the two support rods 24 away from the joint, and the other end is connected to the shell 1.

[0076] The support rod 24 and the support tube 25 are slidably matched, and the support rod 24 can slide in the support tube 25 , wherein the support rod 24 serves as a structural extension of the joint, and the support tube 25 serves as a sliding track of the support tube 25 .

[0077] In this embodiment, a support spring 26 is disposed in the support tube 25 , and two ends of the support spring 26 abut against the end of the support rod 24 and the housing 1 respectively.

[0078] The support spring 26 is used to provide elastic support for the support rod 24. When the ring 3 is squeezed and contracted, the support rod 24 will squeeze the support spring 26 by sliding, and the support spring 26 and the ring 3 will shrink synchronously to elastically store force. When the squeezing force is cancelled, the support spring 26 will push the support rod 24 to assist the ring 3 in resetting through elastic reset.

[0079] In this embodiment, the housing 1 is provided with through holes 27 which are respectively connected to the insides of the two support tubes 25 .

[0080] The through hole 27 can serve as a passage connecting the interior of the support tube 25 with the outside space. When the support rod 24 slides in the support tube 25, the air inside the support tube 25 and the outside air will be discharged or enter the support tube 25 through the through hole 27, avoiding obstruction to the sliding of the support rod 24.

[0081] In this embodiment, the collar 3 is wrapped with a flexible protective layer 28 .

[0082] The flexible protective layer 28 wrapped outside the sleeve 3 can protect the flexible container 2 and reduce the damage to the flexible container 2 caused by the sleeve 3 shrinking and squeezing the flexible container 2.

[0083] An application of intestinal microbial metabolite detection in diagnosing depression, the specific steps of which are:

[0084] Secretory protein gene

[0085] A total of 3946 secretory protein-encoding genes were downloaded from the Human Protein Atlas database (https: / / www.proteinatlas.org / ).

[0086] Weighted gene co-expression network analysis

[0087] WGCNA is used to study the core genes in gene expression data by identifying co-expressed gene modules and exploring the relationship between gene sets and phenotypes. In this study, the WGCNA package was used to construct a scale-free co-expressed gene network for the MDD peripheral blood dataset GSE98793. WGCNA was performed by screening the top 25% of differentially expressed genes, setting the fitting index R2 = 0.9 and the soft threshold β = 10 to ensure that the co-expressed gene network remains scale-independent. After dynamic pruning and TOM hierarchical clustering analysis (minModuleSize = 100), the most important module genes related to the disease were finally determined for subsequent analysis.

[0088] PPI and MCODE analysis

[0089] To investigate the interaction between IBD-related secretory proteins and important genes in MDD. Based on the STRING database (https: / / www.string-db.org), a PPI network related to IBD and MDD was constructed with a medium confidence score of >0.4. Cytoscape software was used to visualize the PPI network, and the MCODE plug-in of Cytoscape was used to calculate the nodes. The parameters were set as follows: DegreeCutoff: 2, Haircut, nodescorecutoff: 0.2, k-core2, MaxDepth100, and the top two modules were finally selected for further analysis.

[0090] Functional enrichment analysis

[0091] In order to clarify the biological functions and pathways of pathogenic genes associated with IBD and MDD, clusterProfiler package was used to perform GO and KEGG enrichment analysis on candidate genes in this study, and ggplot2 package was used to visualize the enrichment analysis results.

[0092] Machine Learning

[0093] To further investigate potential candidate genes for diagnosing IBD-related MDD, the glmnet package was used for the LASSO logistic regression method, while the randomForest package was used for the RF algorithm. Two machine learning methods were used to narrow down the range of candidate biomarkers. Finally, the intersection of the results of the two algorithms was used as candidate genes for diagnosis. In the training set and validation set, the pROC package was used to draw the ROC curve and AUC value of the characteristic genes.

[0094] Constructing nomograms and evaluating prediction models

[0095] To determine the importance of candidate genes for the diagnosis of IBD-related MDD. In this study, the rms package was used to draw a nomogram of characteristic genes. The nomogram consists of "integral" and "total integral", the latter showing the sum of the scores of all genes. The nomogram is an important tool for clinical prediction of IBD-related MDD. The clinical prediction efficiency of the nomogram was subsequently evaluated by calibration curve and decision curve analysis (DCA). The ROC curve was used to evaluate the effect of the nomogram in the prediction of IBD-related MDD, and the AUC value>0.7 was considered clinically significant. Finally, the prediction efficiency of the nomogram was verified using the external MDD dataset GSE39653.

[0096] Validation of expression of signature genes in MDD and IBD

[0097] The characteristic genes obtained after LASSO regression analysis and RF analysis were verified for expression in the MDD and IBD datasets, respectively. The ggplot2 package was used to compare and visualize the expression levels of the characteristic genes, and finally the genes that were consistently upregulated or downregulated in IBD and MDD patients were obtained.

[0098] GSEA

[0099] In the MDD dataset GSE39653, cluster enrichment analysis was performed on each signature gene using the org.Hs.eg.db, clusterProfiler package to compare differential signaling pathways between MDD and healthy controls. The Enrichplot package was used to display the top 10 activated and repressed pathways for each gene in MDD.

[0100] Immune infiltration analysis

[0101] In the MDD dataset GSE39653, the CIBERSORT package was used to assess the number of infiltrating immune cells in the disease. The Wilcoxon test was used to compare the differences in the proportions of 22 immune cells between MDD and healthy control samples, where p<0.05 was statistically significant. The ggplot2 package displayed the infiltration of 22 immune cells. The Spearman rank correlation coefficient was used to analyze the correlation between the expression of diagnostic biomarkers and infiltrating immune cells, where p<0.05 was statistically significant.

[0102] I Sample collection from patients with BD and MDD

[0103] This study included I BD patients who visited the Department of Gastroenterology of Chongqing People's Hospital from January to June 2024. The control group consisted of 20 I BD patients and the experimental group consisted of 20 I BD patients with MDD, totaling 40 patients. I BD patients with MDD were diagnosed according to DSM-5, and the 17-item Hamilton Depression Rating Scale (HAMD-17) was used to assess the patients' depressive symptoms. Patients with other mental disorders other than MDD and those who had taken antidepressants within 2 weeks were excluded. This study was approved by the Clinical Research Ethics Committee of Chongqing People's Hospital, all methods were in compliance with relevant guidelines and regulations, and all participants signed informed consent.

[0104] Evaluation of the diagnostic model in an external cohort

[0105] According to the manufacturer's protocol, serum samples from IBD patients with or without MDD were analyzed using ELISA kits (Wuhan, China) to determine the expression levels of serum HGF, SPARC, ADAM12, and MMP8.

[0106] Statistical analysis

[0107] All data were expressed as mean ± standard deviation, and SPSS was used for statistical analysis. The differences between the two groups were compared by unpaired Student's t-test. P < 0.05 was considered statistically significant.

[0108] Among them, Fig.10As shown in the figure, A is the volcano plot of DEGs in IBD intestinal tissue samples and the heat map of the top 50 up-regulated and down-regulated DEGs. B is the volcano plot of DEGs in IBD peripheral blood samples and the heat map of the top 50 up-regulated and down-regulated DEGs. The up-regulated genes in the volcano plot are represented by red dots, and the down-regulated genes are represented by blue dots. C is the intersection of intestinal tissue and peripheral blood samples with secretory protein genes through the Venn diagram, and 463 IBD intestinal tissue-related secretory proteins and IBD-PBMC-related secretory proteins were obtained. The intersection was further taken to obtain 484 IBD-related secretory protein genes.

[0109] like Fig.11 As shown, A and B are the optimal β values ​​determined by the scale-free topological model, and β=10 is selected as the soft threshold based on the average connectivity and scale independence. C is a network heat map showing the gene dendrogram and module characteristic genes. D is a heat map revealing the relationship between module characteristic genes and MDD status. The correlation (top) and p-value (bottom) of the module characteristic genes and the status of MDD are given. The gray module associated with MDD showed the highest correlation coefficient and was identified as the key module of MDD. E is a correlation diagram of the gray module members and the gene significance of the genes in the gray module.

[0110] like Fig.12 As shown in Figure 1, A is the PPI network of the two module genes with the highest scores based on the Cytoscape plug-in MCODE analysis. B and C are bubble charts showing the results of GO and KEGG enrichment analysis.

[0111] like Fig.13 As shown, A is a Venn diagram of 21 overlapping genes of IBD-related secretory proteins and MDD disease key genes. B and C are MeanDecreaseGin i of 21 genes in MDD using the RF algorithm. D and E are the minimum and l ambda values ​​of diagnostic biomarkers identified using the LASSO logistic regression algorithm. F is the intersection of the candidate genes of the LASSO and RF algorithms, and finally obtained 4 common potential pathogenic characteristic genes HGF, SPARC, ADAM12, and MMP8. G and H are the expression levels of the 4 characteristic genes in the MDD dataset (GSE98793) and IBD dataset.

[0112] like Fig.14As shown, A is a nomogram constructed based on diagnostic biomarkers. B is the calibration curve predicted by the nomogram model in IBD-related MDD. The dotted line is marked as "ideal" and represents the standard curve, representing the perfect prediction of the ideal model. The dotted line is marked as "visual" and represents the uncalibrated prediction curve, while the solid line is marked as "bias-corrected" and represents the calibrated prediction curve. C is the DCA of the nomogram model. The black line is marked as "none" and represents the net benefit of assuming that no patient has MDD. The gray line is marked as "all" and represents the net benefit of assuming that all patients have MDD, and the red line is marked as "nomogram" and represents the hypothetical net benefit of identifying IBD-related MDD based on the MDD diagnostic value predicted by the nomogram model. D is the ROC curve of the diagnostic performance of the nomogram model in predicting MDD in the GEO database internal data set. E is the ROC curve of the diagnostic performance of the nomogram model in predicting MDD in the GEO database external data set. ROC receiver operating characteristic, DCA decision curve analysis.

[0113] like Fig.15 GSEA analysis and KEGG enrichment of the four characteristic genes in A. The violin plot in B shows the comparison of 22 immune cells between the MDD group and the control group. The heat map in C reveals the correlation of the infiltration of 22 immune cells at the p < 0.05 threshold.

[0114] like Fig.16 As shown in Figure 1, the ELISA results in A showed that the levels of HGF and MMP8 in the serum of patients with IBD and MDD were significantly increased, SPARC had an increasing trend, and ADAM12 had no difference. The nomogram in B was developed based on the combination of four diagnostic biomarkers to predict the risk of MDD. The calibration curve of the nomogram prediction in MDD patients in C. The ROC curve of the predictive performance of the four candidate biomarkers and the nomogram in D.

[0115] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A culture device for detecting intestinal microbial metabolites, characterized in that: It comprises a shell and a flexible container for containing excrement arranged in the shell, wherein a collar is arranged outside the flexible container, wherein the collar is formed by an elastic sheet structure spirally wound around and has two end points, the two end points extend in opposite directions, and a telescopic support portion is connected to the shell; A pushing mechanism is arranged on the side of the collar away from the two end points, and the pushing mechanism is used to squeeze and push the collar, and the two end points can slide along the telescopic support part when the collar is squeezed and pushed.

2. The intestinal microbial metabolite detection culture device according to claim 1, characterized in that: The pushing mechanism includes a push rod and a driving part for pushing the push rod. One end of the push rod extends outward through the shell and is connected to the driving part, and the other end is connected to a push plate abutting against the outer wall of the ring. The push rod is slidably matched with the shell.

3. The intestinal microbial metabolite detection culture device according to claim 2, characterized in that: The driving part comprises a sleeve extending parallel to the push rod, the sleeve is arranged outside the push rod, one end of the sleeve is fixedly connected to the housing, and the other end is installed with a sealing cover, and a vent hole is opened on the sealing cover; A piston is slidably and sealingly fitted in the sleeve, and the piston is connected to the end of the push rod.

4. The intestinal microbial metabolite detection culture device according to claim 3, characterized in that: It also includes an air supply mechanism, the air supply mechanism includes a mounting frame and a rotating plate rotatably mounted in the mounting frame, a gas injection hole penetrating along the thickness direction is opened at the center of the top surface of the rotating plate, a gas guide groove is opened at the center of the bottom surface of the rotating plate, the gas guide groove is connected to the gas injection hole, an exhaust hole penetrating along the thickness direction is opened at the bottom of the gas guide groove, an exhaust groove is opened on the top surface of the rotating plate, the exhaust groove extends out of the side of the rotating plate, and the exhaust groove and the exhaust hole are symmetrically arranged on opposite sides of the center of the rotating plate; The mounting frame includes a mounting ring and a cover plate, the mounting ring is sleeved outside the bottom surface of the rotating plate and fixedly mounted on the side wall of the housing, the cover plate covers the top surface of the rotating plate, a plurality of connecting rods are arranged between the edge of the cover plate and the mounting ring, and the two ends of the connecting rods are fixedly connected to the cover plate and the mounting ring respectively; The cover plate is connected with an air inlet joint and an air outlet joint, the air inlet joint is opposite to and connected with the air injection hole, the air outlet joint is located on the rotation track of the air outlet hole and the air outlet groove, and the air outlet joint is connected with the air vent through a pipe; A driving motor for driving the rotating plate to rotate is installed on the shell.

5. The intestinal microbial metabolite detection culture device according to claim 4, characterized in that: The driving motor is installed in the housing, an annular support is provided at the center of the bottom surface of the air guide groove, and a plurality of limiting teeth are provided on the outer circumference of the annular support; A transmission plate is arranged in the annular support platform, the center of the transmission plate is connected to the output shaft of the driving motor, and a plurality of limiting grooves are opened on the outer edge, and the limiting grooves are engaged with the limiting teeth; The transmission plate is provided with a plurality of air guide holes communicating with the air guide grooves and the air injection holes.

6. The intestinal microbial metabolite detection culture device according to any one of claims 1 to 5, characterized in that: At least two of the collars are provided, and the two collars are longitudinally overlapped to form a tubular structure sleeved outside the flexible container.

7. The intestinal microbial metabolite detection culture device according to claim 6, characterized in that: The two telescopic support parts each include a support rod and a support tube connected to a joint, one end of the two support rods are respectively connected to the two joints, and the other end extends backwards, one end of the two support tubes is respectively sleeved on one end of the two support rods away from the joint, and the other end is connected to the shell.

8. The intestinal microbial metabolite detection culture device according to claim 7, characterized in that: A support spring is arranged in the support tube, and two ends of the support spring respectively abut against the end of the support rod and the shell.

9. The intestinal microbial metabolite detection culture device according to claim 8, characterized in that: The shell is provided with through holes which are respectively communicated with the insides of the two support tubes.

10. Use of the intestinal microbial metabolite detection culture device according to any one of claims 1 to 8 in diagnosing depression.

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