Application of kstD-like protein as a molecular marker in the preparation of products for assessing psychological tolerance

Abstract

The present invention provides the use of kstD-like protein as a molecular marker in the preparation of a product for assessing psychological tolerance. The present application found that kstD-like protein is an effective auxiliary molecular marker for depression diagnosis, and the expression of kstD-like protein is negatively correlated with the symptoms in patients with depression. The kstD-like protein is used as a molecular marker to prepare a diagnostic test kit to quickly assess psychological tolerance, with a sensitivity of more than 87.5% and a specificity of more than 75.0%.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the use of kstD-like protein as a molecular marker in the preparation of a product for evaluating psychological endurance. Background Art

[0002] Major depressive disorder (MDD) is widely recognized as one of the most pressing mental health problems. The number of incident cases has increased by nearly 50% worldwide over the past 30 years, and 264 million people of all ages are currently affected. Up to 60% of people with MDD respond to various medications to some degree. After the initial depressive episode, recurrence over the life course is particularly problematic; estimates range as high as 75–90%. According to the World Health Organization, depression is the leading cause of mental and physical disability worldwide and the leading cause of global disease burden.

[0003] Chronic psychological stress is one of the most important risk factors for depression. About 92% of people with depression have experienced stressful events before the onset of the disease. The body activates the hypothalamic-pituitary-adrenal (HPA) axis in response to stress. Cortisol is a stress hormone and the main effector of the HPA axis stress response system. Long-term exposure to cortisol can lead to depressive symptoms. 20-80% of people with depression show some form of elevated cortisol levels. It is worth thinking about that not everyone who faces stress will develop depression. Among people who have experienced similar traumatic or tragic events, only 10-30% of them suffer from depression. People with poor psychological tolerance are more likely to suffer from depression when facing stress.

[0004] Understanding psychological resilience is crucial for the development of strategies for the prevention and treatment of depression. There is currently no marker for assessing psychological resilience, and finding such a marker remains a daunting challenge. Therefore, there is an urgent need to find markers for assessing psychological resilience. Summary of the invention

[0005] The purpose of the present invention is to provide the use of kstD-like protein as a molecular marker in the preparation of a product for assessing psychological tolerance. The present invention has found through experiments that the expression of kstD-like protein is increased in BPH patients and is positively correlated with clinical symptoms.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] In a first aspect of the present invention, there is provided the use of kstD-like protein as a molecular marker in the preparation of a product for assessing psychological endurance.

[0008] Furthermore, compared with healthy people, the expression level of kstD-like protein in the serum of patients with depression was significantly reduced.

[0009] Furthermore, the product for diagnosing prostate cancer includes a detection kit for kstD-like protein.

[0010] The kstD-like protein detection kit includes an ELISA detection kit for kstD-like protein.

[0011] The detection kit of the kstD-like protein includes a real-time fluorescence quantitative PCR kit.

[0012] The detection kit for the kstD-like protein includes an immunohistochemistry detection kit.

[0013] In a second aspect of the present invention, provided is the use of a detection reagent for kstD-like protein in the preparation of a product for assessing psychological endurance.

[0014] The detection reagent of the kstD-like protein includes an immunohistochemical reagent of the kstD-like protein.

[0015] In terms of the sensitivity and specificity of kstD-like protein as a marker for assessing psychological tolerance, the AUC of kstD-like protein was 0.857, the sensitivity was 87.5%, and the specificity was 75.0%.

[0016] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0017] The present invention provides the use of kstD-like protein as a molecular marker in the preparation of a product for evaluating psychological tolerance. The study found that intestinal bacteria expressing kstD-like protein improved cortisol-induced depressive-like behavior. When intestinal bacteria do not express kstD-like protein, mice are susceptible to cortisol and tend to develop depressive-like behavior. It shows that the expression level of kstD-like protein can be used as a marker for diagnosing depressive-like behavior. When the abundance of kstD-like protein is lower than 0.417, it is diagnosed as a stress-susceptible population, indicating that it is easy to develop depression under emotional stress; when it is higher than 0.417, it is diagnosed as a stress-resistant population, indicating that it has a certain degree of resistance to emotional stress and is not easy to develop depression. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0019] Figure 1 The results of the animal experiment on the intestinal flora of healthy people and patients with depression under stress conditions in Example 1;

[0020] Figure 2 The results of the animal experiment on the intestinal flora of different healthy people under stress conditions in Example 1;

[0021] Figure 3 This is the in vitro experiment of healthy human intestinal flora degrading cortisol in Example 1;

[0022] Figure 4 The test results of serum cortisol levels of rats in each group in Example 2;

[0023] Figure 5 The test results of the forced swimming test, tail suspension test, water sprinkling test and novel environment food intake inhibition test of each group of rats in Example 2;

[0024] Figure 6 This is the Venn diagram analysis in Example 3;

[0025] Figure 7 This is the in vitro degradation experiment of cortisol by Escherichia coli expressing kstD-like enzyme in Example 3;

[0026] Schematic diagram of the catalytic reaction of kstD-like enzyme.

[0027] Figure 8 is the LC-MS / MS secondary spectrum of the cortisol degradation product in Example 3;

[0028] Fig. 9 This is a schematic diagram of the biochemical reaction of cortisol catalyzed by the kstD-like enzyme in Example 3;

[0029] Fig.10 The molecular docking analysis in Example 3;

[0030] Fig.11 The test results of serum cortisol levels of rats in each group in Example 4;

[0031] Fig.12 These are the test results of the forced swimming test, tail suspension test, water sprinkling test and novel environment food intake inhibition test for each group of rats in Example 5.

[0032] Fig.13 is the abundance of kstD-like protein in the healthy group and the depression group in Example 6.

[0033] Fig.14 This is the ROC curve of kstD-like for diagnosing depression in Example 6. DETAILED DESCRIPTION

[0034] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0035] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0036] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0037] The overall idea of ​​the present invention is as follows:

[0038] The inventors of the present application have found that the fecal microbiota of healthy volunteers can degrade cortisol, and this degradation ability varies from individual to individual.

[0039] The team then found that the ability of the gut microbiota to protect against cortisol-induced depression-like behavior was related to the cortisol-degrading function of the microbiota. Mice whose gut microbiota lacked the ability to degrade cortisol were prone to depression-like behavior.

[0040] The inventors of the present application have discovered a kstD-like enzyme that can stably bind to cortisol, and intestinal bacteria expressing kstD-like protein can prevent the increase of cortisol levels.

[0041] Then the experiment found that intestinal bacteria expressing kstD-like protein improved cortisol-induced depressive-like behavior. When intestinal bacteria do not express kstD-like protein, mice are susceptible to cortisol and tend to develop depressive-like behavior. This shows that the expression of kstD-like protein can be used as a marker for diagnosing depressive-like behavior. When the abundance of kstD-like protein is lower than 0.417, it is diagnosed as a stress-susceptible population, indicating that it is easy to develop depression under emotional stress; when it is higher than 0.417, it is diagnosed as a stress-resistant population, indicating that it has a certain degree of resistance to emotional stress and is not easy to develop depression.

[0042] The abundance of kstD-like protein refers to the content of kstD-like protein in the proteome. The abundance of kstD-like protein can be determined by methods known in the art, including but not limited to immunohistochemistry, multiplex qPCR, fluorescence quantitative PCR, Panomics or Nanostring technology.

[0043] The following will describe in detail the application of the kstD-like protein of the present application as a molecular marker in the preparation of a product for assessing psychological tolerance in combination with examples and experimental data.

[0044] Example 1 Fecal microbiota of healthy volunteers can degrade cortisol, and this degradation capacity varies from individual to individual

[0045] To explore the impact of the gut microbiota on stress susceptibility in mice, we investigated the effects of fecal microbiota transplantation on cortisol levels in mice exposed to chronic unpredictable mild stress (CUMS), an elevation in cortisol that is a hallmark of psychological stress in humans. CUMS is a classic model of depression, and the stressors involved in this model are mild in nature and reflect the intensity of stress that individuals encounter every day in their daily lives. As expected, mice in the CUMS group had higher cortisol levels than mice in the control group ( Figure 1 ). We transplanted fecal microbiota from healthy individuals (n=5, mixed) or patients with depression (n=5, mixed) into mice by gavage. According to the results of 16S rRNA sequencing, the intestinal microbiota of the mice was similar to that of the corresponding donors, indicating that the microbiota transplantation was successful. Based on liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis, after CUMS modeling, the cortisol levels of mice that received fecal microbiota from patients with depression were not significantly different from those of untreated mice in the CUMS group. In contrast, the cortisol levels of mice that received fecal microbiota from healthy volunteers after CUMS modeling were lower than those of untreated CUMS mice ( Figure 1 These results are similar to those of previous studies showing that the fecal microbiota of healthy people can protect against stress-induced increases in cortisol levels.

[0046] Next, we gavaged the fecal microbiota of five healthy individuals into five groups of mice. After CUMS modeling, LC-MS / MS showed that the fecal microbiota of five healthy individuals had different degrees of protection against the elevated cortisol levels associated with CUMS. There was no significant difference in cortisol levels between mice in the worst protection group and mice in the untreated CUMS group (data not shown), nor between mice in the group with the most effective protection and mice in the control group ( Figure 2 ). These results suggest that the ability of the gut microbiota to modulate stress susceptibility varies among individuals.

[0047] We hypothesized that the protective effect of the fecal microbiota against elevated cortisol levels is related to the ability of bacteria to degrade cortisol. To investigate whether the fecal microbiota of healthy individuals can degrade cortisol, we incubated cortisol (1 g / L) with the fecal microbiota of healthy individuals in vitro. Interestingly, the fecal microbiota of five healthy individuals showed different degrees of cortisol degradation. The gut microbiota of the healthy volunteers with the highest degradation capacity degraded 39% of cortisol after 72 hours of incubation, while the gut microbiome with the lowest degradation capacity did not show significant cortisol degradation ( Figure 3 ). In addition, cortisol was not significantly degraded after incubation with fecal microbiota from patients with depression (data not shown). These results suggest that the fecal microbiota of healthy volunteers can degrade cortisol and that this degradation capacity varies among individuals.

[0048] Example 2 Mice whose intestinal microbiota lacks the ability to degrade cortisol are prone to depressive-like behavior

[0049] To investigate whether stress susceptibility modulated by the gut microbiota depends on the ability to degrade cortisol, we gavaged mice with fecal microbiota from healthy volunteers who had the greatest ability to degrade cortisol (high-performance group) and none (low-performance group). In contrast to the stress-induced increase in cortisol in humans, stress led to a significant increase in corticosterone and a slight increase in cortisol in mice. This increase in corticosterone also led to depressive-like behavior in mice. To avoid interference from corticosterone in behavioral experiments, we applied mouse cortisol to the cortisol-induced depression model instead of the CUMS model. The method of supplementing cortisol to establish a model is well-established. The serum cortisol levels of mice in the high-performance group were not significantly different from those in the control group after administration of cortisol ( Figure 4 ). The low-performance group had higher circulating cortisol levels than the control group, but was not significantly different from the cortisol group ( Figure 4 ). These results suggest that the ability of the gut microbiota of healthy volunteers to protect the host from elevated circulating cortisol levels is related to its ability to degrade cortisol. Mice whose gut microbiota lack the ability to degrade cortisol were susceptible to elevated cortisol levels.

[0050] Next, we performed behavioral experiments in mice to investigate the effects of gut microbiota with different cortisol-degrading abilities on behavior. In the splash test, novelty-suppressed feeding test (NSFT), tail suspension test (TST), and forced swim test (FST), mice treated with cortisol showed depressive-like behaviors similar to those of the control group ( Figure 5 ), indicating that our model was successfully established. Compared with the control group mice, the high-performance group mice did not show depressive-like behavior, while the low-performance group mice showed depressive-like behavior ( Figure 5). These results suggest that the ability of the gut microbiota to protect against cortisol-induced depression-like behavior is associated with the cortisol-degrading effects of the microbiota. Mice whose gut microbiota lack the ability to degrade cortisol are prone to depressive-like behavior.

[0051] Example 3 kstD-like enzyme can stably bind to cortisol

[0052] To investigate the potential mechanism of cortisol degradation, we performed whole genome and transcriptome sequencing. In the annotation of the Swiss-Prot database, 11 genes in the gut microbiota genome were potentially related to steroid metabolism. Transcriptome analysis revealed that a total of 174 genes were upregulated more than 10-fold in the presence of cortisol. Among the 11 genes related to steroid metabolism and the 174 genes upregulated more than 10-fold, one gene was the intersection of the two ( Figure 6 ). We searched the experimentally determined 3D structure database from the Protein Data Bank (PDB) using the protein sequence as a query (Table S4) and found that the most similar structure of the protein expressed by this gene was 3-ketosteroid-Δ1-dehydrogenase (kstD, sequence identity: 34%, coverage: 99%). We named this protein kstD-like enzyme. The amino acid sequence of the kstD-like enzyme is shown in SEQ ID NO.1;

[0053] The sequence of the gene that synthesizes the kstD-like enzyme is shown in SEQ ID NO: 2;

[0054] To experimentally determine the cortisol-degrading function of this kstD-like protein, we introduced the kstD-like gene into the multiple cloning site 5' NcoI--HindIII 3' of the pET28a plasmid, and introduced the recombinant plasmid into E. coli BL21 (DE3) to construct E. coli kstD. After sequence verification, E. coli kstD was incubated with cortisol in vitro. As a control, we introduced the empty vector into E. coli BL21 (DE3) to construct the E. coli vector. LC-MS / MS showed that when incubated with E. coli kstD, cortisol was degraded in a time-dependent manner, while the product increased in a time-dependent manner ( Figure 7 The product was identified as 11β-hydroxyandrostenedione ( Figure 8 When cortisol was incubated with the E. coli vector, no significant degradation was observed (data not shown). These results suggest that the kstD-like protein expressed by the VMR32_13975 gene can convert cortisol into 11β-hydroxyandrostenedione ( Fig. 9 ).

[0055] To investigate the structure of the protein encoded by the VMR32_13975 gene, we simulated the 3D structure of the kstD-like enzyme using the crystal structure of a homologous protein. Molecular docking simulations showed that cortisol forms multiple hydrogen bonds with the catalytic residue and nearby residues, yielding a binding energy of -8.233 kcal / mol ( Fig.10 ), indicating that kstD-like enzymes can stably bind cortisol.

[0056] Example 4 Intestinal bacteria expressing kstD-like protein can prevent elevated cortisol levels

[0057] To investigate whether intestinal bacteria expressing kstD-like proteins can resist elevated cortisol levels, we orally administered E. coli kstD to mice in the kstD-like group. As a control, E. coli vector was orally administered to mice in the vehicle group. LC-MS / MS showed that the serum cortisol level of mice in the kstD-like group was lower than that in the cortisol group and did not increase significantly after cortisol treatment compared with the control group. However, the serum cortisol level of mice in the vehicle group after cortisol treatment was higher than that in the control group and the kstD-like group; however, no significant difference was observed compared with the cortisol group ( Fig.11 These results suggest that gut bacteria expressing kstD-like proteins protect against elevated cortisol levels. When gut bacteria do not express kstD-like proteins, mice are susceptible to elevated cortisol.

[0058] Example 5 Intestinal bacteria expressing kstD-like protein improve cortisol-induced depressive-like behavior

[0059] Behavioral experiments showed that after cortisol treatment, mice in the kstD-like group did not show depressive-like behaviors in the FST, TST, NSFT, and splash tests compared with the control group ( Fig.12 ). However, compared with mice in the control and kstD-like groups, mice in the vehicle group showed depressive-like behaviors after cortisol treatment, and there was no significant difference from mice in the cortisol group ( Fig.12 ). These results suggest that gut bacteria expressing kstD-like proteins improve cortisol-induced depression-like behavior. When gut bacteria do not express kstD-like proteins, mice are susceptible to the effects of cortisol and tend to develop depression-like behaviors.

[0060] Example 6 Diagnostic ability of kstD-like protein in stress-susceptible populations

[0061] In order to accurately study the distribution of kstD-like protein in the human intestine, we used the protein sequence from kstD-like to search for the most similar nucleotide fragments in other bacterial genomes in tblastn. Primers were designed using Primer Blast based on the retrieved nucleotide fragments. The distribution and abundance of the enzyme in human fecal DNA were studied using dye-based multiplex qPCR. A total of three pairs of primers were used in the dye-based multiplex qPCR to study the distribution of kstD-like protein (see the table below). The universal primer EUB was used as an internal reference. 2 -ΔΔCt Methods The relative expression of kstD-like protein gene was evaluated.

[0062] Table 1

[0063]

[0064] The kstD-like protein was detected in 139 healthy volunteers and 100 patients with depression and it was found that the abundance of kstD-like protein in the healthy group was higher than that in the depression group ( Fig.13 ).

[0065] ROC curve analysis showed that the area under the curve of kstD-like protein in diagnosing depression was 0.857 ( Fig.14 ), sensitivity was 0.875, and specificity was 0.750, indicating a high diagnostic ability.

[0066] When the abundance of kstD-like protein is lower than 0.417, people are diagnosed as stress-susceptible, indicating that they are prone to develop depression under emotional stress; when it is higher than 0.417, people are diagnosed as stress-resistant, indicating that they have a certain degree of resistance to emotional stress and are not likely to develop depression.

[0067] The OR value of logistic regression and its 95% confidence interval were 0.187 (0.035 – 0.996), indicating that for every unit increase in the abundance of kstD-like protein, the ability to resist emotional stress increased and the probability of suffering from depression decreased by 81.3%.

[0068] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0069] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0070] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Use of kstD-like protein as a molecular marker in the preparation of a product for diagnosing depression, the amino acid sequence of the kstD-like protein being shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: Compared with the control group, the expression of kstD-like protein in the serum of patients with depression was significantly reduced.

3. The use according to claim 1, characterized in that: The product includes a detection kit for kstD-like protein.

4. The use according to claim 3, characterized in that: The detection kit for the kstD-like protein includes an ELISA detection kit for the kstD-like protein.

5. The use according to claim 3, characterized in that: The detection kit of the kstD-like protein includes a real-time fluorescence quantitative PCR kit.

6. The use according to claim 3, characterized in that: The detection kit for the kstD-like protein includes an immunohistochemistry detection kit.

7. Use of a detection reagent for kstD-like protein in the preparation of a product for diagnosing depression, wherein the amino acid sequence of the kstD-like protein is shown in SEQ ID NO.1.

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