Use of CTRP4 in the assessment and treatment of herpes simplex encephalitis

Abstract

The application relates to application of CTRP4 in condition assessment and treatment of herpes simplex encephalitis, belongs to the technical field of biological medicine, and solves one of the problems that the condition assessment of HSE in the prior art is highly subjective, an imaging examination cannot reflect intracranial core lesions in real time and dynamically, is not related to a core biological mechanism of a disease, brain tissue samples are difficult to obtain, direct and effective indexes of intracranial disease states are scarce, and routine laboratory examinations are not specific for central nervous system pathologies and recovery of nerve functions. The application comprises application of CTRP4 in preparation of a herpes simplex encephalitis diagnostic reagent, and the diagnostic reagent is used for detecting the expression level of CTRP4 in a biological sample of a subject. The application of CTRP4 in preparation of the herpes simplex encephalitis diagnostic reagent can provide a highly specific, non-invasive and dynamic herpes simplex encephalitis condition assessment scheme, and solves the limitation problem of subjective diagnosis by doctors in traditional assessment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of CTRP4 in the assessment and treatment of herpes simplex encephalitis. Background Technology

[0002] Herpes simplex encephalitis (HSE) is a serious central nervous system disease caused by herpes simplex virus infection. It is characterized by rapid disease progression, significant brain tissue damage, and high rates of disability and mortality. Timely and accurate assessment of the severity of the disease and the status of neurological function recovery, as well as the development of targeted treatment plans, are crucial for improving patient prognosis.

[0003] Currently, clinical assessment of HSE primarily relies on patients' clinical symptoms (such as fever, headache, and altered consciousness) and imaging results. However, these assessment methods have significant limitations: clinical symptoms are easily influenced by individual differences and comorbidities, making them highly subjective; imaging examinations cannot reflect the degree of intracranial inflammatory infiltration and subtle changes in neurological function in real time and dynamically, and cannot directly link to the core biological mechanisms of disease progression and recovery. Furthermore, brain tissue samples from HSE patients are extremely difficult to obtain; only in a few special cases, such as cerebral hemorrhage requiring craniotomy, can a small amount of biopsy tissue be obtained. Routine examinations lack effective indicators that directly reflect the state of intracranial disease; and routine laboratory tests such as complete blood counts only reflect systemic inflammatory responses and cannot specifically reflect pathological changes in the central nervous system and the recovery of neurological function. This makes it difficult for clinicians to objectively and accurately determine the stage of the patient's disease, thus affecting the timely adjustment of treatment plans.

[0004] Therefore, finding biomarkers that can reflect the intracranial condition and neurological function recovery status of HSE patients at different disease stages, and exploring treatment strategies, are technical problems that urgently need to be solved by those skilled in the art. Summary of the Invention

[0005] Based on the above analysis, the present invention aims to provide an application of CTRP4 in the assessment and treatment of herpes simplex encephalitis (HSE), in order to solve one of the problems in the prior art: the assessment of HSE is highly subjective; imaging examinations cannot reflect the core intracranial lesions in real time and are not related to the core biological mechanisms of the disease; brain tissue samples are difficult to obtain, resulting in a lack of direct and effective indicators of intracranial disease status; and routine laboratory tests lack specificity for central nervous system pathology and neurological function recovery.

[0006] On one hand, embodiments of the present invention provide an application of CTRP4 in the preparation of a diagnostic reagent for herpes simplex encephalitis, wherein the diagnostic reagent is used to detect the expression level of CTRP4 in a subject's biological sample.

[0007] Furthermore, in the aforementioned applications, the biological sample is selected from serum, cerebrospinal fluid, or brain tissue.

[0008] Furthermore, in the aforementioned applications, the detection method for the diagnostic reagent is one or more of enzyme-linked immunosorbent assay (ELISA), immunofluorescence staining, immunohistochemistry (IHC), Western blot, or real-time quantitative PCR.

[0009] Furthermore, the applications include the use of CTRP4 in diagnosing a mouse model of herpes simplex encephalitis.

[0010] A second aspect of the present invention also provides the use of recombinant CTRP4 protein in the preparation of a therapeutic drug for herpes simplex encephalitis.

[0011] Furthermore, in the aforementioned applications, the therapeutic drug can regulate the TLR2 signaling pathway, reduce the expression of IL-6, IL-2 and TNF-α, and alleviate inflammatory infiltration and neurological damage in brain tissue.

[0012] Furthermore, in the aforementioned applications, the therapeutic drug is in the form of an injection, comprising recombinant CTRP4 protein and a pharmaceutically acceptable carrier. For example, an injectable formulation of the therapeutic drug includes: recombinant CTRP4 protein (final concentration 100 μg / mL), physiological saline (0.9% NaCl), and 5% mannitol (osmotic pressure regulator), with the pH adjusted to 7.3-7.4; the formulation is stored sealed at -20°C.

[0013] Furthermore, in the aforementioned application, the CTRP4 recombinant protein is obtained by purification using a prokaryotic expression vector carrying a His-tag.

[0014] A third aspect of the present invention also provides a method for constructing a cell model for evaluating the effect of CTRP4 on HSE, comprising the following steps:

[0015] (1) An HSE inflammation model was constructed by inducing BV2 cells with HSV-1 virus; (2) Add recombinant CTRP4 protein to the model cells. The recombinant CTRP4 protein... The final concentration was 50 ng / mL; (3) Cell viability was detected by CCK8 assay, and IL-1 levels in cell supernatant were detected by ELISA. 6. TNF-α expression level.

[0016] A fourth aspect of the present invention also provides the application of CTRP4 in the preparation of a TLR2 pathway inhibitor, wherein the inhibitor is used to inhibit excessive inflammatory response in herpes simplex encephalitis, and the CTRP4 interacts with TLR2 and downregulates its expression.

[0017] Compared with the prior art, the present invention can achieve at least one of the following beneficial effects: 1. The application of CTRP4 in the preparation of diagnostic reagents for herpes simplex encephalitis (HSE) as described in this invention provides a highly specific, non-invasive, and dynamic assessment method for HSE, overcoming many limitations of traditional assessments that rely on subjective diagnosis by doctors. CTRP4 is specifically highly expressed in the serum and brain tissue of HSE patients, while its expression level shows no significant difference among patients with bacterial meningitis, tuberculous meningitis, and normal individuals. It can serve as a specific diagnostic marker for HSE, effectively distinguishing different types of meningitis. Its serum concentration is strongly positively correlated with inflammatory factors (IL-6, TNF-α) and strongly negatively correlated with patients' cognitive function (MMSE score). It can dynamically reflect the disease progression of HSE from initiation to progression, plateau, and stabilization, without relying on difficult-to-obtain brain tissue samples. It also avoids the subjectivity of clinical symptom assessment and the lag in imaging examinations, providing accurate evidence for objectively judging the disease stage and assessing the recovery status of neurological function.

[0018] 2. The application of the CTRP4 recombinant protein described in this invention in the preparation of a therapeutic drug for herpes simplex encephalitis (HSE) clarifies the anti-inflammatory and neuroprotective effects of the CTRP4 recombinant protein, providing a new targeted treatment strategy for HSE. This recombinant protein can downregulate the expression of TLR2 through interaction with it, thereby inhibiting the activation of the TLR2 signaling pathway and significantly reducing the expression levels of pro-inflammatory factors such as IL-6, IL-2, and TNF-α. In animal experiments, it can effectively alleviate inflammatory infiltration in the brain of HSE model mice, reduce inflammatory cell aggregation and lymphovascularization, and improve neuronal damage. Open field assays also confirmed its ability to repair neurological function damage and significantly prolong the survival time of model mice. Cellular experiments also showed that it can inhibit excessive activation of microglia and improve cell survival rate. Its therapeutic efficacy has been verified at both the cellular and animal levels, filling the gap in existing targeted therapies for HSE.

[0019] 3. In the application of the CTRP4 recombinant protein described in this invention in the preparation of drugs for the treatment of herpes simplex encephalitis, a stable and reliable technical system and research tools are constructed. On the one hand, the CTRP4 diagnostic reagent can be detected by a variety of mature detection methods such as enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, and real-time quantitative PCR, and is compatible with easily obtainable biological samples such as serum and cerebrospinal fluid, making it convenient to operate and highly practical. On the other hand, an HSV-1-induced BV2 cell HSE inflammation model and a standardized mouse HSE model have been successfully established, clarifying the prokaryotic expression (with His-tag) and purification process of the CTRP4 recombinant protein. This not only provides a reliable tool for the study of the mechanism of action of CTRP4, but also ensures the potential for large-scale preparation of diagnostic reagents and therapeutic drugs, promoting the transformation of the entire technology chain from disease diagnosis to targeted therapy for HSE.

[0020] In this invention, the above-described technical solutions can be combined with each other to achieve more preferred combinations. Other features and advantages of this invention will be set forth in the following description, and some advantages may become apparent from the description or be learned by practicing the invention. The objects and other advantages of this invention can be realized and obtained from what is particularly pointed out in the description and drawings. Attached Figure Description

[0021] The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Throughout the drawings, the same reference numerals denote the same parts. Figure 1 This is a scatter plot showing the serum CTRP4 concentrations of patients with herpes simplex encephalitis (HSE) and the normal control group in Example 1. Figure 2 This is a scatter plot showing the serum CTRP4 concentrations of patients with bacterial meningitis (BM) and the normal control group in Example 1. Figure 3 This is a scatter plot showing the serum CTRP4 concentrations of patients with tuberculous meningitis (TBM) and the normal control group in Example 1. Figure 4 This is a scatter plot showing the correlation between serum CTRP4 concentration and IL-6 concentration in patients with herpes simplex encephalitis (HSE) in Example 1. Figure 5 This is a scatter plot showing the correlation between serum CTRP4 concentration and TNF-α concentration in patients with herpes simplex encephalitis (HSE) in Example 1. Figure 6 The scatter plot shows the correlation between serum CTRP4 concentration and neutrophil-lymphocyte ratio (NLR) in patients with herpes simplex encephalitis (HSE) in Example 1. Figure 7 This is a scatter plot showing the grouped CTRP4 content at different time points in the herpes simplex encephalitis (HSE) mouse model in Example 2; Figure 8 This is a comparison of the head imaging (cross-sectional) lesion areas of the herpes simplex encephalitis (HSE) model in Example 3 after treatment with PBS and CTRP4. Figure 9 Comparison of gross tissue specimens and corresponding pathological sections (including lesion area markings) of the herpes simplex encephalitis (HSE) model in Example 3 after treatment with PBS and CTRP4. Figure 10 This is a statistical chart comparing the total distance of open field tests in mice with herpes simplex encephalitis (HSE) model after treatment with PBS and CTRP4 in Example 3. Figure 11This is a statistical chart comparing the number of times and time it took for mice with herpes simplex encephalitis (HSE) model to enter the central region of the open field after treatment with PBS and CTRP4 in Example 3. Figure 12 A bar chart comparing serum IL-6 and TNF-α concentrations in mice with herpes simplex encephalitis (HSE) model after treatment with PBS and CTRP4 in Example 3; Figure 13 This is a comparison of survival curves of mice with herpes simplex encephalitis (HSE) in Example 3 after treatment with PBS and CTRP4; Figure 14 This is a morphological observation of HSV-1-induced BV2 cells (HSE cell model) after CTRP4 intervention in Example 4; Figure 15 This is a bar chart comparing the CCK8 assay cell viability of HSV-1-induced BV2 cells (HSE cell model) after CTRP4 intervention in Example 4. Figure 16 This is a comparison of the proportion of microglia undergoing amoeboid transformation in HSV-1-induced BV2 cells (HSE cell model) after CTRP4 intervention in Example 4. Figure 17 This is a bar chart comparing the concentration of IL-6 in the supernatant of HSV-1-induced BV2 cells (HSE cell model) after CTRP4 intervention in Example 4. Figure 18 This is a bar chart comparing the concentration of TNF-α in the supernatant of HSV-1-induced BV2 cells (HSE cell model) after CTRP4 intervention in Example 4. Figure 19 The images shown are the electrophoresis diagram of the CTRP4-His prokaryotic expression vector identified by enzyme digestion and the silver staining detection diagram of the recombinant protein in Example 5. Figure 20 This is a peak diagram of the CTRP4 gene sequence in Example 5; Figure 21 The images show the detection of Coomassie brilliant blue staining before and after IPTG induction of CTRP4 recombinant protein in Example 5. Figure 22 This is a Western blot image of the CTRP4 recombinant protein in Example 5; Figure 23 This is a Western blot verification image of the CTRP4 recombinant protein in Example 5; Figure 24 This is a localization map of the plasmid containing the CTRP4-His prokaryotic expression vector insert fragment from Example 5. Figure 25 Immunohistochemical staining image of CTRP4 expression in the brain tissue of normal Balb / c mice in Example 5; Figure 26 This is a multiplex immunofluorescence staining image of endogenous CTRP4 cell localization in the brain tissue of herpes simplex encephalitis (HSE) mice in Example 5; Figure 27 This is a multiplex immunofluorescence staining image of exogenous CTRP4 cell localization in the brain tissue of a mouse model of herpes simplex encephalitis (HSE) in Example 5; Figure 28 This is a graph showing the RNA-seq analysis of TLR2 pathway and inflammatory factor gene expression in the brain tissue of a herpes simplex encephalitis (HSE) model mouse after CTRP4 intervention in Example 5. Figure 29 This is a comparison of TLR2 immunofluorescence staining in the brain tissue of mice with herpes simplex encephalitis (HSE) model after CTRP4 intervention in Example 5. Detailed Implementation

[0022] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, which form part of this application and are used together with the embodiments of the present invention / utility model to illustrate the principles of the present invention / utility model, but are not intended to limit the scope of the present invention / utility model.

[0023] Example 1 1. Sample collection This experiment was approved by the ethics committees of Beijing Tiantan Hospital, Beijing Ditan Hospital, and Capital Medical University, and all participants signed informed consent forms.

[0024] Serum samples: Venous blood was collected from 30 normal donors, 15 patients with herpes simplex encephalitis (HSE), 5 patients with bacterial meningitis (BM), and 6 patients with tuberculous meningitis (TBM). After standing at room temperature for 20 minutes, the blood was centrifuged at 4°C and 1000×g for 20 minutes. The supernatant was collected and stored at -80°C for later use.

[0025] Brain tissue samples: Brain tissue samples were collected from 1 HSE patient (necrotic brain tissue removed by surgery), 1 BM patient, 1 TBM patient and 1 normal donor, and were paraffin-embedded and sectioned for later use.

[0026] 2. Main Reagents and Instruments CTRP4, IL-6, TNF-α ELISA kit (Meimin Biotechnology Co., Ltd., Yancheng, China); CTRP4 rabbit polyclonal antibody (Proteintech, USA, catalog number: 14023-1-AP); Immunohistochemical detection kit (Bioss, China, catalog number: PV-0023). DAB colorimetric reagent kit (BBI, China, catalog number: E670033-0100); microplate reader (Bio-Rad, USA, Hercules).

[0027] 3. Experimental Methods (1) ELISA detection of serum CTRP4, IL-6 and TNF-α levels Follow the instructions in the kit to add the standards (concentration gradient: 4000, 2000, 1000, 500, 250, 125 pg / mL), normal control serum and patient serum samples (100 μL / well) to the ELISA plate, with 3 replicates per group; Add 100 μL of enzyme conjugate and incubate at room temperature for 1 hour; After washing 4 times, add 50 μL of substrate solution A and solution B, and incubate at 37°C for 15 minutes; Add 50 μL of stop solution, mix gently, and then use an ELISA reader to detect the absorbance (OD value) at a wavelength of 450 nm. Calculate the concentrations of CTRP4, IL-6, and TNF-α in the sample based on the standard curve.

[0028] (2) Immunohistochemical (IHC) detection of CTRP4 expression in brain tissue Brain tissue paraffin sections were dewaxed to water, and after antigen retrieval, they were incubated with 3% H2O2 at room temperature for 10 minutes to block endogenous peroxidase. Add blocking buffer and incubate at room temperature for 30 minutes, then add CTRP4 primary antibody (dilution ratio 1:100) and incubate overnight at 4°C; After washing, add secondary antibody, incubate at room temperature for 30 minutes, develop DAB staining, counterstain with hematoxylin, dehydrate and clear, and mount. Observe and photograph under a microscope to assess the expression intensity of CTRP4 (strong positive, weak positive, negative).

[0029] 4. Experimental Results (1) CTRP4 is specifically highly expressed in the serum of HSE patients. ELISA test results show: like Figure 1 As shown, the mean concentration of CTRP4 in the serum of normal controls was significantly lower than that in HSE patients (P<0.001). like Figure 2 and Figure 3 As shown, there was no significant difference in the mean serum CTRP4 concentration between BM patients and TBM patients and the normal control group (P>0.05).

[0030] (2) CTRP4 is highly expressed in the brain tissue of HSE patients. IHC staining results showed: CTRP4 is strongly expressed in the brain tissue of HSE patients, mainly located in glial cells and lymphocytes; CTRP4 showed only weak positive expression in normal brain tissue, brain tissue of BM patients, and brain tissue of TBM patients, and the expression level was significantly lower than that of HSE patients (P<0.05).

[0031] (3) CTRP4 expression level is closely related to the severity of HSE. Correlation analysis shows that: Figure 4 As shown, serum CTRP4 concentration and IL-6 concentration were strongly positively correlated in HSE patients (r=0.937, P<0.001). Figure 5 As shown, it was strongly positively correlated with TNF-α concentration (r=0.945, P<0.001). CTRP4 concentration was strongly negatively correlated with MMSE score (r=-0.937, P<0.001), meaning that the higher the CTRP4 level, the more severe the cognitive impairment in patients. MRI examination showed that before treatment, the lesion area of ​​HSE patients showed long T1 and long T2 signals, accompanied by high expression of CTRP4; after treatment, the patients' clinical symptoms were relieved, the lesions shrank, and the serum CTRP4 level decreased significantly (P<0.01).

[0032] (4) CTRP4 is correlated with the neutrophil-lymphocyte ratio (NLR). like Figure 6 As shown, serum CTRP4 concentration in HSE patients was positively correlated with the neutrophil-lymphocyte ratio (NLR) (r=0.665, P<0.001), suggesting that CTRP4 is involved in the regulation of systemic inflammatory response in HSE.

[0033] 5. Conclusion This experiment confirms that CTRP4 is specifically highly expressed in the serum and brain tissue of HSE patients. The expression level of CTRP4 is closely related to the inflammatory response of HSE. CTRP4 can be used as a diagnostic marker, disease assessment indicator and prognostic indicator for HSE.

[0034] Example 2: Construction of a mouse model of herpes simplex encephalitis (HSE) 1. Experimental animals: 5-week-old female Balb / c mice, weighing 15-17 grams each, were acclimatized in a barrier environment for one week after purchase; 2. Construction process: HSV-1 virus (herpes simplex virus type 1 standard strain, passaged 3 times in Vero cells) was selected and stored at -80℃ (1×10¹). 0 (PFU / mL aliquots, freeze-thaw no more than 3 times); dilute to titer 2 with serum-free DMEM medium before use. 10 9PFU / ml was incubated at 37℃ for 30 minutes to activate the mouse model. After dilution, 4 μl / mouse was injected into the lateral ventricle to induce Balb / c mice to construct an HSE mouse model. The stereotaxic parameters were set as follows: (AP: anterior fontanelle -0.22mm, ML: midline ±1.00mm, DV: subdural -2.50mm). The intracranial HSE model was improved, and a standardized HSE mouse model was established.

[0035] 3. Evaluation of the HSE mouse model: Two to five days after modeling, affected mice exhibited disordered fur, reduced activity, lethargy, and irritability.

[0036] 4. Detection of CTRP4 concentration in mouse serum: The concentration of CTRP4 in mouse serum at different time points was detected using an ELISA method (using the MM-0666M1 mouse C1q tumor necrosis factor-associated protein 4 (CTRP4) ELISA research kit, 96T specification) to analyze the dynamic changes in serum CTRP4 concentration with disease progression after blood-brain barrier disruption.

[0037] After establishing the HSE mouse model, the concentration of CTRP4 in mouse serum was measured at 0 hours (baseline), 6 hours, 12 hours, 24 hours, and 48 hours. Figure 7 As shown.

[0038] 0 hours (baseline): This represents the serum CTRP4 concentration before the HSE model was established. It is the baseline level under normal physiological conditions and serves as the control benchmark for all subsequent time points. It is used to determine whether the "stress-induced changes" in CTRP4 after disease induction are caused by HSE infection. At 6, 12, 24, and 48 hours after infection, the serum CTRP4 concentration in mice decreased progressively.

[0039] Figure 7 The graph shows the dynamic concentration changes of C1Q / TNF-related protein 4 (CTRP4) in the serum of mice with herpes simplex encephalitis (HSE) model. The biological and statistical significance of each time point is as follows: 0 hours (baseline): This represents the normal physiological state before the HSE model was constructed. The corresponding CTRP4 concentration at this time is the baseline level of CTRP4 in a healthy state. 6 hours (inflammation initiation phase): Compared to 0 hours, serum CTRP4 concentration showed a highly significant decrease (P<0.0001), indicating that after HSE virus infection, endogenous CTRP4 has initiated targeted recruitment to intracranial inflammatory sites, participating in early neuroprotection and inflammatory regulation. 12 hours (inflammation progression phase): The difference compared to 0 hours was highly significant (P<0.0001), and the difference compared to 6 hours was significant (P<0.01), indicating that as the degree of intracranial inflammatory infiltration worsens, the consumption / migration rate of serum CTRP4 further increases, and its continuous decrease in serum level is negatively correlated with disease progression. 24 hours (inflammation plateau phase): The difference compared to 0 hours was highly significant (P<0.0001), but there was no statistically significant difference compared to 48 hours (marked "ns", representing P>0.05), indicating that the intracranial inflammatory state entered a plateau phase at this time, and the recruitment / consumption rate of serum CTRP4 tended to stabilize.

[0040] Association between the course of herpes simplex encephalitis (HSE) and serum biomarkers: Dynamic changes in serum CTRP4 can non-invasively reflect the process of intracranial inflammation from initiation to progression to plateau to stability, providing a visual basis for the serological quantitative assessment of the course of HSE. Example 3: The intervention effect of recombinant CTRP4 protein on inflammatory response and neurological function in HSE model mice 1. Experimental Materials Laboratory animals: Balb / c female mice, 5 weeks old, weighing 15-17g, 6 mice per group, purchased from SPF-grade laboratory animal center; Virus and reagents: Herpes simplex virus type 1 (HSV-1) (standard strain of herpes simplex virus type 1, passaged 3 times in Vero cells), viral titer 2×10⁻⁶ 9 PFU / mL; CTRP4 recombinant protein (synthesized by Qingke Company, based on the full-length coding sequence of human CTRP4 with GenBank accession number AF329838.1, with a 6×His-tag at the C-terminus, purified by Ni-NTA affinity chromatography, purity ≥90% as detected by SDS-PAGE, specificity verified by Western blot, and activity confirmed by the BV2 cell viability experiment in Example 4); 10mM PBS buffer; 1% sodium pentobarbital; 4% paraformaldehyde; HE staining kit; IL-6 detection kit (Meimin Biotechnology, model: MM-0049H1), TNF-α ELISA detection kit (Meimin Biotechnology, model: MM-0122H1).

[0041] 2 Experimental Methods 2.1 Construction and grouping of HSE mouse model Mice were randomly divided into 3 groups: Control group: 4 μL PBS injected into the lateral ventricle; HSE+PBS group: The lateral ventricle was located using a stereotaxic instrument (coordinates: AP: anterior fontanelle -0.22mm, ML: midline ±1.00mm, DV: subdural -2.50mm), and 4μL of HSV-1 virus solution was injected at a rate of 1μL / min. The needle was left in place for 2 minutes after injection, and the wound was sutured. HSE+CTRP4 group: The modeling method is the same as that of HSE+PBS group. On days 1, 2 and 3 after modeling, CTRP4 recombinant protein is injected into the tail vein. The HSE+PBS group is injected with the same amount of PBS at the same time.

[0042] 2.2 Assessment of Inflammatory Response MRI examination: On the 3rd day after modeling, mice were anesthetized with 1% sodium pentobarbital and their heads were scanned using a 7.0T small animal MRI machine with T2 sequence to observe the extent of inflammatory infiltration in the brain (the puncture tract area is marked with a red circle). HE staining: Mice were sacrificed after MRI scans, and brain tissue was collected, fixed in 4% paraformaldehyde, embedded in paraffin, and prepared into 4μm thick sections for HE staining. The degree of inflammatory infiltration of the brain parenchyma (including inflammatory cell type, infiltration range, lymphovascular formation and microglial proliferation) was observed under an optical microscope.

[0043] 2.3 Neurological Function and Survival Assessment Open field test: On the 3rd day after modeling, mice were placed in an open field box (40cm×40cm×30cm). The total distance of movement, the total number of times and the total time of entering the central area (10cm×10cm) were recorded within 5 minutes to analyze the improvement of neurological function. Serum inflammatory factor detection: Blood was collected from the orbital cavity of mice, and after standing at room temperature for 20 min, the serum was collected by centrifugation at 1000×g for 20 min at 4℃. The concentrations of IL-6 and TNF-α were detected using an ELISA kit, and the operation was performed according to the kit instructions. Survival statistics: Starting from the day of modeling, the mortality of mice in each group was recorded daily, survival curves were plotted, and survival time and final survival rate were calculated.

[0044] 3 Experimental Results 3.1 Degree of inflammatory infiltration MRI results: Figure 8 As shown, obvious inflammatory infiltration was visible around the viral injection puncture site in the HSE+PBS group (red circle area); the puncture site was clear in the HSE+CTRP4 group with no obvious inflammatory infiltration; the control group showed no abnormal signals. HE staining results: as shown Figure 9As shown, in the HSE+PBS group, a large number of mixed inflammatory cells were infiltrated in the brain parenchyma, with inflammatory cells aggregated under the covering epithelium, neuronal deformation, and typical lymphovascular cuffs and microglial cell proliferation nodules (red arrows). In the HSE+CTRP4 group, the brain tissue showed milder inflammatory invasion, with no obvious glial cell proliferation and inflammatory cell infiltration, no lymphovascular cuffs, and basically normal neuronal morphology. The brain tissue of the control group showed no signs of inflammation.

[0045] 3.2 Neurological function and inflammatory factors Open field test: such as Figure 10 and Figure 11 As shown, compared with the HSE+PBS group, the total distance of movement of mice in the HSE+CTRP4 group was significantly increased, and the total number of times and the total time to enter the central region were significantly increased (P<0.05), suggesting that the neurological function was improved. Serum inflammatory factors: such as Figure 12 As shown, the serum IL-6 and TNF-α concentrations in the HSE+CTRP4 group were significantly lower than those in the HSE+PBS group (P<0.05), indicating a reduced inflammatory response.

[0046] 3.3 Survival period like Figure 13 As shown, mice in the HSE+PBS group began to die on day 3 after modeling, and all mice in the group died on day 6; mice in the HSE+CTRP4 group died on day 4, and 3 mice were still alive on day 6, with a survival rate significantly higher than that of the HSE+PBS group (P<0.05).

[0047] 4 Results Analysis This embodiment confirms that exogenous CTRP4 recombinant protein can reduce inflammatory infiltration in the brain of HSE model mice (reduce inflammatory cell aggregation and inhibit lymphovascular formation), lower serum levels of pro-inflammatory factors IL-6 and TNF-α, improve neurological function damage and prolong survival, thus clarifying the anti-inflammatory and neuroprotective effects of CTRP4 on HSE.

[0048] Example 4: Effects of CTRP4 on HSV-1-induced BV2 cells (HSE cell model) 1. Experimental Materials Cell line: Mouse microglia BV2; Reagent: HSV-1 virus (herpes simplex virus type 1 standard strain, passaged 3 times in Vero cells) (titer 2×10⁻⁶) 9PFU / mL); CTRP4 recombinant protein (synthesized by Qingke Company, based on the full-length coding sequence of human CTRP4 with GenBank accession number AF329838.1, with a 6×His-tag at the C-terminus, purified by Ni-NTA affinity chromatography, with purity ≥90% as detected by SDS-PAGE, and specificity verified by Western blot); DMEM medium (Beijing Wenyuan Tiancheng Biotechnology Co., Ltd., AC-1001120, containing 10% fetal bovine serum); CCK8 kit (Zhongke Maichen (Beijing) Trading Co., Ltd., CTK009.100); IL-6 detection kit (Meimin Biotechnology, model: MM-0049H1), TNF-α ELISA detection kit (Meimin Biotechnology, model: MM-0122H1).

[0049] 2 Experimental Methods 2.1 Construction and Grouping of HSE Cell Model BV2 cells were divided into 3 groups: Control group: Routine culture, no treatment; HSE group: Induction of inflammation model by adding HSV-1 viral fluid (MOI=1); HSE+CTRP4 group: CTRP4 recombinant protein (final concentration 50 ng / mL) was added after HSV-1 induction, while the control group and HSE group were given the same amount of culture medium.

[0050] 2.2 Cell morphology and viability detection Morphological observation: After 24 hours of culture, the morphology of each group of cells was observed under an optical microscope (20x) (including cell density, spacing, survival status and activation characteristics, such as microglia amoeboid degeneration). CCK8 live cell count: After culturing for 24 h, 10 μL CCK8 reagent was added to each well and incubated at 37 ℃ for 2 h. The absorbance (OD value) was detected by microplate reader at 450 nm wavelength. The number of live cells was calculated and the cell viability was analyzed (statistical method: one-way ANOVA, P<0.05 was considered significant).

[0051] 2.3 Detection of inflammatory factors in cell supernatant After 24 hours of culture, cell supernatants from each group were collected, and the concentrations of IL-6 and TNF-α were detected using an ELISA kit. The procedure was performed according to the instructions, and the OD values ​​were read at a wavelength of 450 nm using a microplate reader. The sample concentrations were calculated and statistical analysis was performed.

[0052] 2.4 Experimental Results 2.4.1 Cell morphology and viability Morphology: such as Figure 14 , Figure 16As shown, the BV2 cells in the control group were highly branched with uniform intercellular spacing; the number of cells in the HSE group was significantly reduced, the intercellular spacing was increased, and a large number of dead cells could be seen under high magnification, while the surviving cells showed amoeboid degeneration (enlarged cell bodies and shortened protrusions); the HSE+CTRP4 group had a larger number of cells with uniform intercellular spacing, and the proportion of amoeboid cells was significantly reduced. CCK8 test: such as Figure 15 As shown, compared with the HSE group, the OD values ​​of viable cells in the control group and the HSE+CTRP4 group were significantly increased (P<0.001), and the HSE group had the fewest viable cells and the most severe cell damage.

[0053] 2.4.2 Inflammatory factors in cell supernatant IL-6: such as Figure 17 As shown, the concentration of IL-6 in the cell supernatant of the HSE group was significantly higher than that of the control group and the HSE+CTRP4 group (P<0.001), and the IL-6 level decreased significantly after CTRP4 intervention. TNF-α: such as Figure 18 As shown, the concentration of TNF-α in the cell supernatant of the HSE group was significantly higher than that of the control group and the HSE+CTRP4 group (P<0.01), and the level of TNF-α decreased significantly after CTRP4 intervention.

[0054] 2.5 Results Analysis This embodiment confirms that in the HSV-1-induced BV2 cell HSE model, CTRP4 can exert anti-inflammatory and protective effects at the cellular level by inhibiting microglia overactivation (reducing amoeboid degeneration), improving cell survival, and reducing the secretion of pro-inflammatory factors IL-6 and TNF-α, which is consistent with the animal experiment results in Example 3.

[0055] Example 5: Construction of CTRP4 plasmid and purification of recombinant protein 1. Experimental Materials Genes and vectors: CTRP4 full-length coding sequence (GenBank accession number is AF329838.1); prokaryotic expression plasmid (C-terminus with 6×His-tag tag) (plasmid type pET-28a(+), promoter is T7, resistance marker is kanamycin resistance gene, replicon is pBR322 source); Reagents: Restriction endonuclease; T4 DNA ligase; IPTG (isopropyl-β-D-thiogalactoside); Ni-NTA affinity chromatography column; Coomassie brilliant blue staining kit; silver staining kit; CTRP4 antibody, 6×His-tagged antibody; Western blot detection reagents; Service commissioned: Beijing Qingke Biotechnology Co., Ltd. was commissioned to construct and sequence the CTRP4-His prokaryotic expression vector.

[0056] 2 Experimental Methods 2.1 Construction of CTRP4 prokaryotic expression vector Enzyme digestion and ligation: The CTRP4 coding sequence and the His-tag prokaryotic expression plasmid were double-digested using restriction endonucleases (restriction sites: "NcoI / XhoI" double digestion). After recovering the target fragment, the CTRP4 sequence was inserted into the multiple cloning site of the plasmid (insertion site marked in red) using T4 DNA ligase. Figure 24 As shown.

[0057] Double enzyme digestion reaction system (50 μL): plasmid 2 μg + NcoI 1 μL + XhoI 1 μL + CutSmart Buffer 5 μL + ddH2O to 50 μL, incubate at 37℃ for 3 hours; ligation reaction system (20 μL): target fragment to vector molar ratio 3:1 + T4 DNA ligase 1 μL + T4 Buffer 2 μL + ddH2O to 20 μL, ligate overnight at 16℃ (12 hours).

[0058] Identification and sequencing: The ligation product was transformed into E. coli DH5α competent cells, single clones were selected for culture, plasmids were extracted and identified by enzyme digestion (total plasmid and size of fragments after enzyme digestion were detected), and the sequence was sent for sequencing to verify the correctness of the sequence (comparing the plasmid map with the sequencing results).

[0059] 2.2 Induction and purification of CTRP4 recombinant protein Induction of expression: The correctly identified recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells, and IPTG (final concentration 1 mmol / L) was added and induced at 37℃ for 4 h. The bacterial culture was collected. The control group was the recombinant bacteria without IPTG. Purification: After ultrasonic disruption of the bacterial culture, the supernatant was collected by centrifugation and purified by Ni-NTA affinity chromatography column, and the elution peak was collected. Detection: Coomassie brilliant blue staining was used to detect protein expression before and after IPTG induction and after purification; silver staining was used to detect protein bands in the BV2 cell HSE group, BV2 cell HSE+CTRP4-His group, mouse HSE group, and mouse HSE+CTRP4-His group; Western blot (primary antibody was CTRP4 antibody or 6×His tag antibody, and fluorescent secondary antibody was used for detection) was used to verify the target protein.

[0060] 3 Experimental Results 3.1 Plasmid Construction Enzyme digestion identification: such as Figure 19 As shown, both the total plasmid band (Lane1) and the fragment after enzyme digestion (Lane2) are located in the range of 5000-8000 bp, with no extraneous bands, which is consistent with the expected size; Sequencing verification: such as Figure 20 As shown, the sequencing results showed no extraneous peaks and were consistent with the CTRP4 gene sequence and plasmid map, confirming that the recombinant plasmid was constructed correctly.

[0061] 3.2 Protein Expression and Purification Coomassie brilliant blue staining: such as Figure 21 As shown, no target band was observed before IPTG induction, but a CTRP4-His protein band appeared after induction. The band was strongest after purification with Ni column, indicating high purity. Silver staining and Western blot: such as Figure 22 and Figure 23 As shown, silver staining revealed clear CTRP4-His target bands (marked with red arrows) in each group; Western blot detected a band of approximately 35.2 kDa on the left side using CTRP4 antibody and a band of approximately 38 kDa (containing His-tag) on ​​the right side using 6×His antibody, both of which were specific fluorescent bands, confirming that the purified product was the CTRP4-His recombinant protein.

[0062] Western blot validation conditions: CTRP4 rabbit polyclonal antibody (1:1000 dilution) and 6×His-tagged antibody (1:5000 dilution) were incubated overnight at 4°C; TBST washing buffer (20 mmol / L Tris-HCl, 150 mmol / L NaCl, 0.1% Tween-20, pH 7.4) were used for washing three times (10 minutes each time); fluorescent secondary antibody (1:10000 dilution) was incubated at room temperature for 1 hour; ECL chemiluminescence solution was used for development, and ImageJ software was used to analyze the band intensity.

[0063] 4 Results Analysis This embodiment successfully constructed the CTRP4-His prokaryotic expression vector and purified the CTRP4 recombinant protein, providing a qualified reagent basis for the CTRP4 intervention experiments in Examples 1 and 2. At the same time, it confirms that the expression and purification method of the recombinant protein is stable and reliable and can be used for subsequent HSE-related research and applications.

[0064] Example 6: Tissue localization of CTRP4 and its regulation of the TLR2 pathway in HSE mice 1. Experimental Materials Experimental materials: normal Balb / c mouse brain tissue; HSE mouse brain tissue from each group in Example 1; CTRP4 antibody, Iba1 antibody (microglia marker), Neun antibody (neuronal marker), TLR2 antibody; DAPI staining solution; immunofluorescence staining kit; RNA extraction kit; high-throughput sequencing (RNA-seq) service; protein co-precipitation kit; mass spectrometry detection service.

[0065] 2 Experimental Methods 2.1 Localization of CTRP4 in normal brain tissue Brain tissue from normal Balb / c mice was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with immunohistochemical stain (primary antibody was CTRP4 antibody, DAB staining). The distribution of CTRP4 was observed under 10x and 20x objectives.

[0066] 2.2 Cellular localization of CTRP4 (endogenous and exogenous) Endogenous CTRP4: Brain tissue sections from HSE mice were subjected to multiple immunofluorescence staining (primary antibodies: CTRP4 antibody (red), Neun antibody (green, neuronal marker); secondary antibody: fluorescently labeled secondary antibody, DAPI (blue) staining of cell nuclei). The secretory cells and subcellular localization of endogenous CTRP4 were observed under a 40x fluorescence microscope. Exogenous CTRP4: Brain tissue sections from HSE+CTRP4 group mice were subjected to multiple immunofluorescence staining (primary antibodies: CTRP4 antibody (red), Iba1 antibody (green, microglia marker); DAPI staining of cell nuclei). The cells and subcellular localization of exogenous CTRP4 were observed under a 40x fluorescence microscope.

[0067] 2.3 Regulation of the TLR2 pathway by CTRP4 Grouping and RNA-seq: A control group, an HSE group (6h, 12h, 24h, and 48h after modeling), and an HSE+CTRP4 group (48h after modeling) were set up, with 3 mice in each group. Total RNA was extracted from brain tissue and subjected to conventional transcription library sequencing (RNA-seq) to analyze the expression changes of inflammatory factors (IL-6, IL-2) and TLR2 pathway genes. Protein coprecipitation and mass spectrometry: Brain tissue from HSE mice was collected, total protein was extracted, and protein coprecipitation was performed using CTRP4 antibody. The precipitate was sent for mass spectrometry to identify proteins that interact with CTRP4 (with a focus on the expression abundance of TLR2). Immunofluorescence verification: Brain tissue sections from mice in the control group, HSE group, and HSE+CTRP4 group were taken and TLR2 immunofluorescence staining was performed (TLR2 antibody labeling, fluorescent secondary antibody detection, DAPI staining of nuclei) to observe changes in TLR2 expression.

[0068] 3 Experimental Results 3.1 Tissue and cellular localization of CTRP4 Normal brain tissue: such as Figure 25 As shown, under 10x and 20x magnification, CTRP4 (brown) is mainly distributed around neurons, confirming the presence of CTRP4 expression in neuron-related regions of normal brain tissue. Endogenous CTRP4: such as Figure 26As shown, red fluorescence (CTRP4) and green fluorescence (Neun, neurons) co-localize, and CTRP4 is located in the cytoplasm (blue DAPI is the cell nucleus), confirming that endogenous CTRP4 is secreted by neurons and located in the cytoplasm; Exogenous CTRP4: such as Figure 27 As shown, red fluorescence (CTRP4) and green fluorescence (Iba1, microglia) colocalize, and CTRP4 is located in the cytoplasm of microglia, confirming that exogenous CTRP4 mainly acts on microglia.

[0069] 3.2 TLR2 pathway regulation RNA-seq: such as Figure 28 As shown, compared with the HSE group, the expression levels of IL-6, IL-2 and TLR2 pathway genes in the brain tissue of the HSE+CTRP4 group (48h) were significantly decreased, suggesting that CTRP4 can inhibit TLR2 pathway activation. Mass spectrometry detection: As shown in Table 1, mass spectrometry identification of HSE mouse brain tissue proteins after co-precipitation showed that TLR2 (Toll-like receptor 2 protein) had a unique peptide of 21, an abundance score of 90.7, and a sequest value of 1.79E+09, indicating high expression abundance, which confirmed the interaction between CTRP4 and TLR2.

[0070] Table 1. Identification of the top 10 proteins co-precipitated with CTRP4 in Balb / c mice after HSV-1 stimulation.

[0071] Immunofluorescence: such as Figure 29 As shown, the fluorescence intensity of TLR2 in the brain tissue of mice in the HSE group was significantly higher than that in the control group; the fluorescence intensity of TLR2 in the HSE+CTRP4 group was significantly lower than that in the HSE group, confirming that CTRP4 can reduce the expression of TLR2 in the HSE state.

[0072] 4 Results Analysis This embodiment clarifies that: ① Endogenous CTRP4 is secreted by neurons and located in the cytoplasm, while exogenous CTRP4 mainly acts on microglia; ② CTRP4 can interact with TLR2 to downregulate the expression of the TLR2 pathway and downstream inflammatory factors (IL-6, IL-2), which is the key mechanism by which CTRP4 exerts its anti-inflammatory effect on HSE, providing a mechanistic basis for CTRP4 as a therapeutic target for HSE.

[0073] The above description is merely a preferred embodiment of the present invention / utility model, but the protection scope of the present invention / utility model is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the protection scope of the present invention / utility model.

Claims

1. The application of CTRP4 in the preparation of diagnostic reagents for herpes simplex encephalitis, characterized in that, The diagnostic reagent is used to detect the expression level of CTRP4 in the subject's biological sample.

2. The application according to claim 1, characterized in that, The biological samples are selected from serum, cerebrospinal fluid, or brain tissue.

3. The application according to claim 1, characterized in that, The detection method for the diagnostic reagent is one or more of the following: enzyme-linked immunosorbent assay (ELISA), immunofluorescence staining, immunohistochemistry, Western blot, or real-time quantitative PCR.

4. The application according to claim 1, characterized in that, This includes the application of CTRP4 in the diagnosis of a mouse model of herpes simplex encephalitis.

5. Application of a recombinant CTRP4 protein in the preparation of a drug for the treatment of herpes simplex encephalitis.

6. The application according to claim 5, characterized in that, The therapeutic drug can regulate the TLR2 signaling pathway, reduce the expression of IL-6, IL-2 and TNF-α, and alleviate inflammatory infiltration and neurological damage in brain tissue.

7. The application according to claim 5, characterized in that, The therapeutic drug is in the form of an injection, and the therapeutic drug includes recombinant CTRP4 protein and a pharmaceutically acceptable carrier.

8. The application according to claim 7, characterized in that, The recombinant CTRP4 protein was obtained by purification using a prokaryotic expression vector carrying a His-tag.

9. A method for constructing a cell model to evaluate the effect of CTRP4 on herpes simplex encephalitis, characterized in that, Includes the following steps: (1) An HSE inflammation model was constructed by inducing BV2 cells with HSV-1 virus; (2) Add CTRP4 recombinant protein to the model cells to a final concentration of 50 ng / mL; (3) Detect cell viability by CCK8 assay and detect the expression levels of IL-6 and TNF-α in the cell supernatant by ELISA.

10. An application of CTRP4 in the preparation of TLR2 pathway inhibitors, characterized in that, The inhibitor is used to suppress the excessive inflammatory response in herpes simplex encephalitis, and CTRP4 interacts with TLR2 and downregulates TLR2 expression.

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