Objective: Construction of a recombinant subunit vaccine against rat hepatitis E virus (Rat HEV) and evaluation of its antiviral immunoprotective efficacy. Methods: Rat HEV-ORF3 and Rat HEV-ORF2 truncated fragments (413-419 aa, 413-436 aa, 432-436 aa, 404-594 aa, 357-594 aa) were amplified by PCR, and Overlap PCR was utilized to construct ORF3-ORF2 fusion genes and induce expression of recombinant vaccine proteins. NTA affinity chromatography purification and immunization of Wistar rats, ELISA was used to dynamically monitor the serum-specific IgG potency and binding effect to the virus, and nested PCR was used to detect the fecal viral load after the attack to evaluate the protective effect. Results: Six groups of recombinant proteins were successfully constructed and expressed. Among them, the Rat HEV-ORF2 (P239) protein induced the highest antibody potency (1∶204 800), which was significantly better than the other fusion protein groups (P＜0.001). Attack experiments showed that no viral RNA was detected in the feces of rats in the P239 group at 8-21 days post-infection, whereas the other groups (containing the ORF3 fusion truncate) continued to excrete the virus. Structural analysis showed that P239 retained the intact P structural domain of ORF2 (containing immunodominant epitopes) and the synergistic conformation of the M-P structural domain, which was the key to its efficient protection. Conclusion: Rat HEV-ORF2 (P239) shows significant potential for vaccine development by maintaining the natural conformation and conserved epitopes of ORF2, which can induce potent humoral immunity and completely block viral replication. This study provides an experimental basis for the design of Rat HEV subunit vaccines.

Objective: To evaluate the efficacy and safety of the nourishing yin for lowering fire Chinese herbal formula Shuangbai Wugu decoction combined with standard quadruple Western medicine in the treatment of pulmonary tuberculosis of syndrome of hyperactivity of fire due to yin deficiency. Methods: A total of 60 patients with pulmonary tuberculosis syndrome of hyperactivity of fire due to yin deficiency were randomly divided into observation group and control group. The observation group received standard quadruple regimen combined with Shuangbai Wugu decoction, while the control group only received standard quadruple regimen. The course of treatment in both groups was 2 months. The erythrocyte sedimentation rate recovery, chest CT lesion absorption, TCM syndrome score and drug-induced liver function injury rate were compared between the two groups. Results: The total effective rate of treatment in the observation group was 75%, which was significantly higher than that in the control group. The effective rate of lesion absorption, the recovery rate of TCM syndromes and the recovery rate of erythrocyte sedimentation rate in the observation group were 100% and 90.6%, respectively, which were significantly better than those in the control group. The rate of drug-induced liver injury in the observation group was 3%, which was significantly lower than 44% in the control group, and the differences between the groups were statistically significant. Conclusion: Shuangbai Wugu decoction combined with standard quadruple western medicine in the treatment of pulmonary tuberculosis syndrome of hyperactivity of fire due to yin deficiency can synergistically enhance the curative effect, promote symptom relief and lesion absorption, and significantly reduce the risk of liver injury caused by anti-tuberculosis drugs. It is safe and worthy of clinical application.

Objective: To explore the molecular mechanism of the neuroprotective effect of hypoxic preconditioning at the cellular level, and to provide experimental basis and target strategy for the prevention and treatment of hypoxic/ischemic diseases. Methods: The data of stroke, remote ischemic preconditioning (RIPC), hypoxic preconditioning (HPC), and oxygen-glucose deprivation/reoxygenation (OGD/R) injury models were collected. Network pharmacology was used to analyze the intersection of potential targets in related databases by Kyoto Encyclopedia of Genes and Genomes (KEGG). It was found that cyclin-dependent kinase 2 (CDK2) may be a potential target for neuroprotection of hypoxia/ischemic preconditioning. In SH-SY5Y cells, CDK2 gene was overexpressed and knocked down by transfection of CDK2 overexpression plasmid and CDK2-siRNA. Cell apoptosis and viability were detected by flow cytometry and CCK8 method to evaluate the neuroprotective effect. Western Blotting was used to verify the effects of overexpression/knockdown of CDK2 on mammalian target of rapamycin (mTOR) and hypoxia inducible factor-1α (HIF-1α) proteins. Results: In the OGD/R injury model of SH-SY5Y cells, compared with the OGD/R group, the apoptosis of HPC+OGD group was decreased (P=0.033 9), and the cell viability was increased (P=0.001 6). After knocking down CDK2, compared with the control group, the apoptosis of CDK2 knockdown group was decreased (P=0.007 3), and the cell viability was increased (P=0.002 1). After overexpression of CDK2, compared with the control group, the apoptosis of CDK2 overexpression group increased (P=0.008 8), and the cell viability decreased (P＜0.000 1). Knockdown of CDK2 gene up-regulated the expression of p-mTOR and HIF-1α (P=0.0003, P=0.008 3). Overexpression of CDK2 down-regulated the expression of p-mTOR and HIF-1α (P=0.036 1, P=0.006 9). Conclusion: Hypoxic preconditioning can exert neuroprotective effects, which may be achieved by regulating the CDK2/mTOR/HIF-1α pathway.

Objective: To investigate the correlation between the methylation status of the catechol-o-methyltransferase (COMT) gene promoter region and its mRNA expression level in peripheral blood and the occurrence and development of Parkinson's disease (PD). Methods: Peripheral blood samples from 80 PD patients and 61 healthy controls in our hospital were collected as the PD group and the control group, respectively. The COMT mRNA expression level in peripheral blood was detected by quantitative real-time PCR (Q-PCR). The methylation status of the COMT gene promoter region was detected by methylation-specific PCR. Results: The methylation level of COMT promoter region in peripheral blood of PD group was significantly lower than that of control group (P＜0.05), and the mRNA level was significantly higher than that of control group (P＜0.05). Mid-to-late-stage PD patients had a lower degree of COMT promoter methylation and higher mRNA expression than early-stage patients, and the differences were statistically significant (P＜0.05). In the PD group, a significant negative correlation was found between peripheral blood COMT mRNA and the methylation status of its promoter region (r=-0.316, P=0.004); COMT mRNA was positively correlated with the total UPDRS score (r=0.274, P=0.014), UPDRS motor symptom score (r=0.328, P=0.003), and UPDRS non-motor symptom score (r=0.256, P=0.022). Binary logistic regression analysis showed that increased COMT mRNA expression level was a risk factor for PD onset (OR=2.583, 95%CI: 1.106-6.033, P=0.028), while a hypermethylated state of the COMT promoter region and hypercholesterolemia were protective factors against PD onset (OR=0.266, 95%CI: 0.117-0.607, P=0.002; OR=0.470, 95%CI: 0.252-0.876, P=0.018, respectively). Conclusion: PD patients exhibit decreased methylation levels and upregulated mRNA expression of the COMT promoter in peripheral blood, which dynamically evolve with the worsening of Hoehn-Yahr stage. Hypomethylation of the COMT promoter may participate in the pathological process of Parkinson's disease by promoting its gene transcription.

Objective: In order to meet the requirements of high solubility and purity of recombinant antigens in vaccine preparation, this study modified the expression vector by genetic engineering technology, aiming at constructing a recombinant vector that can effectively promote the soluble expression of proteins and realize the isolation and purification of high purity. Methods: The original vector was modified by molecular cloning technique, using Msc Ⅰ and BamH Ⅰ as the enzymatic sites: (1)Excise non-essential sequences such as enterokinase site and S-tag; (2)Addition of specific coagulase cleavage site and 6×His purification tag; subsequently, the target fragment was amplified by using botulinum toxin type A heavy chain carboxyl-terminal (BONT/A-Hc) gene as a template, and recombinant expression vector pET-32a(+)-BoNT/A-Hc was constructed. pET-32a(+)-BoNT/A-Hc was transformed into Escherichia coli BL21 (DE3) for induced expression, and the protein was analyzed by SDS-PAGE. expression form, and protein purification was performed using Ni-NTA affinity chromatography combined with gradient imidazole elution strategy. Results: (1)The expression vector was successfully constructed, and the insertion of each element was verified by sequencing; (2)In the study of the expression of the target protein, the results of SDS-PAGE showed that the target protein was mainly expressed as inclusion bodies under the induction condition at 37 ℃; Western blot results showed that the expressed protein was the target protein; (3)After modifying the vector, recombinant BoNT/A-Hc protein with high purity was obtained. Conclusion: The vector modification strategy established in this study not only improves the purification efficiency of the target protein, but also significantly improves the stability of protein expression, which provides key technical support for large-scale research of recombinant subunit vaccines.

Objective: To investigate the effect of the active components of royal jelly on the expression of squalene epoxidase (SE) in HepG2 cells, and to clarify the potential biological mechanism of lipid-lowering effect of royal jelly. Methods: The original royal jelly was separated into macromolecular, small molecular, liposoluble and water-soluble components by active fractionation method, and then each component was applied to HepG2 cells. The effect on the expression of SE gene was detected by real-time quantitative PCR (Real-time PCR), and the effect on cell activity was monitored by CCK-8. Results: All components of royal jelly could inhibit cell activity and reduce the expression of SE gene to varying degrees, among which the water-soluble component (＜10 kDa) had the most significant effect on inhibiting cell activity and reducing the expression of SE gene. Conclusion: The active components of royal jelly can down-regulate the expression of SE gene in HepG2 cells, suggesting that royal jelly may reduce the body's cholesterol level through this pathway, and the water-soluble substances (＜10 kDa) are the main active components to reduce cholesterol.

Objective: To investigate the temporal pattern and and related regulation mechanism of angiogenesis after cerebral ischemia-reperfusion injury, and to find the best time window for the intervention of ischemic brain injury. Methods: A total of 120 SD male rats were randomly divided into ischemia-reperfusion group (I/R) and sham operation group (Sham) with 60 rats in each group. Each group was divided into 1 d group, 3 d group, 7 d group and 14 d group according to the postoperative sampling time, with 5 rats in each group. The ischemia-reperfusion model was established by occluding the middle cerebral artery. The expression of vascular endothelial growth factor (VEGF) positive cells in the ischemic penumbra was detected by immunohistochemical staining. FITC-dextran was used to label plasma, and the perfusion volume, microvascular diameter and morphology of brain plasma were observed under laser confocal microscope. Western Blotting was used to detect the expression of VEGF and notch intracellular domains (NICD) protein in cortical ischemic penumbra. Results: (1) After cerebral ischemia-reperfusion injury in I/R group, the plasma perfusion of ischemic penumbra in 7 d group was higher than that in 3 d group (P＜0.05); (2) Compared with the Sham group, the proportion of microvessels with a diameter of >2 and ≤4 μm in the ischemic penumbra of the I/R group increased at all time points after surgery, while the proportion of microvessels with a diameter of >4 and ≤6 μm decreased. The expression of VEGF in the I/R group was significantly higher than that in the Sham group at all time points after surgery (P＜0.05), and reached a peak in the 7 d group; (3) The expression of NICD and VEGF protein in I/R group reached the highest in 3 d group and 7 d group, respectively. Conclusion: After cerebral ischemia-reperfusion injury in rats, cerebral plasma perfusion and microvascular number in the ischemic penumbra show an increasing trend followed by a plateau, while the expression of NICD and VEGF shows a phase-specific increase followed by a decrease. The VEGF-Notch signaling pathway plays an important regulatory role in this process.

Objective: To investigate the protective of dexmedetomidine (Dex) preconditioning on cerebral ischemia-reperfusion injury (CIRI) in rats through PI3K/AKT/NF-κB signaling pathway. Methods: Wistar rats were randomly divided into the Sham group, the Model group was further divided into 24 h, 72 h and 120 h reperfusion subgroups according to the reperfusion time and the Dex group was further divided into 24 h, 72 h and 120 h reperfusion subgroups according to the reperfusion time, after successful modeling of left middle cerebral artery occlusion, resulting in a total of 7 groups, with 12 rats in each group. The Zea-Longa score and open field test were used to evaluate the neuromotor function of rats. The structure and morphology of Nissl bodies were observed by Nissl staining. Western blot was used to detect the expression levels of PI3K/AKT/NF-κB signaling pathway related proteins. Results: Compared with the Sham group, the Zea-Longa score was increased, the total distance of open field exercise was increased, the structure of Nissl bodies was damaged, the expression levels of PI3K and AKT proteins were decreased and the expression of NF-κB was upregulated in the Model group (all P＜0.05). Compared with the Model group, Zea-Longa score was decreased, the total distance of open field exercise was reduced, the structure of Nissl bodies was intact, the protein expression levels of NF-κB was decreased and the protein expression levels of PI3K and AKT were increased in the Dex group (all P＜0.05). Conclusions: Dex pretreatment alleviate brain injury in CIRI model rats by reducing inflammatory response through PI3K/AKT/NF-κB signaling pathway.

Objective: To investigate the differential expression of plasma miR-145 in patients with coronary atherosclerotic heart disease (CHD) and to evaluate its feasibility as an indicator for assessing disease severity and plaque vulnerability in patients with acute coronary syndrome (ACS). Methods: A total of 120 patients admitted to the Department of Cardiology, Baotou Central Hospital from November 2023 to November 2024 and undergoing coronary imaging were included. The ACS group consisted of 30 patients with acute myocardial infarction (AMI) and 30 with unstable angina (UA). The control group comprised 30 patients with stable angina pectoris (SAP) and 30 individuals with no vascular abnormalities. The relative expression of plasma miR-145 was measured by real-time quantitative polymerase chain reaction (RT-qPCR). Optical coherence tomography (OCT) was utilized to observe and characterize culprit plaques. Results: (1) Compared with the control group, plasma miR-145 expression was significantly decreased in both the SAP and ACS groups (P＜0.001); Furthermore, miR-145 expression was significantly lower in the ACS group than in the SAP group (P＜0.001). (2) MiR-145 expression was positively correlated with the minimum fibrous cap thickness of culprit plaques (P＜0.001), and negatively correlated with maximum lipid arc and maximum fibrous plaque length (P＜0.001), while no significant correlation was observed with minimum lumen area (P＞0.05). Conclusion: Plasma miR-145 shows predictive value for evaluating disease severity and plaque vulnerability in patients with ACS.

Objective: To explore the application value of body composition phenotype based on computed tomography (CT) in the evaluation of clinical and imaging severity of acute pancreatitis (AP). Methods: The clinical and CT imaging data of 206 patients with AP were retrospectively analyzed. The L3 skeletal muscle mass index at the third lumbar vertebra level (L3 SMI), intermuscular adipose tissue (IMAT) and visceral-to-subcutaneous fat ratio (VSR) were measured, and four body composition phenotypes were constructed accordingly. The bedside index for severity in acute pancreatitis (BISAP) and modified CT severity index (MCTSI) were used to evaluate the severity of AP patients. Spearman correlation analysis, Logistic regression analysis and receiver operating characteristic (ROC) curve were used to explore the relationship between body composition parameters and their phenotypes and the severity of AP. Results: ①Clinical severity (BISAP stratification): Univariate analysis showed that VSR, IMAT and sarcopenia (SP) were significantly associated with AP severity (all P＜0.05), while L3 SMI was not statistically significant. Among them, phenotype D had the highest risk of severe AP (OR=10.5, P=0.001). Multivariate regression showed that age, neutrophil-to-lymphocyte ratio (NLR) and C-reactive protein (CRP) were independent risk factors (all P＜0.05), and body composition phenotype had no independent predictive value. ROC analysis showed that the area under the receiver operating characteristic curve (AUC) of the combined prediction model constructed by age, inflammation index and body composition parameters was 0.883 (95%CI: 0.816-0.951), and the prediction efficiency was significantly better than that of single index (VSR was better than CRP in single index).②Imaging severity (MCTSI stratification): Univariate analysis showed that only phenotype D showed a significant correlation (OR=3.333, P=0.046), and the other single items did not show statistical significance (all P＞0.05). Multivariate analysis showed that age (P=0.008) and CRP (P=0.027) were independent risk factors, and there was no significant difference in body composition phenotype and phenotype D (all P＞0.05). ROC analysis showed that the combined model of age and CRP had a certain predictive ability (AUC=0.679), and the age performance was the most stable (AUC=0.643, P=0.001). There was no significant difference between VSR and CRP (P＞0.05). Conclusion: Phenotype D (sarcopenia and visceral fat dominance) based on CT evaluation has important value in the progression of AP patients. Although multivariate analysis shows that its independent predictive value is limited, the body composition phenotype can be combined with independent risk factors such as age, CRP and NLR in clinical evaluation to construct a multi-dimensional evaluation system to achieve early and accurate management of AP patients.

Objective: To investigate the clinical efficacy of abdominal douche during laparoscopic appendectomy in the treatment of pediatric acute suppurative appendicitis complicated with peritonitis. Methods: The clinical data of 61 children undergoing laparoscopic appendectomy were retrospectively analyzed. They were divided into study group (22 children) and control group (39 children) according to whether abdominal douche was performed during operation. The hospitalization time, postoperative complications, perioperative inflammation and immune related indexes were compared between the two groups. Results: The hospitalization time of the study group was shorter than that of the control group[9(7, 9)d vs 10(9, 12)d, P=0.004]; there was no significant difference in the incidence of postoperative complications between the two groups. At 12 h after operation, the level of serum C-reactive protein (CRP) in the study group was lower than that in the control group (P=0.009). At 72 h after operation, the white blood cell count (WBC), neutrophil percentage (NEUT) and CRP levels in the study group were better than those in the control group (all P＜0.05), but there was no significant difference in procalcitonin (PCT) levels between the two groups (all P＞0.05). In terms of immune indexes, there was no significant difference in preoperative indexes between the two groups (all P＞0.05). At 72 h after operation, the levels of IgA (P＜0.001) and IgM (P＜0.001) in the study group were higher than those in the control group, and there was no significant difference in other immune indexes (all P＞0.05). Conclusion: Abdominal douche during laparoscopic appendectomy can effectively reduce the early inflammatory response, improve the immune function and shorten the hospitalization time.

Objective: To analyze the impact of different degrees of meconium-stained amniotic fluid on neonatal outcomes, so as to provide guidance for clinical diagnosis and treatment. Methods: Clinical data of 482 parturients with meconium-stained amniotic fluid who delivered in the Third Hospital of Baogang Group from January 2023 to January 2024 were collected. They were divided into degree Ⅰ (n=292), degree Ⅱ (n=132) and degree Ⅲ (n=58) groups according to the degree of contamination. Clinical data of parturients and neonates were collected; neonates were divided into the good outcome group (n=429) and the adverse outcome group (n=53) according to their outcomes, and the characteristics of the two groups were compared. The neonates with adverse outcomes were divided into mild and severe pollution subgroups, and the risk factors of adverse outcomes were analyzed by Logistic regression. Results: There were statistically significant differences in maternal age, gestational age, AFI and umbilical artery blood flow parameters among the three groups (all P＜0.05). The incidences of gestational hypertension increased with the severity of contamination, and the degree Ⅲ group had the highest vaginal delivery rate. No neonatal death occurred in the three groups, and the incidence of adverse neonatal outcomes increased with the severity of contamination (P＜0.001). The degree Ⅲ group had lower Apgar scores and higher inflammatory markers. The adverse outcome group had higher rates of advanced maternal age, gestational hypertension and vaginal delivery, as well as more obvious abnormalities in maternal age, amniotic fluid and umbilical artery blood flow-related indicators. The mother-infant related abnormal indicators were more prominent in the severe contamination subgroup. Logistic regression showed that maternal age, degree of amniotic fluid contamination, AFI and AFV were independent risk factors for adverse neonatal outcomes (all P＜0.001). Conclusion: The severity of meconium-stained amniotic fluid is closely related to multiple maternal and neonatal indicators. Severe contamination, advanced maternal age and abnormal amniotic fluid are independent risk factors for adverse neonatal outcomes. Clinical attention should be paid to evaluation and individualized management should be implemented.

Objective: The pathological mechanism of ischemic stroke (IS) involves mitochondrial dysfunction induced by cerebral hypoxia, which leads to neuronal energy metabolism disorders and apoptosis. Studies have shown that tunneling nanotubes (TNTs), as intercellular channels composed of F-actin, can mediate the transfer of functional mitochondria from bone marrow mesenchymal stem cells (BMSCs) to damaged neurons, thereby rebuilding their oxidative phosphorylation capacity. Hypoxic preconditioning (HPC) can significantly enhance the mitochondrial biosynthesis and anti-apoptotic ability of BMSCs by inducing sublethal hypoxic stress, and improve their survival rate and proliferative potential. This pretreatment method not only promotes the directional migration of BMSCs to the ischemic region, but also increases the mitochondrial transfer efficiency, thereby improving neuronal ATP synthesis, alleviating reactive oxygen species (ROS) damage, and ultimately achieving neurological protection. This article systematically reviews the latest research progress and molecular mechanism of HPC regulation of BMSCs-mediated mitochondrial transplantation in the treatment of IS.

Upper limb dysfunction after stroke seriously affects the daily living ability and quality of life of patients, and its rehabilitation treatment is the clinical focus. Based on the theory of brain plasticity, the brain-limb collaborative rehabilitation model integrates central and peripheral intervention techniques to promote neurological function reorganization and motor function recovery through multi-target collaborative stimulation. According to the intervention technology, the model can be divided into three categories: modern rehabilitation, traditional Chinese medicine rehabilitation and integrated traditional Chinese and Western medicine. According to the timing, it can be divided into synchronous and non-synchronous strategies. This paper systematically reviews the basic connotation, classification methods and clinical application of brain-limb collaborative rehabilitation technology (BLCRM) in upper limb rehabilitation of stroke, covering the combined application of repetitive transcranial magnetic stimulation, transcranial direct current stimulation, brain-computer interface, mirror therapy and scalp acupuncture, in order to provide more accurate rehabilitation programs for patients.

Photodynamic therapy (PDT) and sonodynamic therapy (SDT) are two non-invasive cancer treatment methods based on reactive oxygen species (ROS) killing tumor cells. PDT is activated by light-activated photosensitizer and SDT is activated by ultrasound-activated sonosensitizer. Both of them have the advantages of strong tissue penetration, high targeting and combined radiotherapy and chemotherapy. In the past two decades, significant progress has been made in the development, mechanism analysis and clinical application of sensitizers, but they are still limited by the poor biocompatibility of sensitizers, low penetration efficiency of deep tumors and insufficient individualized treatment options. In the future, it is necessary to focus on the design of new sensitizers, parameter optimization and multi-therapy combination to promote them to become an important choice for the treatment of malignant tumors. Through literature review and data analysis, this paper systematically summarizes the research status and development trend of PDT and SDT, and provides a theoretical basis for their clinical transformation.