To compare the effects of upper limb remote ischemic preconditioning (RIPC) with different pressure modes on enhancing high-altitude acclimatization in healthy adult males rapidly exposed to high-altitude environments.

In May 2024, 86 male adult residents living plain areas planning a high-altitude travel were recruited through advertisements in Chongqing by Department of Anesthesiology at the Second Affiliated Hospital of Army Medical University. The participants were randomly divided into a variable pressure training group (n=29), a constant pressure training group (n=29), and a control group (n=28). The variable pressure training group underwent RIPC training in a variable pressure mode [occlusion pressure was set at systolic blood pressure (SBP) + 40 mmHg], while the constant pressure training group received RIPC training in a constant pressure mode (fixed occlusion pressure of 200 mmHg). Both groups completed a bilateral upper limb RIPC training (twice daily, 5 cycles per time of 10-min occlusion followed by 5-min reperfusion) for 14 d. The control group received no such training. On the 3rd day post-training, all participants entered a simulated 4 500 m altitude chamber for 7 h. The incidence and severity of acute mountain sickness (AMS) were observed and evaluated, and the vital signs and cerebral tissue oxygenation index (CTOI) were recorded.

The incidence of AMS was 23.1% in the variable pressure training group (RR=0.4, 95%CI: 0.2~0.8, Chi-square=9.433, P=0.002) and 16.0% in the constant pressure training group (RR=0.2, 95%CI: 0.1~0.6, Chi-square=12.833, P<0.001), and both incidences were significantly lower than that in the control group (65.4%). The AMS symptom score in the variable pressure training group [1.5 (0.8, 3.0) vs (3.1±1.9), P=0.018] and the score in the constant pressure training group [1.0 (1.0, 2.0) vs (3.1±1.9), P=0.001] were significantly lower than that in the control group. The dizziness score was obviously lower in the variable pressure training group [0 (0, 1.0) vs 1.0 (1.0, 1.0), P=0.001] and the constant pressure training group [1.0 (0,1.0) vs 1.0 (1.0,1.0), P=0.003] than the control group, so was the fatigue/weakness score in the variable pressure training group [0 (0, 0.3) vs 1.0 (0,1.0), P=0.006], the constant pressure training group [0 vs 1.0(0,1.0), P<0.001], and the control group. The change of CTOI (ΔCTOI) in the variable pressure training group (P=0.010) and the constant pressure training group (P=0.042) was significantly lower than that in the control group. There were no statistical differences in the 3 groups in terms of SpO₂, HR, SBP or DBP (P>0.05). What’s more, no significant differences were observed in the incidence of AMS, AMS score, dizziness score, fatigue/weakness score, or ΔCTOI between the variable pressure training group and the constant pressure training group (P>0.05).

Both upper limb RIPC protocols, variable-pressure (SBP+40 mmHg) and fixed-pressure (200 mmHg), can effectively enhance high-altitude acclimatization by reducing AMS incidence, symptom severity, and cerebral oxygen desaturation.

To investigate the characteristic changes in motor function and psychological factors among Parkinson’s disease (PD) patients with different phenotypic patterns of freezing of gait (FOG), and to explore the trends in phenotypic heterogeneity of these features.

A cross-sectional study was conducted on 17 PD patients without FOG (PD-NFOG) and 25 PD patients with FOG (PD-FOG) [including 13 cases of trembling-in-place (TP) and 12 cases of shuffling-with-small-steps (SS)]. Another 28 demographically matched (age, gender, education level and others) healthy individuals were recruited from community and served as healthy controls (HC). Motor function was assessed using quantitative gait analysis and clinical scales, including the unified Parkinson’s disease rating scale part Ⅲ (UPDRS-Ⅲ) and H&Y staging. Psychological evaluations were carried out with Hospital Anxiety and Depression Scale (HADS-A/HADS-D), Generalized Anxiety Disorder-7 (GAD-7), Patient Health Questionnaire-9 (PHQ-9), Snaith-Hamilton Pleasure Scale (SHAPS), Reading the Mind in the Eyes Test (RMET), Montreal Cognitive Assessment (MoCA), and digital symbol substitution test (DSST).

① There were significant differences between the PD-FOG and PD-NFOG groups in terms of motor parameters (stride width, length, height and cadence) and cognitive function (MoCA) score (P<0.05). The largest effect sizes were found for cognitive function scores of RMET [η²=0.716 (95%CI:0.611~0.761), P<0.05], DSST [η²=0.667 (95%CI:0.553~0.744), P<0.05], and MoCA [η²=0.597 (95%CI:0.425~0.750), P<0.05], all of which were classified as medium effect sizes. ② Associative characteristics of motor and psychological factors in the PD-FOG group: H&Y stage was positively correlated with HADS-A [r=0.470 (95%CI:0.080~0.735), P=0.018] and HADS-D [r=0.560 (95%CI:0.199~0.787), P=0.004], while the total score of UPDRS-Ⅲ was negatively correlated with DSST [r=-0.574 (95%CI: -0.794~-0.219), P=0.003]. ③Subgroup analysis revealed significant differences (P<0.05) between the TP and SS patterns in motor parameters (stride length and cadence). Moderate effect sizes were observed for HADS-A [δ=0.752 (95%CI: -0.070~1.558), P=0.072], SHAPS [δ=0.512 (95%CI: -0.291~1.305), P=0.132] and HADS-D [δ=0.481 (95%CI: -0.321~1.273), P=0.253], with mean increase by 39.0%, 8.3%, and 24.5%, respectively, in the TP subgroups when compared to the SS subgroups.

PD-FOG patients exhibit characteristic changes in motor function and psychological factors. Cognitive impairment in PD patients may serve as a characteristic marker for FOG progression. Additionally, psychological features in PD-FOG patients with different motor phenotypes show trends of phenotypic heterogeneity.

To evaluate the resuscitation efficacy of novel lyophilized platelets (LP, thrombin-stimulated platelets) combined with lactated Ringer’s (LR) solution in rabbits with hemorrhagic shock and seawater immersion.

Fifty rabbits were randomly assigned to 5 groups (Groups A, B, C, D and E, n=10). After all rabbits were anesthetized with 3% pentobarbital sodium at a dose of 1 mL/kg, soft tissue injury was inflicted in the left lower limb. The blood loss from the soft tissue injury was quantified after gauze hemostasis. The right lower limb was subjected to femoral artery catheterization, followed by blood withdrawal equivalent to 26% of the total blood volume of the rabbit. The rabbits were then vertically immersed in 3% artificial seawater, with the water level reaching above the xiphoid process, and were retrieved in 15 min later. Resuscitation strategies were applied to the rabbits from different group: Group A (no resuscitation), Group B (resuscitation with LR solution), Group C (resuscitation with LR solution and fresh platelets), Group D (resuscitation with LR solution and LP), and Group E (resuscitation with LR solution and novel LP). Coagulation function test, routine blood test, blood gas analysis, and thromboelastography were conducted at baseline and at 1, 2 and 4 h after injury.

The LP and rabbit model of hemorrhagic shock and seawater immersion were successfully prepared. At 1 h after injury, the mean arterial pressure (MAP) of Groups C, D and E (infused with platelet preparations) was significantly higher than that of Group A (without resuscitation, P<0.05); the lactate (Lac) content of Group C was obviously lower than that of Groups A and B (P<0.05); the base excess (BE) and blood urea nitrogen (BUN) levels of Groups C, D and E were notably lower than those of Groups A and B (P<0.05); and the prothrombin time (PT) of Group A was significantly longer than that of before injury (P<0.05). At 2 h after injury, the MAP of Groups C and D was significantly higher than that of Groups A and B, and that of Group E was notably higher than that of Group A (P<0.05); the Lac content of Groups C and E was obviously lower than that of Groups A and B, while that of Group D was also lower than that of Group A (P<0.05); the BE and BUN levels of Groups C, D and E were remarkably lower than those of Groups A and B (P<0.05); the maximum amplitude (MA) value of Group C was higher than that of Group A, while the value of Groups A and D at this time was significantly lower than the corresponding value before injury (P<0.05); and the activated partial clotting time (APTT) of Groups A and D was statistically longer than the corresponding baseline time (P<0.05). At 4 h after injury, the MAP of Groups C, D, and E was higher than that of Groups A and B, and that of Group B was higher than that of Group A (P<0.05); the Lac and BUN levels of Groups C, D, and E were lower than those of Groups A and B (P<0.05); the BE level of Groups C and D were lower than those of Groups A and B (P<0.05); the MA value of Groups B, C, and E were higher than those of Group A (P<0.05), while the MA value and APTT value of Groups A and D were significantly lower than their corresponding baseline values (P<0.05).

For rabbits with hemorrhagic shock and seawater immersion, the novel LP combined with LR solution can significantly increase the MAP level, reduce Lac content, and sustainably maintain blood clot firmness and coagulation function.

To explore the expression profile of adenosine A1 receptor in the paraventricular thalamic nucleus (PVT) in wakefulness/sleep state and its role in regulating sleep.

Male C57BL/6J mice (6~12 weeks old, weighing 22~28 g) were randomly divided into ZT0, ZT6, ZT10, ZT12, and sleep deprivation and recover0y sleep groups (n=16 or n=4). RT-qPCR and fluorescence in situ hybridization were used to observe the changes of adenosine A1 receptor in PVT. Whole-cell patch-clamp recording was conducted to determine the effects of adenosine on the membrane potential and discharge frequency of PVT neurons. The experimental animals were divided into adenosine A1 receptor interference group (n=8) and interference control group (n=7). After RNA interference, electroencephalography (EEG) and electromyography (EMG) were applied simultaneously to observe the changes in wake and sleep time between the 2 groups during sleep deprivation for 6 h and sleep recovery for 4 h.

RT-qPCR and fluorescence in situ hybridization confirmed that the expression of adenosine A1 receptor in PVT was affected by wakefulness/sleep state, with the level in the ZT0 group (active stage) significantly higher than that in the ZT12 group (non-active stage, P<0.05). Whole-cell patch clamp recording indicated that adenosine exerted inhibitory effects on PVT neurons through 2 distinct response types (P<0.05), and the inhibition was in a diminishing trend with a decrease in the expression level of adenosine A1 receptor. After sleep deprivation, the expression level of adenosine A1 receptor showed significant intergroup differences: the level was significantly higher in the sleep deprivation group than the recovery sleep group (P<0.05), while that of the recovery sleep group was higher than that of the ZT6 group and that of the ZT10 group (P<0.05). After knocking down adenosine A1 receptor (shRNA-A1R) in the PVT, the wakefulness timing of the shRNA-A1R group was significantly increased, while the sleep timing was significantly decreased within 1 h after sleep recovery when compared with the interference control group (P<0.05).

The expression of adenosine A1 receptor in the PVT is dynamically regulated by sleep pressure, which was increasing as sleep pressure rises.

To investigate whether aberrant DNA methylation of ubiquinol-cytochrome C reductase core protein 1 (UQCRC1) is related to cognitive impairment caused by neonatal sevoflurane exposure.

A total of 94 SPF C57 mice of either sex, aged 6 d, and weighing 4~6 g, were randomly divided into 7 groups: control group (Con, n=6), sevoflurane-6 and -24 h exposure groups (Sev-6 and -24 h, n=6), control+DMSO group (Con+DMSO, n=19), control+5-aza-2'-deoxycytidine (5-AZA, methylation inhibitor) group (Con+5-AZA, n=19), sevoflurane+DMSO group (Sev+DMSO group, n=19), and sevoflurane+5-AZA group (Sev+5-AZA group, n=19). From 6 to 8 d after birth, the mice of the Sev-6 and -24 h exposure groups were exposed to 3% sevoflurane daily (with 97% oxygen, 2 L/min, 2 h per day), while those from the Con groups were given exposure of 100% oxygen (2 L/min, 2 h per day). For the mice of the 5-AZA and DMSO groups, 1 mg/kg of 5-AZA or an equal volume of DMSO was injected intraperitoneally 30 min before daily exposure. In 6 and 24 h after the last exposure to sevoflurane, 6 mice from the Con, Sev-6 h, and Sev-24 h groups were euthanized for biochemical analysis, and in 24 h post-exposure, 6 mice from the Con+DMSO, Con+5-AZA, Sev+DMSO, and Sev+5-AZA groups were randomly selected for biochemical analysis, while another 3 mice from above each group were also randomly selected for morphological analysis. The remaining 10 mice in these groups underwent behavioral testing (open field test, novel object test, and Y-maze test) at 30~33 d after birth to assess cognitive function, and were euthanized in 24 h after the final behavioral test. RT-qPCR and Western blotting were used to detect the hippocampal expression of UQCRC1, DNA methyltransferases (Dnmts), and methyl CpG binding protein 2 (Mecp2) at mRNA and protein levels, respectively. Immunofluorescence assay was employed to observe the distribution and expression of UQCRC1 in the hippocampus. Bisulfite sequencing PCR (BSP) was applied to measure the methylation in the UQCRC1 promoter region.

Compared with the Con group, the mRNA and protein levels of UQCRC1 were down-regulated (P<0.05), and the mRNA level of Dnmts was up-regulated (P<0.05) in both the Sev-6 h and Sev-24 h exposure groups, while the methylation level in the UQCRC1 promoter region was enhanced in the Sev-24 h exposure group (P<0.05). Additionally, the Sev+5-AZA group had obviously increased mRNA and protein levels of UQCRC1 (P<0.05), and notable improvement in cognitive impairment (P<0.05) when compared with the Sev+DMSO group.

Hypermethylation of UQCRC1 promoter region and thus down-regulating its mRNA and protein expression might be the main mechanism by which repeated neonatal sevoflurane exposure induces cognitive impairment later in life.

To prepare tubeimoside Ⅲ nanoemulsion (TBMⅢ-NE) and evaluate its adjuvant effect in vaccines.

TBMⅢ-NE was prepared using low-energy emulsification. Dynamic light scattering was used to characterize the particle size and polydispersity index of the obtained TBMⅢ-NE, and transmission electron microscopy (TEM) was employed to observe the morphology. CCK-8 assay was utilized to determine the cytotoxicity of TBMⅢ-NE on bone marrow-derived dendritic cells (BMDCs). The in vitro safety of TBMⅢ-NE was evaluated using a hemolysis assay. The ability of TBMⅢ-NE to promote the phagocytosis of antigens by DC2.4 cells was observed using confocal laser microscopy. After co-incubation of TBMⅢ-NE with BMDCs, the expression levels of CD40, CD86, MHC-Ⅰ, and CCR7 on the surface of BMDCs were detected using flow cytometry, and the levels of cytokines in the supernatant of BMDCs were measured using enzyme-linked immunosorbent assay (ELISA). After female BALB/c mice were immunized with the SARS-CoV-2 antigen RBD in combination with TBMⅢ-NE, ELISA was conducted to determine the serum levels of specific IgG, IgG2a, and IgG1 antibodies. The number of specific IFN-γ-secreting cells in mouse splenocytes was detected using enzyme-linked immunospot (ELISpot) assay.

The prepared blank nanoemulsion (BNE) and TBMⅢ-NE were in a particle size of 25.46 and 25.89 nm, and a polydispersity index of 0.214 and 0.125, respectively. TEM displayed that TBMⅢ-NE was in uniform sphere and well dispersed. When the TBMⅢ-NE adjuvant was diluted by 400-fold, the survival rate of BMDCs was approximately 86%. Compared with free TBMⅢ, the hemolytic toxicity of TBMⅢ-NE was significantly reduced (P<0.01). TBMⅢ-NE promoted the phagocytosis of antigens by DC2.4 cells and significantly increased the expression of CCR7 on the surface of BMDCs (P<0.05), indicating its potential to promote more dendritic cells to effectively migrate to lymph nodes. TBMⅢ-NE also promoted the expression of IL-6 and IL-1β in the supernatant of BMDCs (P<0.05). When combined with RBD, TBMⅢ-NE significantly increased the levels of specific IgG, IgG2a, and IgG1 antibodies in mouse serum (P<0.01) and promoted the secretion of specific IFN-γ in splenocytes (P<0.01), indicating that TBMⅢ-NE could enhance specific cellular immune responses.

A stable and highly effective TBMⅢ-NE that can induce humoral and cellular immune responses is successfully prepared.

To investigate the role of transcription factor EB (TFEB) in endotoxin-tolerant macrophages.

The RAW264.7 cells were divided into blank group (DMEM medium), LPS 5 group (5 ng/mL LPS treatment for 4 h), LPS 100 group (100 ng/mL LPS treatment for 4 h), and tolerance group (5 ng/mL LPS for 12 h followed by 100 ng/mL LPS for 4 h). The releases of inflammatory factors TNF-α and IL-6 were measured using ELISA. Western blotting and immunofluorescence assay were used to evaluate the distribution of autophagy-related proteins LC3 and P62, as well as TFEB in the cytoplasm and nucleus. Lentiviral overexpression of TFEB or siRNA-mediated knockdown of TFEB were performed to observe the changes in autophagy levels and bacterial clearance ability in the tolerant cells.

The cells in the tolerance group had significantly lower contents of TNF-α and IL-6, as well as reduced bacterial clearance ability (P<0.01), down-regulated LC3 expression while up-regulated P62 level, and decreased expression of TFEB in both the cytoplasm and nucleus (P<0.01) when compared with the cells of the LPS 100 group. Overexpression of TFEB significantly increased LC3 level, reduced P62 level, and enhanced bacterial clearance ability in the endotoxin-tolerant cells (P<0.01). In contrast, siRNA-mediated knockdown of TFEB had no significant impacts on LC3 and P62 expression levels or bacterial clearance ability.

‍Overexpression of TFEB can restore the autophagy of endotoxin-tolerant cells and enhance their bacterial clearance capacity, thereby alleviating the immunosuppressive state of sepsis. These findings suggest that TFEB holds promise as a potential therapeutic target for the prevention and treatment of sepsis.

To explore the role of X chromosome encoded epigenetic regulator UTX in NK cell-mediated anti-tumor activity.

Male Ncr1-iCre mice were crossed with female UTX^fl/fl mice to generate F1 Ncr1-iCre⁺UTX^fl/- male mice, which were further crossed with female UTX^fl/fl mice to obtain male Ncr1-iCre^-UTX^fl/-control mice (M-Con) and NK-specific deletion of UTX male mice Ncr1-iCre⁺UTX^fl/- (M-KO), as well as female Ncr1-iCre^-UTX^fl/fl control mice (F-Con) and UTX-deficient female mice Ncr1-iCre⁺UTX^fl/fl (F-KO). UTX-deficient mice were injected with melanoma cell line B16F10 via tail vein to observe pulmonary metastatic tumor nodules. Moreover, flow cytometry was applied to detect the proportion and quantity of pulmonary NK cells (CD3^-CD19^-NK1.1⁺), maturation makers KLRG1 and CD11b, activation receptors NKG2D and CD69, and effector molecules, including perforin, granzyme B, CD107a, and IFN-γ. Then pulmonary NK cells were sorted and co-cultured with B16F10 cells, and the apoptosis of the melanoma cells was measured with flow cytometry.

Compared with the M-Con mice, the M-KOmice presented less number of pulmonary tumor nodules (P<0.05), increased proportion and quantity of NK cells in the tumor microenvironment (P<0.01), though no obvious changes in the ratio of NK maturation makers KLRG1 to CD11b, enhanced expression level of cytotoxic molecule perforin (P<0.01), but no changes in the expression of effector molecule granzyme B, degranulation marker CD107a and cytokine IFN-γ in NK cells. Co-culture of NK cells and B16F10 cells promoted the apoptosis of tumor cells (P<0.05). Compared with the F-Con mice, the F-KO mice had no statistical difference in the number of pulmonary tumor nodules, but larger proportion and number of NK cells (P<0.05), decreased ratio of KLRG1 to CD11b (P<0.01), elevated level of perforin but decreased levels of granzyme B, CD107a and IFN-γ in NK cells (P<0.01). The co-culture of NK cells and B16F10 cells reduced the apoptosis of tumor cells in F-KOfemale mice (P<0.05).

NK-specific deletion of UTX regulates pulmonary metastasis of melanoma in a sex-dependent manner.

To investigate the molecular mechanism by which receptor activator of nuclear factor-κB ligand (RANKL) regulates autophagy and osteoclast differentiation through the ROS-HIF-1α signaling axis.

Mouse monocytic/macrophage RAW264.7 cells were randomly divided into control group, RANKL group, and 3 combined intervention groups, that is, RANKL+CQ group (intervention with autophagy inhibitor, CQ), RANKL+FG-4592 group (intervention with HIF-1α stabilizer, FG-4592), and RANKL+NAC group (intervention with ROS scavenger, NAC). The number of mature multinucleated osteoclasts was quantitatively detected with tartrate-resistant acid phosphatase (TRAP) staining; the protein levels of HIF-1α and LC3 were detected by Western blotting and the mRNA levels of osteoclast differentiation-related genes (TRAP, Cath K and MMP-9) were analyzed by qRT-PCR; autophagy flux and activity were observed by adenovirus mRFP-GFP-LC3 labeling system combined with confocal laser microscopy; and the production of intracellular reactive oxygen species (ROS) was measured by DCFH-DA probe.

Compared with the control group, the RANKL group obtained significantly increased number of TRAP-positive multinucleated osteoclasts (P<0.05), up-regulated mRNA levels of osteoclast differentiation-related genes, activated autophagy pathway, as evidenced by the increased LC3-Ⅱ/LC3-Ⅰ ratio (P<0.05) and number of autophagic lysosomes (P<0.05), elevated intracellular ROS generation (P<0.05), and down-regulated HIF-1α protein level (P<0.05). NAC intervention significantly inhibited intracellular ROS accumulation (P<0.05), reversed the lowered expression of HIF-1α protein (P<0.05), decreased the LC3-Ⅱ/LC3-Ⅰ ratio (P<0.05) and autophagy flux and activity (P<0.05), and reduced expression levels of osteoclast differentiation-related genes and the number of mature osteoclasts (P<0.05). In the RANKL+FG-4592 group, HIF-1α protein level was significantly increased (P<0.05), autophagy activation was inhibited [with decreased LC3-Ⅱ/LC3-Ⅰ ratio (P<0.05) and autophagy flux and activity (P<0.05)], and the expression of osteoclast differentiation-related genes and number of mature osteoclasts were decreased (P<0.05) when compared with the RANKL group. While CQ treatment resulted in inhibited autophagy flux and activity (P<0.05), declined expression levels of osteoclast differentiation-related genes, and reduced number of mature osteoclasts (P<0.05).

RANKL activates autophagy through ROS-mediated down-regulation of HIF-1α, and thereby promotes osteoclast differentiation.