To investigate the mechanism by which angiotensin Ⅱ type 1 receptor autoantibody (AT1-AA) promotes phenotypic switch of vascular smooth muscle cells (VSMCs) and vascular fibrosis through abnormal expression of circadian clock protein BMAL1.

Twelve male SD rats (6~8 weeks old, weighing 180~220 g) were randomly divided into (n=6) a control group and an AT1-AA-positive group [established by active immunization of SD rats with AT1R extracellular loop Ⅱ peptide (AT1R-ECLⅡ)]. HE and Masson stainings were used to observe structural changes and fibrosis in the thoracic aorta (n=3). Western blotting was performed to detect the expression of Collagen I, phenotypic switch-related proteins (SM22, α-SMA, OPN and MMP2) in vascular tissues and primary VSMCs (n=4), as well as the expression of BMAL1 at CT0, CT4, CT8, CT12, CT16, and CT20. Transwell and scratch assays were used to assess the proliferation and migration of VSMCs (n=3). si-RNA was employed to knock down Bmal1, followed by detection of BMAL1, Collagen I, and phenotypic conversion-related protein expression (n=3). Additionally, AT1-AA-positive AT1R-knockout (AT1R-KO) rats were constructed to measure BMAL1 expression in thoracic aortic tissues (n=4).

The AT1-AA-positive rats had significantly thickened thoracic aortic vessel wall [(140±9)% vs (120±5)%, P<0.05], badly arranged VSMCs, obvious blue Masson staining, and up-regulated Collagen I expression (P<0.05). In the thoracic aorta of AT1-AA-positive rats and AT1-AA-treated VSMCs, the expression of contractile phenotype-related proteins (α-SMA, SM22) was decreased (P<0.05), while the expression of synthetic phenotype-related proteins (OPN, MMP2) was increased (P<0.05). AT1-AA enhanced the scratch healing ability and migration ability of VSMCs. Furthermore, both mRNA and protein levels of Bmal1 were significantly up-regulated at CT12 (P<0.05), and the rhythmicity of Bmal1 was lost. Knockdown of Bmal1 partially ameliorated AT1-AA-induced phenotypic switch of VSMCs. Compared with AT1-AA-positive WT rats, AT1-AA-positive AT1R-KO rats showed significantly reduced BMAL1 expression in the thoracic aorta (1.35±0.06 vs 0.86±0.07，P<0.001). At the cellular level, AT1-AA-induced phenotypic switch and high Collagen I expression in VSMCs were partially improved in AT1R-KO VSMCs.

AT1-AA promotes VSMCs phenotypic conversion and vascular fibrosis through the AT1R-Bmal1 axis.

To modify YC-1, an inhibitor of hypoxia-inducible factor-1α (HIF-1α) into nanoparticles and explore its effect on reversing chemoresistance of colorectal cancer cells.

Nano-inhibitor mPEG₃₅₀-YC-1 (MYC-1) was prepared by the carboxyl condensation reaction of the active functional group hydroxyl (-OH) in the YC-1 molecule with methoxy polyethylene glycol carboxylic acid, and was verified by ¹H nuclear magnetic resonance (¹H-NMR). Its morphology was analyzed by transmission electron microscope (TEM), and its particle size distribution was analyzed by dynamic light scattering (DLS). By interacting FITC-labeled MYC-1 (FYC-1) with HCT15 cells, the uptake ability of FYC-1 by the cells was observed using laser confocal microscopy. The cytotoxicity of MYC-1 was measured with CCK-8 assay. The sensitization effect of MYC-1 on the chemotherapy drug 5-Fu was detected through toxicity tests of resistant HCT15 cells (resistant HCT15, R-HCT15) and live/dead cell staining. The mechanism of MYC-1 reversing drug resistance was determined with immunofluorescence staining of HIF-1α and western blotting. Finally, a subcutaneous transplanted tumor model of R-HCT15 cells was constructed. The tumor bearing mice were randomly divided into PBS, 5-Fu, MYC-1, and MYF groups, with 3 mice in each group. The tumor volume and weight were observed after treatment in each group to evaluate the ability of MYC-1 to reverse 5-Fu resistance in colorectal cancer.

The nano-inhibitor MYC-1 was successfully prepared. TEM and DLS showed that MYC-1 could self-assemble into nanoparticles with a diameter of approximately 19.96±2.97 nm in aqueous solution. FYC-1 was also successfully prepared. When FYC-1 was interacted with HCT15 cells, FYC-1 could be better taken up by the cells, indicating that the amphiphilic MYC-1 could be better endocytosed into the cells to exert its function. When MYC-1 and 5-Fu acted in combination in colon cancer R-HCT15 cells, the resistance index (RI) was decreased from 7.99 to 1.55, and the relative reversal rate (RRR) of RI was 80.6%. Live (green)/dead (red) cell staining revealed that MYC-1 increased the toxicity of 5-Fu to R-HCT15 cells. Immunofluorescence staining (P<0.01) and Western blotting indicated that MYC-1 effectively inhibited the intracellular expression of HIF-1α. The combined action of MYC-1 and 5-Fu on mice with R-HCT15 subcutaneous transplanted tumors had the best therapeutic effect when compared with PBS (P<0.001), 5-Fu (P<0.01), and MYC-1 (P<0.01). Immunofluorescence staining of HIF-1α in tumor tissues displayed that the expression of HIF-1α was decreased in the MYC-1 and MYF groups. HE staining showed that there was no obvious damage to the important organs in each treatment group.

Nano-inhibitor MYC-1 can reverse the drug resistance of colorectal cancer to 5-Fu chemotherapy by reducing the expression of HIF-1α protein in colorectal cancer cells, thereby effectively improving the therapeutic effect of 5-Fu.

To explore the role of mitophagy in pancreatic cancer cachexia-induced muscle atrophy and its underlying mechanism.

Six male C57BL/6J mice (8 weeks old, weighing 20~30 g) were equally and randomly divided into a control group (intrapancreatic injection of normal saline) and a cachexia group (orthotopic pancreatic injection of KPC1199 cells). After successful model establishment, gastrocnemius muscles were harvested for transmission electron microscopy (TEM) to assess mitochondrial ultrastructure. Western blotting was performed to quantify mitochondrial respiratory chain complexes (Ⅰ~Ⅳ) and autophagy-related proteins, while immunofluorescence staining was conducted to evaluate mitochondrial-lysosomal colocalization. In in vitro experiments, C2C12 myoblasts were differentiated into myotubes, and then divided into a control group (standard culture) and a cachexia group (co-cultured with KPC1199 cells for 48 h using transwell chambers). Mitochondrial-lysosomal colocalization and autophagy-related protein expression were analyzed with immunofluorescence assay and Western blotting. The mitochondrial division inhibitor Mdivi-1 (20 μmol/L) was added to the co-culture system to assess its myotube diameter.

Compared to the control mice, the cachectic mice exhibited mitochondrial swelling, reduced cristae density, and significantly increased mitochondrial-lysosomal colocalization in gastrocnemius muscle (P<0.05). Western blotting revealed the expression levels of mitochondrial respiratory chain proteins complexⅠ (1.00±0.04 vs 0.51±0.04, P<0.05), complexⅡ (1.00±0.13 vs 0.73±0.15, P<0.05), complexⅢ (1.00±0.20 vs 0.64±0.01, P<0.05), complexⅣ (1.00±0.06 vs 0.65±0.02, P<0.05) and PGC1α (1.00±0.03 vs 0.62±0.06, P<0.05) were decreased, and the levels of mitophagy markers, LC3-Ⅰ/Ⅱ (1.00±0.14 vs 1.65±0.25, P<0.05), PINK1 (1.00±0.11 vs 1.51±0.05, P<0.05), and BNIP3 (1.00±0.22 vs 2.02±0.10, P<0.05) were elevated when compared to the control. In the C2C12 myotube model, tumor cell co-culture increased mitochondrial-lysosomal colocalization and upregulated mitophagy-related protein expression (P<0.05), consistent with the in vivo findings. Mdivi-1 treatment increased myotube diameter from 220.6±35.5 μm to 315.0±39.1 μm (R²=0.666 5, P<0.05).

Mitophagy is activated in pancreatic cancer cachexia-induced muscle atrophy. Inhibiting mitophagy can effectively alleviate muscle atrophy induced by pancreatic cancer cachexia.

To assess the therapeutic efficacy of low-intensity focused ultrasound (LIFU) on knee arthrofibrosis in rats and explore its underlying mechanisms.

Fifteen male SD rats (8 weeks old, weighing 250~300 g) were randomly divided into a blank control group, a model group, and an ultrasound treatment group, with 5 animals in each group. Rat model of knee joint stiffness was established in the model and ultrasound treatment group. The rats in the ultrasound group received LIFU intervention (frequency: 1 MHz, power: 1.5 W, 20 min per session, 5 times/week) for 4 weeks. The knee ranges of motion (ROM, flexion and extension) were measured in all 3 groups at 0, 14 and 28 d after intervention. Histological analysis was conducted for the morphological changes in the posterior capsular area of the knee. After the synovial fibroblasts were primary isolated from rat knee joints, the cells were divided into blank control, LIFU, TGF-β (10 ng/mL), and TGF-β+LIFU groups. Changes in fibrosis-related indicators and the TGF-β/Smad signaling pathway were assessed in each group.

In the animal experiments, LIFU intervention for 14 d resulted in significantly improved flexion-extension ROM in knee joints when compared with the rats in the model group (P<0.05), and the improvement was more obvious at 28 d after intervention (P<0.05). After 28 d of LIFU intervention, the fibrosis of the posterior capsule of the knee joint was notably improved in the ultrasound group than the model group, and the expression levels of fibrosis-related indicators (α-SMA，TGF-β) were decreased (P<0.05). In the cellular experiments, the TGF-β group exhibited remarkable up-regulation of fibrosis-related molecules (α-SMA，COL1A1，COL3A1) at both protein and mRNA levels (P<0.05), and activation of the TGF-β/Smad signaling pathway when compared to the blank control group. While LIFU intervention inhibited the expression of TGF-β-induced upregulation of fibrotic indicators (α-SMA，COL1A1，COL3A1) and the activation of TGF-β/Smad signaling pathway (P<0.05).

LIFU can effectively improve knee arthrofibrosis in rats, which potentially through the inhibition of fibroblast activation and modulation of the TGF-β/Smad signaling pathway.

To investigate the protective effect of verapamil on hyperuricemia nephropathy (HN) in mice through modulating TXNIP/NLRP3 inflammasome signaling pathway.

Thirty-two male C57BL/6J mice (8 weeks old, weighing 18~22 g) were randomly divided into a blank control group, a model group, an allopurinol group (10 mg/kg), and a verapamil group (40 mg/kg), with 8 animals in each group. Except for the control mice, the other mice were given 10% fructose water and adenine to establish a mouse model of HN. After successful establishment of model mice, the corresponding interventions were administered to the mice of the other 3 groups for 4 consecutive weeks. The levels of serum uric acid (UA), creatinine (Cr), urea (UREA), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured. HE staining was used to assess the alterations in renal morphology and the infiltration of inflammatory cells, while Masson’s staining was employed to evaluate renal fibrosis. Moreover, ELISA was employed to measure the contents of IL-1β and IL-6 in kidney tissue, while serum levels of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) were detected by colorimetric assay. Furthermore, immunohistochemical staining and Western blot analysis were conducted to examine the expression of TXNIP, NLRP3, IL-1β, MMP7, FN1, CD68, and MPO proteins in the kidney.

Compared to the control group, HN mice exhibited increased serum UA, Cr, and UREA levels (P<0.05), renal pathological changes including renal tubular regeneration, interstitial or periglomerular fibrosis and prominent infiltration of inflammatory cells, and significantly increased renal contents of IL-1β and IL-6 and serum MDA level (P<0.05), while reduced serum SOD and GSH-Px contents (P<0.05), as well as up-regulation of kidney proteins TXNIP, NLRP3, IL-1β, CD68, MPO, FN1 and MMP7 (P<0.01). Verapamil treatment notably reduced serum UA and Cr levels (P<0.01), improved kidney lesions to some extents, decreased collagen volume fraction (CVF) (P<0.01), and restored pro-inflammatory cytokines and oxidative stress markers (P<0.05) when compared with the levels in the model group. Further research found that the expression of kidney proteins TXNIP, NLRP3, IL-1β, CD68, MPO, FN1, and MMP7 was significantly down-regulated by verapamil treatment (P<0.05).

Verapamil exhibits a renal protective effect on HN mice through its anti-inflammatory, antioxidant, and antifibrotic properties, and its mechanism may be related to the inhibition of the TXNIP/NLRP3 inflammasome signaling pathway.

To investigate the spatiotemporal expression pattern of metabotropic glutamate receptor 4 (mGluR4) in the mouse cochlear basilar membrane and its dynamic changes during postnatal development until hair cell synaptic maturation.

Ten healthy 3-month-old C57BL/6L wild-type mice (5 males, 5 females, 25～35 g) were bred to obtain offspring. Cochlear samples from postnatal days 1~30 (P1~P30) were analyzed. Frequency-specific auditory brainstem response (ABR) thresholds were measured at 45.2, 32.0, 22.6, 16.0, 11.3, 8.0, 5.6, and 4.0 kHz in P12, P14, P16, P21, and P30 mice. Immunofluorescence assay combined with confocal laser scanning microscopy was used to scan cochlear whole-mount preparations. Spatiotemporal co-localization profiling of mGluR4 and synaptic markers Ctbp2 and Snap25 was observed, and quantitative comparisons of fluorescent puncta density were performed.

ABR testing revealed undifferentiated waveforms in all P12 mice, while 70% of P14 mice exhibited well-defined Wave I. In P14, P16, P21 and P30, mice undergoing ABR testing, 50% failed to exhibit ABR waveforms when stimulated by 45.2 kHz tones, and 87.5% showed no response to 32.0 kHz stimuli. Auditory response thresholds for other frequencies progressively decreased with increasing postnatal age. Immunolocalization demonstrated stronger mGluR4 expression in inner hair cells (IHCs) versus outer hair cells (OHCs). Distinct mGluR4 puncta were observed at IHC presynaptic active zones (co-localized with Snap25) as early as P11, but showed minimal overlap with CtBP2-labeled ribbons. Quantitative analysis revealed significantly higher mGluR4 puncta density at P30 compared to P14 synapses (P<0.05).

Postnatal upregulation of mGluR4 at IHC synaptic regions correlates with functional maturation. These findings suggest mGluR4 may modulate vesicular exocytosis regulation and exert pharmacological inhibition on glutamate release, potentially influencing synaptic plasticity during auditory pathway refinement.

To investigate whether there exist differences in the improvement of left ventricular diastolic function between angiotensin receptor-neprilysin inhibitor (ARNI) and angiotensin-converting enzyme inhibitor (ACEI)/angiotensin receptor blocker (ARB) in patients with hypertensive heart disease (HHD).

A retrospective cohort study was conducted on the HHD patients admitted in Department of Cardiovascular Diseases of our hospital from January 2021 to December 2024. The general information, echocardiographic parameters before and after treatment, and results of routine tests were collected. Finally, 517 HHD patients were subjected, including 117 receiving ARNI treatment (ARNI group) and 400 getting ACEI/ARB treatment (ACEI/ARB group). A 1∶1 propensity score matching (PSM) was performed with a caliper value of 0.02, resulting in 89 matched cases for each group. Electrocardiography was performed to assess left ventricular diastolic dysfunction (LVDD), with various parameters, including left atrial volume index, early diastolic peak velocity at the interventricular septal portion of mitral annulus (septal e’), early diastolic peak velocity at the lateral wall portion of mitral annulus (lateral e’), tricuspid regurgitation velocity, and E/e’. These parameters were followed up and reassessed during the treatment period. Kaplan-Meier survival curve was plotted to compare the incidence of LVDD between the 2 groups. Multivariable logistic regression model was employed to identify the risk factors contributing to LVDD.

The median follow-up time was 412 (309, 736) d in the whole cohort, and was 409 (300, 729) d for the patients after PSM. Kaplan-Meier survival analysis demonstrated that the incidence of LVDD was lower in the ARNI group than the ACEI/ARB group both before and after PSM (P<0.05). After treatment, the ARNI group obtained lower lateral e’ [8.00 (7.00, 9.40) vs 9.00 (7.10, 10.30) cm/s, P<0.001], thinner left ventricular posterior wall thickness [12.20 (10.80, 12.80) vs 12.30 (11.20, 12.90) mm, P<0.048] when compared with the ACEI/ARB group. After adjusting for confounding factors, multivariable logistic regression analysis revealed that advanced age (OR=1.082, P<0.001), increased systolic blood pressure (OR=1.009, P=0.005), thicker left ventricular posterior wall thickness (OR=1.462, P<0.001), left atrial enlargement (OR=1.081, P<0.001), and use of calcium channel blocker (OR=1.548, P=0.006) were independent risk factors for LVDD, and positively correlated with the risk of LVDD. While, male (OR=0.709, P=0.043) and BMI (OR=0.933, P=0.006) were protective factors, which were negatively correlated with LVDD risk.

In HHD patients, ARNI is superior to ACEI/ARB in reducing the incidence of LVDD.

To explore the clinical characteristics of patients with heat illness and the related factors for progression to heatstroke so as to provide a basis for early clinical intervention.

A case-control study design was adopted. A total of 164 patients with heat illness admitted to a hospital in Hainan Province from January 2018 to December 2023 were included, and divided into a heatstroke group (21 cases) and a non-heatstroke group (143 cases). Univariate and multivariate logistic regression analyses were used to evaluate the associations of body temperature, heart rate and mean arterial pressure at admission with the progression to heatstroke.

The incidence of organ dysfunction in the patients with heat illness was 45.12% (74/164), and 12.80% (21/164) progressed to heatstroke. Multivariate logistic regression analysis showed that at admission, body temperature >40 ℃ (OR=10.11, 95%CI: 2.86~35.78), heart rate >100 beats/min (OR=9.07, 95%CI: 2.75~29.94), and mean arterial pressure <70 mmHg (OR=9.05, 95%CI: 2.75~29.75) were the related factors affecting the progression of heat illness patients to heatstroke.

The incidence of organ dysfunction in heat illness patients is relatively high. Body temperature, heart rate and mean arterial pressure at admission may be the related factors for predicting the progression of heat illness to heatstroke.

To establish a mouse model of bronchiectasis with acute infection and evaluate the immunogenicity and protective effect of a self-assembling Pseudomonas aeruginosa (PA) nanoparticle vaccine rePO-FN based on fusion of PcrV-OprI (rePO) protein with self-assembling ferritin (Ferritin).

①SPF-grade female C57BL/6 mice (aged 6~8 weeks, weighing 18~20 g) were randomly allocated into normal saline group, and low-, medium- and high-dose elastase groups (n=6). A mouse model of bronchiectasis was established via intratracheal instillation of different doses of elastase (30 μL of normal saline containing 0.65, 1.30 and 2.60 IU elastase) for 3 consecutive days. At 14 and 21 d after modeling, ELISA and HE staining were performed respectively to detect the concentration of IL-6 and to observe pathological changes in lung tissue in order to confirm the modeling. ②A recombinant plasmid encoding the gene of fusion protein rePO-FN was constructed and expressed in E. coli. The target protein was purified via affinity chromatography and renatured to obtain the desired protein. The physicochemical properties of the rePO-FN protein were characterized using SDS-PAGE protein gel electrophoresis, dynamic light scattering, molecular sieve chromatography, and transmission electron microscopy. ③ C57BL/6J mice were randomly divided into PBS group, rePO group, rePO-FN group, and Ferritin group (n=10). The mice in the above groups were immunized intramuscularly with 100 μL PBS buffer alone or containing 10 μg of corresponding proteins on days 0, 7, and 14. ELISA was used to measure the specific antibodies in serum. In 7 d after the final immunization, an acute PA infection model was used to compare the survival rates and bacterial colonization among the PBS, rePO, and rePO-FN groups. After establishing a bronchiectasis model by intratracheal instillation of 2.60 IU of elastase in C57BL/6J mice as described above, the mice were randomly divided into bronchiectasis PBS group, bronchiectasis rePO group, and bronchiectasis rePO-FN group (n=10). Immunization was conducted at the same dose and procedure as described above, in 21 d after bronchiectasis modeling. At the 7^th d after the final immunization, an acute PA infection model was used to compare the survival rates and bacterial colonization among the groups.

①Repeated intratracheal instillation of elastase significantly increased the concentration of IL-6 in the lung tissue when compared to the content of the normal saline group (P<0.05). Pathological observations revealed varying degrees of bronchial wall destruction, alveolar fusion, edema, neutrophil infiltration, and hemorrhage, with the severity increasing with elastase dose, which confirming successful establishment of the mouse model of bronchiectasis. ② Well-dispersed rePO-FN nanoparticles were successfully prepared, with an average particle size of 91.28 nm, a Zeta potential of approximately -6.5 mV, and a polydispersity index (PDI) of 0.306. Molecular sieve chromatography determined the elution volume of rePO-FN protein to be 8.80 mL, corresponding to a molecular weight of approximately 1 400 kDa. ③Under acute PA XN-1 strain infection, the survival rate of the rePO-FN immunization group and the bronchiectasis rePO-FN immunization group were significantly higher than that of the PBS control group (P<0.05). Additionally, bacterial colonization in the lung tissues was significantly lower in the rePO-FN immune group and the bronchiectasis rePO-FN immune group under acute PA XN-1 strain infection than that in the rePO group and the bronchiectasis rePO group (P<0.05).

Our vaccine rePO-FN can effectively trigger a strong humoral immune response and provide significant protection against PA infection in a mouse bronchiectasis model.