ObjectiveTo investigate the influence of histone deacetylase 3 (HDAC3) on the occurrence, development of psoriasis-like inflammation in mice, and the relative immune mechanisms. MethodsHealthy C57BL/6 mice aged 6-8 weeks were selected and randomly divided into 3 groups: control group (Control), psoriasis model group (IMQ), and HDAC3 inhibitor RGFP966-treated psoriasis model group (IMQ+RGFP966). One day prior to the experiment, the back hair of the mice was shaved. After a one-day stabilization period, the mice in Control group was treated with an equal amount of vaseline, while the mice in IMQ group was treated with imiquimod (62.5 mg/d) applied topically on the back to establish a psoriasis-like inflammation model. The mice in IMQ+RGFP966 group received intervention with a high dose of the HDAC3-selective inhibitor RGFP966 (30 mg/kg) based on the psoriasis-like model. All groups were treated continuously for 5 d, during which psoriasis-like inflammation symptoms (scaling, erythema, skin thickness), body weight, and mental status were observed and recorded, with photographs taken for documentation. After euthanasia, hematoxylin-eosin (HE) staining was used to assess the effect of RGFP966 on the skin tissue structure of the mice, and skin thickness was measured. The mRNA and protein expression levels of HDAC3 in skin tissues were detected using reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot (WB), respectively. Flow cytometry was employed to analyze neutrophils in peripheral blood and lymph nodes, CD4⁺ T lymphocytes, CD8⁺ T lymphocytes in peripheral blood, and IL-17A secretion by peripheral blood CD4⁺ T lymphocytes. Additionally, spleen CD4⁺ T lymphocyte expression of HDAC3, CCR6, CCR8, and IL-17A secretion levels were analyzed. Immunohistochemistry was used to detect the localization and expression levels of HDAC3, IL-17A, and IL-10 in skin tissues. ResultsCompared with the Control group, the IMQ group exhibited significant psoriasis-like inflammation, characterized by erythema, scaling, and skin wrinkling. Compared with the IMQ group, RGFP966 exacerbated psoriasis-like inflammatory symptoms, leading to increased hyperkeratosis. The psoriasis area and severity index (PASI) skin symptom scores were higher in the IMQ group than those in the Control group, and the scores were further elevated in the IMQ+RGFP966 group compared to the IMQ group. Skin thickness measurements showed a trend of IMQ+RGFP966>IMQ>Control. The numbers of neutrophils in the blood and lymph nodes increased sequentially in the Control, IMQ, and IMQ+RGFP966 groups, with a similar trend observed for CD4⁺ and CD8⁺ T lymphocytes in the blood. In skin tissues, compared with the Control group, the mRNA and protein levels of HDAC3 decreased in the IMQ group, but RGFP966 did not further reduce these expressions. HDAC3 was primarily located in the nucleus. Compared with the Control group, the nuclear HDAC3 content decreased in the skin tissues of the IMQ group, and RGFP966 further reduced nuclear HDAC3. Compared with the Control and IMQ groups, RGFP966 treatment decreased HDAC3 expression in splenic CD4⁺ and CD8⁺ T cells. RGFP966 treatment increased the expression of CCR6 and CCR8 in splenic CD4⁺ T cells and enhanced IL-17A secretion by peripheral blood and splenic CD4⁺ T lymphocytes. Additionally, compared with the IMQ group, RGFP966 reduced IL-10 protein levels and upregulated IL-17A expression in skin tissues. ConclusionRGFP966 exacerbates psoriatic-like inflammatory responses by inhibiting HDAC3, increasing the secretion of the cytokine IL-17A, and upregulating the expression of chemokines CCR8 and CCR6.

Aim To observe the effect of corticotropin-releasing factor ( CRF) -expressing neurons on presympathetic neurons in hypothalamic paraventricular nucleus ( PVN) of normotensive Wistar Kyoto ( WKY) rats or spontaneously hypertensive rats (SHR) , and to elucidate the underlying neuronal circuit mechanism of central sympathetic hyperexcitability. Methods The expression levels of CRF protein in WKY rats and SHR PVN were determined by Western blot. Meanwhile, the WKY and SHR PVN CRF-expressing neurons and presympathetic neurons were observed by immunofluo-rescent staining. Adult WKY rats and SHR were used in this study. By microinjection of Cre-dependent ade-no-associated viruses ( AAV) that specifically recognized the CRF promoter and AAV of chemogenetics into the PVN, CRF-expressing neurons expressed designer receptors exclusively activated by designer drugs (DREADDs). Human M3 muscarinic DREADD coupled to Gq receptor ( hM3 Dq) was specifically expressed in PVN CRF-expressing neurons in WKY rats, while human M4 muscarinic DREADD coupled to Gi receptor ( hM4Di) was specifically expressed in PVN CRF-expressing neurons in SHR. Clozapine-N-oxide (CNO) , as a designer ligand, would couple to excitatory hM3Dq or inhibitory hM4Di to regulate the excitability of PVN CRF-expressing neurons. Then the PVN presympathetic neurons were retrogradely labeled by microinjection of fluosecent tracer into the intermedio-lateral column (IML) of spinal cord. Lastly, whole cell patch clamp was used to determine the effect of CNO (10 jjumol L~ ) on spontaneous excitatory postsynaptic currents ( sEPSCs) and current-evoked firing of PVN presympathtic neurons of WKY rats and SHR. Results The expression of CRF protein in the PVN of SHR was significantly higher than that of WKY rats, and the activity and number of CRF-expressing neurons in the PVN of SHR were increased. PVN CRF-expressing neurons were expressed with chemogenetic DREADDs and PVN presympathetic neurons were retrogradely labeled with fluorescent tracer in WKY rats and SHR. In SHR expressed with chemogenetic inhibitory hM4Di-mCherry of PVN CRF-expressing neurons, bath application of CNO to the brain slices resulted in a significant decrease in sEPSCs frequency, but no change in their amplitude of labeled PVN presympathetic neurons. In contrast, in WKY rats expressed with excitatory hM3Dq-eGFP of PVN CRF-expressing neurons, CNO had no obvious effect on the sEPSCs frequency and amplitude in PVN presympathetic neurons. Furthermore, bath application of CNO had no significant effect on current-evoked firing of PVN presympathetic neurons of either WKY rats with hM3Dq-eGFP expression in CRF neurons or SHR with hM4Di-mCherry expression in CRF neurons. Conclusions The activity and number of PVN CRF-expressing neurons are increased in SHR, and CRF-expressing neurons enhance the excitability of presympathetic neurons, which acts as a regulatory neuronal microcircuit between CRF neurons and presympathetic neurons in the PVN.

Objective: To explore the basic characteristics of conventional echocardiography of apical hypertrophic cardiomyopathy (ApHCM) patients complicating with left ventricular apical aneurysm (LVAA). Methods: This is a retrospective study. Patients who underwent echocardiography and cardiac magnetic resonance (CMR) and were diagnosed with ApHCM complicated with LVAA by CMR at Fuwai Hospital, Chinese Academy of Medical Sciences from August 2012 to July 2017 were enrolled. According to whether LVAA was detected by echocardiography, the enrolled patients were divided into two groups: LVAA detected by echocardiography group and LVAA not detected by echocardiography group. Clinical data of the two groups were compared to analyze the causes of missed diagnosis by echocardiography. Results: A total of 21 patients were included, of whom 67.0% (14/21) were males, aged (56.1±16.5) years. Patients with chest discomfort accounted for 81.0% (17/21), palpitation 38.1% (8/21), syncope 14.3% (3/21). ECG showed that 21 (100%) patients had ST-T changes and 18 (85.7%) had deep T-wave invertion. Echocardiography revealed ApHCM in 17 cases (81.0%) and LVAA in 7 cases (33.3%). The mean left ventricular apical aneurysm diameter was 33.0 (18.0, 37.0) mm, and left ventricular ejection fraction was (66.5±6.6) %, and left ventricular apex thickness was (21.0±6.3) mm. Left ventricular outflow tract obstruction was presented in 4 cases and middle left ventricular obstruction in 10 cases. The mean left ventricular apical aneurysm diameter of LVAA detected by echocardiography was greater than that of LVAA not detected by echocardiography (25.0 (18.0, 28.0) mm vs. 16.0 (12.3, 21.0) mm, P=0.006). Conclusions: Conventional echocardiography examination has certain limitations in the diagnosis of ApHCM. Smaller LVAA complicated with ApHCM is likely to be unrecognized by echocardiography. Clinicians should improve their understanding of this disease.
Male ; Humans ; Female ; Apical Hypertrophic Cardiomyopathy ; Retrospective Studies ; Stroke Volume ; Cardiomyopathy, Hypertrophic/diagnostic imaging* ; Ventricular Function, Left ; Echocardiography ; Heart Aneurysm/diagnostic imaging*

This study explores the effect of total flavonoids of Rhododendra simsii(TFR) on middle cerebral artery occlusion(MCAO)-induced cerebral injury in rats and oxygen-glucose deprivation/reoxygenation(OGD/R) injury in PC12 cells and the underlying mechanism. The MCAO method was used to induce focal ischemic cerebral injury in rats. Male SD rats were randomized into sham group, model group, and TFR group. After MCAO, TFR(60 mg·kg~(-1)) was administered for 3 days. The content of tumor necrosis factor-α(TNF-α), interleukin-1(IL-1), and interleukin-6(IL-6) in serum was detected by enzyme-linked immunosorbent assay(ELISA). The pathological changes of brain tissue and cerebral infarction were observed based on hematoxylin and eosin(HE) staining and 2,3,5-triphenyltetrazolium chloride(TTC) staining. RT-qPCR and Western blot were used to detect the mRNA and protein levels of calcium release-activated calcium channel modulator 1(ORAI1), stromal interaction molecule 1(STIM1), stromal intera-ction molecule 2(STIM2), protein kinase B(PKB), and cysteinyl aspartate specific proteinase 3(caspase-3) in brain tissues. The OGD/R method was employed to induce injury in PC12 cells. Cells were randomized into the normal group, model group, gene silencing group, TFR(30 μg·mL~(-1)) group, and TFR(30 μg·mL~(-1))+gene overexpression plasmid group. Intracellular Ca~(2+) concentration and apoptosis rate of PC12 cells were measured by laser scanning confocal microscopy and flow cytometry. The effect of STIM-ORAI-regulated store-operated calcium entry(SOCE) pathway on TFR was explored based on gene silencing and gene overexpression techniques. The results showed that TFR significantly alleviated the histopathological damage of brains in MCAO rats after 3 days of admini-stration, reduced the contents of TNF-α, IL-1, and IL-6 in the serum, down-regulated the expression of ORAI1, STIM1, STIM2, and caspase-3 genes, and up-regulated the expression of PKB gene in brain tissues of MCAO rats. TFR significantly decreased OGD/R induced Ca~(2+) overload and apoptosis in PC12 cells. However, it induced TFR-like effect by ORAI1, STIM1 and STIM2 genes silencing. However, overexpression of these genes significantly blocked the effect of TFR in reducing Ca~(2+) overload and apoptosis in PC12 cells. In summary, in the early stage of focal cerebral ischemia-reperfusion injury and OGD/R-induced injury in PC12 cells TFR attenuates ischemic brain injury by inhibiting the STIM-ORAI-regulated SOCE pathway and reducing Ca~(2+) overload and inflammatory factor expression, and apoptosis.
Animals ; Male ; Rats ; Apoptosis ; Brain Ischemia/metabolism* ; Caspase 3 ; Interleukin-1 ; Interleukin-6 ; Rats, Sprague-Dawley ; Reperfusion Injury/metabolism* ; Tumor Necrosis Factor-alpha/genetics* ; Flavonoids/pharmacology* ; Rhododendron/chemistry*

Humans ; Joint Dislocations/surgery* ; Spinal Fractures/surgery* ; Anesthesia, General ; Cervical Vertebrae/injuries* ; Spinal Fusion

OBJECTIVES: To explore the application of whole-genome sequencing (WGS) in the rapid clinical diagnosis of critically ill neonates. METHODS: The critically ill neonates who admitted to the neonatal intensive care unit of Children's Hospital of Fudan University and underwent WGS from August to September, 2019 were enrolled in this prospective study. The genetic testing results and clinical outcome were analyzed with reference to the sequencing data and clinical features of the neonates. RESULTS: A total of 15 neonates were tested, among whom there were 9 boys and 6 girls. The main reason for hospitalization included abnormal breathing in 7 neonates, poor response in 2 neonates, feeding difficulty in 2 neonates, fever in 1 neonate, hypothermia in 1 neonate, preterm birth in 1 neonate, and convulsion in 1 neonate. The mean turn-around time was 4.5 days for WGS. Finally a genetic diagnosis was obtained for 3 neonates, with a positive diagnostic rate of 20% (3/15). Among the 3 neonates, 2 neonates were withdrawn from the treatment due to severe conditions and 1 neonate died on the day when the sample was sent for genetic testing, whose etiology could be explained by the results of genetic testing. CONCLUSIONS WGS technique can provide a timely and effective diagnosis for critically ill neonates suspected of genetic diseases and provide genetic evidence for clinical treatment of critically ill cases.
Infant, Newborn ; Male ; Child ; Female ; Humans ; Critical Illness ; Prospective Studies ; Premature Birth ; Dyspnea ; Fever

Objective: To establish and optimize PCR methods for the gene encoding of Clostridium perfringens β₂ toxin (cpb₂) and atypical-cpb₂ (aty-cpb₂), analyze the epidemiological characteristics and genetic polymorphism of the cpb₂ of Clostridium perfringens in 9 Chinese areas from 2016 to 2021. Methods: The cpb₂ of 188 Clostridium perfringens strains were examined by PCR; the cpb₂ sequences were acquired by whole-genome sequencing to analyze the genetic polymorphism. Using Mega 11 and the Makeblastdb tool, a phylogenetic tree, and cpb₂-library based on 110 strains carrying the cpb₂ were produced. Using the Blastn technique, a comparison was made to discover sequence similarity between consensus-cpb₂ (con-cpb₂) and aty-cpb₂. Results: The specificity of PCR assay for the cpb₂ and aty-cpb₂ was verified. The PCR results for cpb₂ amplification were highly consistent with the whole-genome sequencing approach (Kappa=0.946, P<0.001). A total of 107 strains from nine regions in China carried cpb₂, 94 types A strains carried aty-cpb₂, 6 types A strains carried con-cpb₂, and 7 types F strains carried aty-cpb₂. The nucleotide sequence similarity between the two coding genes was 68.97%-70.97%, and the similarity between the same coding genes was 98.00%-100.00%. Conclusions: In this study, a specific PCR method for cpb₂ toxin was developed, and the previous PCR method for detecting aty-cpb₂ was improved. aty-cpb₂ is the primary gene encoding of β₂ toxin. There is a significant nucleotide sequence variance between the various cpb₂ genotypes.
Humans ; Clostridium perfringens/genetics* ; Clostridium Infections ; Bacterial Toxins/genetics* ; Phylogeny ; Polymerase Chain Reaction ; Polymorphism, Genetic

The aim of this study is to explore the mechanism of ligustilide, the main active constituent of essential oils of traditional Chinese medicine Angelicae Sinensis Radix, on alleviating oxygen-glucose deprivation/reperfusion(OGD/R) injury in PC12 cells from the perspective of ferroptosis. OGD/R was induced in vitro, and 12 h after ligustilide addition during reperfusion, cell viability was detected by cell counting kit-8(CCK-8) assay. DCFH-DA staining was used to detect the level of intracellular reactive oxygen species(ROS). Western blot was employed to detect the expression of ferroptosis-related proteins, glutathione peroxidase 4(GPX4), transferrin receptor 1(TFR1), and solute carrier family 7 member 11(SLC7A11), and ferritinophagy-related proteins, nuclear receptor coactivator 4(NCOA4), ferritin heavy chain 1(FTH1), and microtubule-associated protein 1 light chain 3(LC3). The fluorescence intensity of LC3 protein was analyzed by immunofluorescence staining. The content of glutathione(GSH), malondialdehyde(MDA), and Fe was detected by chemiluminescent immunoassay. The effect of ligustilide on ferroptosis was observed by overexpression of NCOA4 gene. The results showed that ligustilide increased the viability of PC12 cells damaged by OGD/R, inhibited the release of ROS, reduced the content of Fe and MDA and the expression of TFR1, NCOA4, and LC3, and improved the content of GSH and the expression of GPX4, SLC7A11, and FTH1 compared with OGD/R group. After overexpression of the key protein NCOA4 in ferritinophagy, the inhibitory effect of ligustilide on ferroptosis was partially reversed, indicating that ligustilide may alleviate OGD/R injury of PC12 cells by blocking ferritinophagy and then inhibiting ferroptosis. The mechanism by which ligustilide reduced OGD/R injury in PC12 cells is that it suppressed the ferroptosis involved in ferritinophagy.
Animals ; Rats ; PC12 Cells ; Ferroptosis/genetics* ; Reactive Oxygen Species ; Transcription Factors ; Glutathione

A fluorescence endoscopic laser confocal microscope(FELCM) was used to direct the injection of sinomenine solid lipid nanoparticles(Sin-SLN) into the joint, and the in vitro effectiveness of Sin-SLN in the treatment of rheumatoid arthritis(RA) was evaluated. Sin-SLN was prepared with the emulsion evaporation-low temperature curing method. The Sin-SLN prepared under the optimal conditions showed the encapsulation efficiency of 64.79%±3.12%, the drug loading of 3.84%±0.28%, the average particle size of(215.27±4.21) nm, and the Zeta potential of(-32.67±0.84) mV. Moreover, the Sin-SLN demonstrated good stability after sto-rage for 30 days. The rabbit model of RA was established by the subcutaneous injection of ovalbumin and complete Freund's adjuvant. Five groups were designed, including a control group, a model group, a Sin(1.5 mg·kg~(-1)) group, a Sin-SLN(1.5 mg·kg~(-1)) group, and a dexamethasone(positive drug, 1.0 mg·kg~(-1), ig) group. The control group and the model group only received puncture treatment without drug injection. After drug administration, the local skin temperature and knee joint diameter were monitored every day. The knee joint diameter and the local skin temperature were lower in the drug administration groups than in the model group(P<0.05, P<0.01). FELCM recorded the morphological alterations of the cartilage of knee joint. The Sin-SLN group showed compact tissue structure and smooth surface of the cartilage. Enzyme-linked immunosorbent assay(ELISA) was employed to determine the serum le-vels of interleukin-1(IL-1) and tumor necrosis factor-α(TNF-α). The findings revealed that the Sin-SLN group had lower IL-1 and TNF-α levels than the model group(P<0.05, P<0.01). Hematoxylin-eosin(HE) staining was employed to reveal the pathological changes of the synovial tissue, which were significantly mitigated in the Sin-SLN group. The prepared Sin-SLN had uniform particle size and high stability. Through joint injection administration, a drug reservoir was formed. Sin-SLN effectively alleviate joint swelling and cartilage damage of rabbit, down-regulated the expression of inflammatory cytokines, and inhibited the epithelial proliferation and inflammatory cell infiltration of the synovial tissue, demonstrating the efficacy in treating RA.
Animals ; Rabbits ; Tumor Necrosis Factor-alpha ; Fluorescence ; Arthritis, Rheumatoid/drug therapy* ; Interleukin-1 ; Arthritis, Experimental/drug therapy*

Objective: To explore the basic characteristics of conventional echocardiography of apical hypertrophic cardiomyopathy (ApHCM) patients complicating with left ventricular apical aneurysm (LVAA). Methods: This is a retrospective study. Patients who underwent echocardiography and cardiac magnetic resonance (CMR) and were diagnosed with ApHCM complicated with LVAA by CMR at Fuwai Hospital, Chinese Academy of Medical Sciences from August 2012 to July 2017 were enrolled. According to whether LVAA was detected by echocardiography, the enrolled patients were divided into two groups: LVAA detected by echocardiography group and LVAA not detected by echocardiography group. Clinical data of the two groups were compared to analyze the causes of missed diagnosis by echocardiography. Results: A total of 21 patients were included, of whom 67.0% (14/21) were males, aged (56.1±16.5) years. Patients with chest discomfort accounted for 81.0% (17/21), palpitation 38.1% (8/21), syncope 14.3% (3/21). ECG showed that 21 (100%) patients had ST-T changes and 18 (85.7%) had deep T-wave invertion. Echocardiography revealed ApHCM in 17 cases (81.0%) and LVAA in 7 cases (33.3%). The mean left ventricular apical aneurysm diameter was 33.0 (18.0, 37.0) mm, and left ventricular ejection fraction was (66.5±6.6) %, and left ventricular apex thickness was (21.0±6.3) mm. Left ventricular outflow tract obstruction was presented in 4 cases and middle left ventricular obstruction in 10 cases. The mean left ventricular apical aneurysm diameter of LVAA detected by echocardiography was greater than that of LVAA not detected by echocardiography (25.0 (18.0, 28.0) mm vs. 16.0 (12.3, 21.0) mm, P=0.006). Conclusions: Conventional echocardiography examination has certain limitations in the diagnosis of ApHCM. Smaller LVAA complicated with ApHCM is likely to be unrecognized by echocardiography. Clinicians should improve their understanding of this disease.
Male ; Humans ; Female ; Apical Hypertrophic Cardiomyopathy ; Retrospective Studies ; Stroke Volume ; Cardiomyopathy, Hypertrophic/diagnostic imaging* ; Ventricular Function, Left ; Echocardiography ; Heart Aneurysm/diagnostic imaging*