Periodontitis is a chronic inflammatory disease, and its occurrence and development are closely related to the imbalance of local innate immune responses. The caspase family plays a crucial role in regulating inflammatory responses and cell death pathways in periodontal innate immune cells (such as gingival epithelial cells, neutrophils, macrophages, dendritic cells, and natural killer cells). These proteases exhibit a dual regulatory effect on cellular functions. On one hand, apoptotic pathways mediated by caspase-3/7/9 enable the programmed clearance of senescent or damaged cells, while pyroptosis pathways mediated by caspase-1/4 contribute to immune defense and pathogen elimination, collectively helping to maintain tissue homeostasis. On the other hand, excessive activation of the caspase-1/gasdermin D pathway, as well as inflammatory amplification pathways involving caspase-4/6/8, promotes the release of inflammatory cytokines such as IL-1β and IL-18, leading to the disruption of the epithelial barrier and exacerbation of periodontal tissue damage. Caspase regulation exhibits both commonality and cell specificity. In gingival epithelial cells, caspase-1 mediates pyroptosis and inflammation activation, caspase-3 regulates apoptosis and proliferation signaling, and caspase-4 participates in differentiation regulation and pathogen-selective immune responses, collectively adapting to physiological and pathological changes. Neutrophils can utilize the caspase-1/gasdermin D signaling pathway to drive the release of neutrophil extracellular traps without triggering typical pyroptosis. In macrophages, caspase-1 and caspase-8 synergistically promote polarization toward the M1 phenotype, while caspase-3 acts as an apoptosis executor to facilitate macrophage transition to the M2 phenotype in specific microenvironments. This article reviews caspase’s specific mechanism of action in periodontal innate immune-related cells, aiming to provide a new theoretical basis for targeted regulation of caspase in the treatment of periodontitis.

ObjectiveThe study aims to investigate the intervention effect of modified Xiangsha Liujunzitang （M-XSLJZ） on intestinal-derived lipopolysaccharide （LPS）-activated Kupffer cell inflammation in rats with hyperlipidemia spleen deficiency syndrome. MethodsSeventy male SD rats were randomly divided into seven groups （n=10）： blank control （CON）， high-fat diet without spleen deficiency （HFD）， high-fat diet with spleen deficiency （SD-HFD）， M-XSLJZ low-， medium-， and high-dose groups （XS-L， XS-M， XS-H）， and western medicine control （R）. Spleen deficiency was induced in SD-HFD， XS-L， XS-M， XS-H， and R groups via irregular diet combined with exhaustive swimming for 15 days. The CON group received a standard diet， while other groups were fed a high-fat diet for 10 weeks to establish the hyperlipidemia model. After successful modeling， rats were treated for 8 weeks： M-XSLJZ was administered at 3.51， 7.02， 14.04 g·kg^-1 in XS-L， XS-M， and XS-H groups， respectively. The R group received 9×10^-4 g·kg^-1 of a reference drug. D-xylose excretion rate was measured by the phloroglucinol method. Blood lipids were assessed using an automated biochemical analyzer. Hematoxylin-eosin （HE） staining was used to evaluate the pathological conditions of the liver， and oil red O staining was used to observe the lipid deposition in the liver. The levels of LPS， portal vein serum LPS， LPS-binding protein （LBP）， serum interleukin-6 （IL-6）， IL-1β， and tumor necrosis factor-α （TNF-α） were detected by enzyme-linked immunosorbent assay （ELISA）. Immunofluorescence was used to evaluate CD86 expression and CD68/TLR4 co-localization in the liver. Protein levels of TLR4， MyD88， NF-κB p65， and p-NF-κB p65 in Kupffer cells were analyzed via Western blot automated protein analysis. Hepatic IL-6， TNF-α， and IL-1β mRNA and protein levels were measured using Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot. ResultsCompared with the CON group， the SD-HFD group showed a decrease in D-xylose excretion （P<0.01）. TC， TG， HDL-C， and LDL-C increased （P<0.05， P<0.01）. A large number of hepatic lipid vacuoles and orange-red lipid droplet deposition appeared in the liver. Ileal LPS， portal LPS， and LBP increased （P<0.05， P<0.01）. The levels of serum IL-6， TNF-α， and IL-1β increased （P<0.01）. The expression of CD86 was upregulated （P<0.01）， and the co-expression of CD68 and TLR4 was enhanced. The protein levels of TLR4， MyD88， and p-p65 in Kupffer cells increased （P<0.01）. The mRNA and protein levels of IL-6， TNF-α， and IL-1β increased （P<0.05， P<0.01）. Compared with the HFD group， the SD-HFD group exhibited decreased D-xylose excretion （P<0.01）， higher HDL-C， LDL-C （P<0.05）， increased portal LBP and LPS （P<0.05）， increased serum IL-6 and TNF-α （P<0.01）， upregulated CD86 （P<0.01）， enhanced CD68/TLR4 co-expression， and higher TNF-α mRNA/protein （P<0.05）. Compared with the SD-HFD group， all M-XSLJZ treatment groups showed reduced TC， TG， and LDL-C （P<0.05， P<0.01）. XS-H and R groups displayed improved hepatic lipid deposition. XS-H and R groups had lower ileal LPS， portal LPS， and LBP levels （P<0.05， P<0.01）. All M-XSLJZ treatment groups exhibited reduced serum IL-6， IL-1β， and TNF-α （P<0.01）. The XS-H group showed downregulated CD86 （P<0.01） and weakened CD68/TLR4 co-expression. The XS-H group had reduced TLR4， MyD88， and p-NF-κB p65 in Kupffer cells （P<0.01）. XS-H and R groups showed lower IL-6， TNF-α， and IL-1β mRNA/protein （P<0.05， P<0.01）. ConclusionM-XSLJZ may exert its lipid-lowering effects by inhibiting intestinal-derived LPS and alleviating Kupffer cell inflammation in the liver.

OBJECTIVE: To explore the improvement effect of electroacupuncture (EA) based on Xingnao Kaiqiao acupuncture (acupuncture for regaining consciousness and opening orifices) on cognitive impairment in mice with Parkinson's disease (PD), and to explore its regulatory mechanisms on the kinesin family member 5A (KIF5A)/mitochondrial Rho GTPase 1 (Miro1) pathway and mitophagy in prefrontal cortical neurons. METHODS: A total of 70 male C57BL/6J mice of clean grade were randomly divided into a normal group (12 mice), a sham operation group (12 mice), and a model pre-screening group (46 mice). Unilateral stereotaxic injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle was adopted to establish the PD model in the model pre-screening group. Twenty-four mice after successful modeling were randomly selected and divided into a model group and an EA group, 12 mice in each one. In the EA group, acupuncture was applied at "Shuigou" (GV26) and bilateral "Sanyinjiao" (SP6) and "Neiguan" (PC6), ipsilateral "Sanyinjiao" (SP6) and "Neiguan" (PC6) were connected to EA respectively, with disperse-dense wave, 5 Hz/20 Hz in frequency, 0.5 mA in current intensity, 20 min a time, 6 times a week for 30 days. Cognitive function was assessed by Y-maze and Morris water maze tests; morphology of prefrontal cortex was observed by H.E. staining; reactive oxygen species (ROS) level in prefrontal cortex was detected by fluorescence probe method; mitochondrial morphology and autophagosome ultrastructure were observed by transmission electron microscopy; the mRNA expression of tyrosine hydroxylase (TH) was detected by quantitative real-time PCR; the protein expression of TH, KIF5A, Miro1, p62, Parkin and PTEN induced kinase 1 (PINK1) was detected by Western blot. RESULTS: Compared with the sham operation group, both the model group and the EA group exhibited increased rotation number of per minute (P<0.001). Compared with the sham operation group, in the model group, the novel arm exploration time of Y-maze test was shortened (P<0.001), the escape latency of Morris water maze test was prolonged (P<0.05) and the platform crossing number of Morris water maze test was reduced (P<0.01); in the prefrontal cortex, the number of cellular vacuole and neurons with karyopyknosis was increased (P<0.001), and mitochondrial autophagosomes could be observed; in the prefrontal cortex, the relative expression of ROS was increased (P<0.001), the protein and mRNA expression of TH was decreased (P<0.001), the protein expression of Miro1, PINK1, Parkin was increased (P<0.001, P<0.01), the protein expression of KIF5A and p62 was decreased (P<0.001). Compared with the model group, in the EA group, the novel arm exploration time of Y-maze test was prolonged (P<0.01), the escape latency of Morris water maze test was shortened (P<0.05) and the platform crossing number of Morris water maze test was increased (P<0.05); in the prefrontal cortex, the number of cellular vacuole and neurons with karyopyknosis was decreased (P<0.001), and the number of mitochondrial autophagosomes reduced and the mitochondrial morphology was improved; in the prefrontal cortex, the relative expression of ROS was decreased (P<0.01), the protein and mRNA expression of TH was increased (P<0.001, P<0.01), the protein expression of Miro1, PINK1, Parkin was decreased (P<0.001, P<0.01, P<0.05), the protein expression of KIF5A and p62 was increased (P<0.01, P<0.05). CONCLUSION Xingnao Kaiqiao electroacupuncture effectively alleviates cognitive impairment and damage of neuronal function in PD mice, its mechanism may be related to the regulation of KIF5A/Miro1 pathway, hence reducing the mitophagy in prefrontal cortical neurons.
Animals ; Electroacupuncture ; Male ; Mice ; Parkinson Disease/physiopathology* ; Cognitive Dysfunction/psychology* ; Kinesins/genetics* ; Humans ; Mitophagy ; Mice, Inbred C57BL ; rho GTP-Binding Proteins/genetics* ; Mitochondria/genetics* ; Prefrontal Cortex/metabolism*

OBJECTIVE: To investigate the effect of stretch on long non-coding RNA taurine upregulated gene 1 (TUG1)-mediated miR-545-3p/cannbinoida receptor 2 (CNR2) pathway regulating bone regeneration in the distraction area of rats during distraction osteogenesis. METHODS: Thirty-six 10-week-old male Sprague Dawley rats were randomly divided into 3 groups ( n=12 in each group): group A (femoral fracture+injection of interfering RNA), group B (distraction osteogenesis+injection of interfering RNA), and group C (distraction osteogenesis+injection of TUG1). Groups A and B were injected with 60 μg of interfering RNA at the beginning of incubation period (immediate after operation), the beginning of distraction phase (7 days after operation), and the end of distraction phase (21 days after operation), and group C was injected with 60 μg of synthetic TUG1 in vivo interfering sequence at the same time. The general situation of rats in each group was observed during the experiment. The mineralization of fracture space or distraction area was observed by X-ray films at 21, 35, and 49 days after operation. At 49 days after operation, the samples of the distraction area were taken for HE staining to observe the mineralization, and real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expressions of osteoblast-related genes such as TUG1, miR-545-3p, CNR2, alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). Blood samples were collected from the abdominal aorta of the rats, and the expressions of ALP and C terminal telopeptide of type Ⅰ (CTX-Ⅰ) protein were detected by ELISA assay. RESULTS: The results of X-ray film and HE staining observations showed that osteogenesis in group C was superior to groups A and B at the same time point. The results of qRT-PCR showed that the relative mRNA expressions of TUG1, CNR2, ALP, OCN, and OPN in group C were significantly higher than those in group A and group B, and the relative mRNA expression of miR-545-3p in group C was significantly lower than that in group A and group B ( P<0.05). The relative mRNA expressions of TUG1 and ALP in group B were significantly higher than those in group A, and the relative mRNA expression of miR-545-3p in group B was significantly lower than that in group A ( P<0.05). There was no significant difference in the relative mRNA expressions of CNR2, OCN, and OPN between group A and group B ( P>0.05). The results of ELISA showed that the expressions of ALP and CTX-Ⅰ protein were significantly higher in group C than in group A and group B, and in group B than in group A ( P<0.05). CONCLUSION Under the action of stretch, the expression of TUG1 in the femoral distraction area of rats increases, which promotes the expression of CNR2 by inhibiting the expression of miR-545-3P, which is helpful to the mineralization of the extension area and osteogenesis.
Animals ; MicroRNAs/genetics* ; Rats, Sprague-Dawley ; Male ; Osteogenesis, Distraction/methods* ; Rats ; RNA, Long Noncoding/metabolism* ; Osteopontin/genetics* ; Osteogenesis ; Bone Regeneration ; RNA, Small Interfering/genetics* ; Osteocalcin/genetics* ; Alkaline Phosphatase/metabolism* ; Osteoblasts/cytology* ; Signal Transduction ; Femoral Fractures/surgery*

Viruses constitute a significant group of pathogens that have caused numerous fatalities and substantial economic losses in recent years, particularly with the emergence of coronaviruses. While the impact of SARS-CoV-2 appears to be diminishing in daily life, only a limited number of drugs have received approval or emergency use authorization for its treatment. Given the high mutation rate of viral genomes, host-directed agents (HDAs) have emerged as a preferred choice due to their broad applicability and lasting effectiveness. In contrast to direct-acting antivirals (DAAs), HDAs offer several advantages, including broad-spectrum antiviral activities, potential efficacy against future emerging viruses, and a lower likelihood of inducing drug resistance. In our review article, we have synthesized known host-directed antiviral targets that span diverse cellular pathways and mechanisms, shedding light on the intricate interplay between host cells and viruses. Additionally, we have provided a brief overview of the development of HDAs based on these targets. We aim for this comprehensive analysis to offer valuable perspectives and insights that can guide future antiviral research and drug development efforts.

Objective To explore the application effect of immersive virtual reality(IVR)technology and different viewing content in pediatric patients with general anesthesia during elective surgery,and to provide references for clinical implementation.Methods A total of 180 pediatric patients who underwent elective surgery under general anesthesia in a tertiary hospital in Qingdao from February to October 2023 were selected as study population using convenient sampling method.According to the operation time,60 pediatric patients who underwent surgery from May to July 2023 were included in the immersive panoramic surgical education group,and they could watch the panoramic surgical education video immersively on the basis of routine care.A total of 60 pediatric patients who underwent surgery from August to October 2023 were included in the immersive animation group to watch cartoons immersively on the basis of routine care.A total of 60 pediatric patients who underwent surgery from February to April 2023 were included in the control group and routine preoperative care was implemented.The preoperative anxiety levels,anesthesia induction compliance,and incidence of emergence agitation were compared in the 3 groups by the modified Yale Preoperative Anxiety Scale-Short Form(mYPAS-SF),anesthesia induction cooperation grade,and the Pediatric Anesthesia Emergence Delirium scale(PAED).Results There were statistically significant differences in preoperative anxiety level,anesthesia induced compliance and incidence of emergence agitation during awakening between the 3 groups(P＜0.001).Among them,the preoperative anxiety level of the immersive panoramic surgical education group and the immersive animation group was lower than that of the control group,and the difference was statistically significant(P＜0.001).The anesthesia induced compliance degree of the immersive panoramic surgical education group and the immersive animation group was better than that of the control group,and the difference was statistically significant(P＜0.017),and the incidence of emergence agitation in the immersive panoramic surgical education group was lower than that of the control group,and the difference was statistically significant(P=0.004).Conclusion The use of IVR technology to watch panoramic surgical education videos and cartoons can help reduce the preoperative anxiety level and improve anesthesia induction cooperation degree of pediatric patients with general anesthesia during elective surgery,but the intervention effect of panoramic surgical education videos is better in improving the emergence agitation.

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

Objective To investigate the potential mechanism by which dihydromyricetin(DHM)ameliorates diabetic nephropathy(DN),and to screen and validate its possible key molecular targets.Methods A DN model was established using db/db mice,and 100 mg/(kg·d)DHM was administered via gavage 5 d per week for totally 10 weeks.Renal morphological changes were observed after staining to evaluate the effects of DHM.GSE161885 and GSE270526 datasets were obtained from the Gene Expression Omnibus(GEO)database and analyzed in combination with the GeneCards database to screen for DN-related differentially expressed genes(DEGs).Protein-protein interaction(PPI)network and molecular docking were employed to predict potential DHM targets.Western blotting and immunofluorescence staining were performed to detect the effects of DHM on pyroptosis-related pathways in the renal tissues of db/db mice and in high glucose(HG)-induced human renal tubular epithelial cells(HK-2).The specific NLR family pyrin domain containing protein 3(NLRP3)inhibitor MCC950 was also used to validate the predicted mechanism.Results In vivo experiments showed that DHM significantly ameliorated renal pathological damage in db/db mice,alleviated glomerular hypertrophy and mesangial expansion,and markedly reduced Paller scores(P<0.001).Immunofluorescence staining revealed significantly weakened fluorescence signals for α-smooth muscle actin(α-SMA),fibronectin,and collagen Ⅰ in renal tissues.Western blot results showed that the expression levels of collagen Ⅰ,collagen Ⅲ,α-SMA,and transforming growth factor beta 1(TGF-β1)were significantly decreased(P<0.05).A total of 16 DN-related DEGs were identified.Enrichment analysis revealed that these genes were primarily enriched in pathways such as viral protein interactions,cytokine-cytokine receptor interaction,and the AGE-RAGE signaling pathway in diabetic complications,and were primarily involved in gene functions such as the positive regulation of lymphocyte-mediated immunity,positive regulation of adaptive immune response,and chemokine activity.Molecular docking confirmed NLRP3 as a potential target of DHM.In vivo validation showed that DHM significantly down-regulated gasdermin-D(GSDMD)fluorescence signals and inhibited the expression of pyroptosis-related proteins including NLRP3,Caspase 1,Cleaved-Caspase 1,interleukin 18(IL-18),and GSDMD(P<0.05).In vitro studies further confirmed that both DHM and the specific NLRP3 inhibitor MCC950 alleviate high glucose-induced fibrosis and pyroptosis in HIC-2 cells.Conclusion DHM can ameliorate the progression of DN,and its mechanism is related to inhibiting NLRP3 inflammasome-mediated pyroptosis,thereby alleviating renal inflammation and fibrosis.

AIM:To explore suitable methods for the induction and cryopreservation of mouse bone marrow-derived macrophages(BMDMs).METHODS:Mouse fibroblasts(L929 cells)were cultured under varying conditions of temperature,seeding density,and serum concentration.The concentration of macrophage colony-stimulating factor(M-CSF)in the cell supernatant was measured using ELISA to determine the optimal conditions.Mouse bone marrow cells were extracted,and a differential adherence method was employed to pre-culture the bone marrow cells for 4 h,followed by flow cytometry to assess the proportion of resident macrophages.Flow cytometry was utilized to assess the ratio of F4/80 positive cells among the suspended and adherent cells.Induction of BMDMs was conducted using L929 cell supernatant or recombinant M-CSF for 7 d,and flow cytometry was applied to evaluate the proportion of F4/80 and CD11b double-positive cells.The morphologic changes during cell induction were observed under an inverted microscope,and the phagocytic ca-pacity and inflammatory response levels of BMDMs derived from C57BL/6N and C57BL/6J mice were evaluated using neu-tral red and ELISA methods.The cells were immediately cryopreserved after extraction,and then induced after recovery,or cryopreserved after successful induction and recovered.The cell morphology was observed under an inverted micro-scope,cell viability was assessed using the CCK-8 method,and phagocytic ability was measured using the neutral red method.RESULTS:The M-CSF concentration in the supernatant from L929 cells cultured at 33℃,10%fetal bovine se-rum(FBS)for 7 d was rich.Following 4 h of pre-culture,the proportion of F4/80 positive cells in adherent cells was sig-nificantly higher than that in suspended cells(P＜0.01).After 7 d of induction with L929 cell supernatant or recombinant M-CSF,the proportions of F4/80+CD11b+cells showed no significant difference(P＞0.05).Compared with the BMDMs derived from C57BL/6J mice,those from C57BL/6N mice exhibited stronger phagocytic capacity(P＜0.01),and released lower levels of TNF-α(P＜0.01)and IL-6(P＜0.05),and higher levels of IL-1β(P＜0.05).Compared with the BMDMs that were induced after recovery from initial cryopreservation,those cryopreserved immediately after extraction and in-duced upon recovery exhibited better macrophage morphology,higher cell viability(P＜0.01),and enhanced phagocytic ability(P＜0.01).CONCLUSION:The supernatant from L929 cells cultured at 33℃,10%FBS for 7 d is rich in M-CSF,successfully inducing bone marrow cells to differentiate into mouse BMDMs.The differential adherence method for pre-culturing can eliminate resident macrophages from the original bone marrow.The phagocytic capacity and inflammato-ry response levels differ between BMDMs derived from the C57BL/6N and C57BL/6J mouse subtypes.Cryopreserving bone marrow cells immediately after extraction and subsequently inducing them upon recovery is a preferable method for BMDM cryopreservation.

OBJECTIVE To design and synthesize rifamycin S derivatives and load them into milk exosomes to evaluate their in vitro antimicrobial activity.METHODS Rifamycin S derivatives were synthe-sized and characterized by mass spectrometry and NMR.Using the dilution assay method,the inhibitory activity of each rifamycin S derivatives molecule against Staphylococcus aureus and Pseudomonas aerugi-nosa was determined,and the IC50 was calculated.Derivatives molecules with excellent antimicrobial activity were selected and loaded into milk exosomes using the ultrasonication method,resulting in the preparation of milk exosome-loaded rifamycin S derivatives.The antimicrobial activity against Staphylo-coccus aureus was determined using the dilution assay method.The inhibitory effect of the exosome-loaded rifamycin S derivatives on Staphylococcus aureus residing within macrophages was detected using the plate colony counting method.RESULTS Three rifamycin S derivatives were successfully designed and synthesized,which demonstrated superior antimicrobial activity against Staphylococcus aureus(the parent compound's antimicrobial activity is merely from 1/20 to 1/80 of that of the three rifamycin S derivatives)and Pseudomonas aeruginosa(the parent compound's antimicrobial activity is only 1/14 and 1/9 of that of compound 1 and compound 3)compared to the parent compound.The loading of milk exosomes with the rifamycin S derivatives compound 3 was successfully achieved,with a loading efficiency of 10.9％.The antimicrobial activity of the compound after exosome loading was significantly enhanced against Staphylococcus aureus in vitro and against Staphylococcus aureus residing within macrophages(P＜0.01).CONCLUSION The designed and synthesized derivatives of rifamycin S possess stronger anti-microbial activity,and their antibacterial efficacy against both extracellular and intracellular bacteria can be further enhanced after loading into exosomes.