OBJECTIVE To investigate the effects and mechanism of the Yishen paidu formula on renal fibrosis in rats with chronic renal failure （CRF） through the reactive oxygen species （ROS）/thioredoxin-interacting protein （TXNIP）/NOD-like receptor thermal protein domain associated protein 3 （NLRP3） pathway. METHODS Rats were randomly divided into control group， model group， Yishen paidu formula low-dose （Yishen paidu formula-L） group， Yishen paidu formula high-dose （Yishen paidu formula- H） group， Yishen paidu formula-H+pcDNA-NC group， and Yishen paidu formula-H+ pcDNA-TXNIP group， with 10 rats in each group. Except for control group， all other rats were fed a diet containing 0.5% adenine to establish a CRF model； the rats were then administered corresponding drugs or normal saline intragastrically or via tail vein， once daily， for 8 consecutive weeks. After the last administration， the levels of serum creatinine （Scr）， blood urea nitrogen （BUN）， ROS， superoxide dismutase （SOD）， malondialdehyde （MDA）， tumor necrosis factor-α （TNF-α）， interleukin （IL）-6， and IL-1β were measured in each group. Pathological changes in renal tissue were observed， and the protein expression levels of Collagen Ⅲ， α-smooth muscle actin （α-SMA）， transforming growth factor-β1 （TGF-β1）， TXNIP and NLRP3 in renal tissue were detected. RESULTS Compared with model group， the renal histopathological damage and fibrosis of rats in Yishen paidu formula-L group and Yishen paidu formula-H group were significantly alleviated. The levels of Scr， BUN， ROS， MDA， TNF- α， IL-6 and IL-1β， and the protein expressions of Collagen Ⅲ， α-SMA， TGF-β1， TXNIP and NLRP3 were significantly decreased， while SOD levels were significantly increased （P＜0.05）. Moreover， the changes were more pronounced in the Yishen paidu formula-H group （P＜0.05）. Compared with Yishen paidu formula-H+pcDNA-NC group， above indexes of rats in Yishen paidu formula-H+pcDNA-TXNIP group were reversed significantly （P＜0.05）. CONCLUSIONS Yishen paidu formula can inhibit renal fibrosis in CRF rats by suppressing the ROS/TXNIP/NLRP3 pathway.

BACKGROUND:Acupuncture is an effective method for lumbar pain in lumbar disc herniation,but its mechanism has not yet been clarified.Factors related to the JAK2/STAT3 signaling pathway regulate the body's inflammatory response and are involved in the process of neuropathic pain. OBJECTIVE:To study the mechanism of acupuncture on lumbar disc herniation in a rat model based on the JAK2/STAT3 signaling pathway. METHODS:Forty Sprague-Dawley rats were randomly divided into four groups:sham operation group,model group,acupuncture group,and acupuncture+agonist group,with 10 rats in each group.Animal models of L5 lumbar disc herniation was constructed through autologous disc cell transplantation in the model group,acupuncture group,and acupuncture+agonist group.Rats in the acupuncture group and the acupuncture+agonist group received acupuncture treatment(Yanglingquan,Shenshu,Huantiao,and Dachangshu acupoints)at 3 days after modeling,and acupuncture treatment was given once a day,20 minutes each,for 15 consecutive days.Rats in the acupuncture+agonist group were injected intrathecally with coumermycin A1,a JAK2 agonist,into the L4/L5 intervertebral space,once a day,20 minutes each,prior to the acupuncture at 6,12,and 18 days after modeling.Paw withdrawal mechanical threshold was detected before and 3,6,9,12,15,and 18 days after modeling.At 18 days after modeling,serum inflammatory factor levels were detected,hematoxylin-eosin staining was performed to observe the morphology of L5-L6 tissues,RT-PCR was performed to detect the expression of JAK2 and STAT3 mRNAs in L5-L6 tissues,and western blot was performed to detect the expression of JAK2,p-JAK2 and p-STAT3 proteins in L5-L6 tissues. RESULTS AND CONCLUSION:The paw withdrawal mechanical thresholds of rats in the model group at different time points after modeling were lower than those in the sham operation group(P<0.05),the paw withdrawal mechanical thresholds of rats in the acupuncture group were higher than those in the model group at 9,12,15,and 18 days after modeling(P<0.05),and the paw withdrawal mechanical thresholds of rats in the acupuncture+agonist group were lower than those in the acupuncture group at 9,12,15,and 18 days after modeling(P<0.05).The levels of interleukin 6,tumor necrosis factor α,neurotransmitter substance P,and brain neuropeptide Y were elevated in the model group compared with the sham operation group(P<0.05);the levels of all four inflammatory factors were reduced in the acupuncture group compared with the model group(P<0.05);and the levels of all four inflammatory factors were elevated in the acupuncture+agonist group compared with the acupuncture group(P<0.05).Hematoxylin-eosin staining showed that lumbar degeneration was obvious in the model group but reduced in the acupuncture group and the acupuncture+agonist group.Moreover,the reduction was more obvious in the acupuncture group compared with the acupuncture+agonist group.The JAK2 and STAT3 mRNA expression as well as the p-JAK2 and p-STAT3 protein expression were elevated in the model group compared with the sham operation group(P<0.05),were decreased in the acupuncture group compared with the model group(P<0.05),and were increased in the acupuncture+agonist group compared with the acupuncture group(P<0.05).To conclude,acupuncture can alleviate inflammation to exert analgesic effects in the rat model of lumbar disc herniation,and its mechanism of action may be related to the inhibition of the JAK2/STAT3 signaling pathway.

Objective To explore the construction of α-1,3-galactosyltransferase (GGTA1) gene-knockout (GTKO) Diannan miniature pigs and the kidney xenotransplantation from pigs to rhesus macaques, and to assess the effectiveness of GTKO pigs. Methods The GTKO Diannan miniature pigs were constructed using the CRISPR/Cas9 gene-editing system and somatic cell cloning technology. The phenotype of GTKO pigs was verified through polymerase chain reaction, Sanger sequencing and immunofluorescence staining. Flow cytometry was used to detect antigen-antibody (IgM) binding and complement-dependent cytotoxicity. Kidney xenotransplantation was performed from GTKO pigs to rhesus macaques. The humoral immunity, cellular immunity, coagulation and physiological indicators of the recipient monkeys were monitored. The function and pathological changes of the transplanted kidneys were analyzed using ultrasonography, hematoxylin-eosin staining, immunohistochemical staining and immunofluorescence staining. Results Single-guide RNA (sgRNA) targeting exon 4 of the GGTA1 gene in Diannan miniature pigs was designed. The pGL3-GGTA1-sgRNA1-GFP vector was transfected into fetal fibroblasts of Diannan miniature pigs. After puromycin selection, two cell clones, C59# and C89#, were identified as GGTA1 gene-knockout clones. These clones were expanded to form cell lines, which were used as donor cells for somatic cell nuclear transfer. The reconstructed embryos were transferred into the oviducts of trihybrid surrogate sows, resulting in 13 fetal pigs. Among them, fetuses F04 and F11 exhibited biallelic mutations in the GGTA1 gene, and F04 had a normal karyotype. Using this GTKO fetal pig for recloning and transferring the reconstructed embryos into the oviducts of trihybrid surrogate sows, seven surviving piglets were obtained, all of which did not express α-Gal epitope. The binding of IgM from the serum of rhesus monkey 20# to GTKO pig PBMC was reduced, and the survival rate of GTKO pig PBMC in the complement-dependent cytotoxicity assay was higher than that of wild-type pig. GTKO pig kidneys were harvested and perfused until completely white. After the left kidney of the recipient monkey was removed, the pig kidney was heterotopically transplanted. Following vascular anastomosis and blood flow restoration, the pig kidney rapidly turned pink without hyperacute rejection (HAR). Urine appeared in the ureter 6 minutes later, indicating successful kidney transplantation. The right kidney of the recipient was then removed. Seven days after transplantation, the transplanted kidney had good blood flow, the recipient monkey's serum creatinine level was stable, and serum potassium and cystatin C levels were effectively controlled, although they increased 10 days after transplantation. Seven days after transplantation, the levels of white blood cells, lymphocytes, monocytes and eosinophils in the recipient monkey increased, while platelet count and fibrinogen levels decreased. The activated partial thromboplastin time, thrombin time and prothrombin time remained relatively stable but later showed an upward trend. The recipient monkey survived for 10 days. At autopsy, the transplanted kidney was found to be congested, swollen and necrotic, with a small amount of IgG deposition in the renal tissue, and a large amount of IgM, complement C3c and C4d deposition, as well as CD68⁺ macrophage infiltration. Conclusions The kidneys of GTKO Diannan miniature pigs may maintain normal renal function for a certain period in rhesus macaques and effectively overcome HAR, confirming the effectiveness of GTKO pigs for xenotransplantation.

Male factors contribute to 50% of infertility cases, with 20%-30% of cases being solely attributed to male infertility. Helicase for meiosis 1 ( HFM1 ) plays a crucial role in ensuring proper crossover formation and synapsis of homologous chromosomes during meiosis, an essential process in gametogenesis. HFM1 gene mutations are associated with male infertility, particularly in cases of non-obstructive azoospermia and severe oligozoospermia. However, the effects of intracytoplasmic sperm injection (ICSI) in HFM1 -related infertility cases remain inadequately explored. This study identified novel biallelic HFM1 variants through whole-exome sequencing (WES) in a Chinese patient with severe oligozoospermia, which was confirmed by Sanger sequencing. The pathogenicity of these variants was assessed using real-time quantitative polymerase chain reaction (RT-qPCR) and immunoblotting, which revealed a significant reduction in HFM1 mRNA and protein levels in spermatozoa compared to those in a healthy control. Transmission electron microscopy revealed morphological abnormalities in sperm cells, including defects in the head and flagellum. Despite these abnormalities, ICSI treatment resulted in a favorable fertility outcome for the patient, indicating that assisted reproductive techniques (ART) can be effective in managing HFM1 -related male infertility. These findings offer valuable insights into the management of such cases.
Humans ; Male ; Sperm Injections, Intracytoplasmic ; Oligospermia/therapy* ; Adult ; Spermatozoa/ultrastructure* ; Exome Sequencing ; Mutation

OBJECTIVES: To investigate the therapeutic mechanism of Huoxue Shufeng Granules (HXSFG) for alleviating central sensitization in a mouse model of chronic migraine (CM). METHODS: We analyzed the main chemical components of HXSFG through literature review and explored their pharmacological mechanisms by bioinformatics analyses. In a male C57BL/6J mouse model of CM established by intraperitoneal injections of nitroglycerin (10 mg/kg) every other day (5 injections), the effects of gavage with low, and high doses of HXSFG or intraperitoneal injections of topiramate for ameliorating central sensitization were evaluated using Von Frey test and a hot plate apparatus; the changes in expressions of inflammatory factors, the proteins in the TLR4/NF‑κB signaling pathway, and activation of c-Fos and CGRP were detected using RT-qPCR, Western blotting and immunofluorescence staining. RESULTS: Network pharmacology analysis suggested that the main active components in HXSFG for alleviating CM included formononetin, paeoniflorin, quercetin, and tanshinone. Gene Ontology (GO) enrichment analysis identified 492 GO entries, comprising 366 biological processes, 46 cellular components, and 80 molecular functions. KEGG pathway enrichment analysis indicated that the Toll-like receptor and NF‑κB signaling pathways were crucial in mediating the therapeutic effects of HXSFG on CM. In the mouse models of CM, both topiramate and HXSFG treatments alleviated the symptoms of central sensitization, evidenced by improved mechanical and thermal pain thresholds in the mice. HXSFG significantly reduced the expression of c-Fos and CGRP, improved inflammatory markers, and downregulated the expressions of TLR4, p-NF‑κB, IL-1β, and TNF‑α proteins in the mouse models. CONCLUSIONS HXSFG effectively alleviates central sensitization in CM mice by modulating the inflammatory pathways and inhibiting the TLR4/ NF-κB signaling pathway, suggesting its potential as a therapeutic option for CM.
Animals ; Toll-Like Receptor 4/metabolism* ; NF-kappa B/metabolism* ; Drugs, Chinese Herbal/therapeutic use* ; Mice ; Male ; Mice, Inbred C57BL ; Signal Transduction/drug effects* ; Migraine Disorders/metabolism* ; Disease Models, Animal ; Inflammation

OBJECTIVES: To investigate the mechanisms of Modified Chaihu Guizhi Decoction (MCGD) for ameliorating anxiety- and depression-like behaviors in a mouse model of chronic unpredictable mild stress (CUMS). METHODS: The main chemical constituents of MCGD were identified through literature review, and network pharmacology analysis was performed to predict the potential pharmacological mechanisms of MCGD. For in vivo validation, male C57BL/6J mice were randomized into control group, CUMS model group, fluoxetine (FLX) treatment group, and low- and high-dose MCGD treatment groups (n=15), and in all but the control group, CUMS models were established by daily exposure to two randomized stressors for 28 consecutive days. Starting from 3 days prior to modeling, MCGD and fluoxetine treatments were administered daily via gavage and intraperitoneal injection, respectively. Depression- and anxiety-like behaviors of the mice were assessed using sucrose preference test, forced swim test, open field test and elevated plus maze test. The changes in mRNA expressions of the clock genes and inflammatory markers and expressions of the JAK2/STAT3 signaling proteins were detected using RT-qPCR and Western blotting, and immunofluorescence staining was used to detect microglia activation in the mice. RESULTS: The key active compounds in MCGD identified by network pharmacology analysis included quercetin, acacetin, formononetin, nobiletin, and baicalein. GO analysis identified 607 enriched pathways, and KEGG pathway enrichment revealed significant involvement of the JAK2/STAT3 and NF-κB signaling pathways. In the mouse models of CUMS, treatment with both fluoxetine and MCGD significantly alleviated anxiety- and depression-like behaviors. MCGD treatment significantly reduced Iba1 expression, improved the inflammatory markers, reversed the decrease in clock gene circadian rhythm amplitude, and obviously downregulated the expressions of JAK2, p-STAT3, p-NF-κB, IL-1β, and IL-6 proteins. CONCLUSIONS MCGD effectively alleviates anxiety- and depression-like behaviors in CUMS mice by modulating the inflammatory pathways and inhibiting the JAK2/STAT3 signaling pathway.
Animals ; Janus Kinase 2/metabolism* ; STAT3 Transcription Factor/metabolism* ; Signal Transduction/drug effects* ; Depression/metabolism* ; Drugs, Chinese Herbal/pharmacology* ; Mice ; Male ; Mice, Inbred C57BL ; Anxiety/drug therapy* ; Stress, Psychological ; Disease Models, Animal

Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) cause a severe neurodevelopmental disorder, yet the impact of truncating mutations remains unclear. Here, we introduce the Cdkl5^492stop mouse model, mimicking C-terminal truncating mutations in patients. 492stop/Y mice exhibit altered dendritic spine morphology and spontaneous seizure-like behaviors, alongside other behavioral deficits. After creating cell lines with various Cdkl5 truncating mutations, we found that these mutations are regulated by the nonsense-mediated RNA decay pathway. Most truncating mutations result in CDKL5 protein loss, leading to multiple disease phenotypes, and offering new insights into the pathogenesis of CDKL5 disorder.
Animals ; Disease Models, Animal ; Mice ; Protein Serine-Threonine Kinases/deficiency* ; Mutation/genetics* ; Epileptic Syndromes/genetics* ; Humans ; Dendritic Spines/pathology* ; Spasms, Infantile/genetics* ; Male ; Seizures/genetics* ; Mice, Inbred C57BL

Extensive epigenetic reprogramming involves in memory CD8+ T-cell differentiation. The elaborate epigenetic rewiring underlying the heterogeneous functional states of CD8+ T cells remains hidden. Here, we profile single-cell chromatin accessibility and map enhancer-promoter interactomes to characterize the differentiation trajectory of memory CD8+ T cells. We reveal that under distinct epigenetic regulations, the early activated CD8+ T cells divergently originated for short-lived effector and memory precursor effector cells. We also uncover a defined epigenetic rewiring leading to the conversion from effector memory to central memory cells during memory formation. Additionally, we illustrate chromatin regulatory mechanisms underlying long-lasting versus transient transcription regulation during memory differentiation. Finally, we confirm the essential roles of Sox4 and Nrf2 in developing memory precursor effector and effector memory cells, respectively, and validate cell state-specific enhancers in regulating Il7r using CRISPR-Cas9. Our data pave the way for understanding the mechanism underlying epigenetic memory formation in CD8+ T-cell differentiation.
CD8-Positive T-Lymphocytes/metabolism* ; Cell Differentiation ; Chromatin/immunology* ; Animals ; Mice ; Immunologic Memory ; Epigenesis, Genetic ; SOXC Transcription Factors/immunology* ; NF-E2-Related Factor 2/immunology* ; Mice, Inbred C57BL ; Gene Regulatory Networks ; Enhancer Elements, Genetic

The pursuit of healthy aging has long rendered aging and senescence captivating. Age-related ailments, such as cardiovascular diseases, diabetes, and neurodegenerative disorders, pose significant threats to individuals. Recent studies have shed light on the intricate mechanisms encompassing genetics, epigenetics, transcriptomics, and metabolomics in the processes of senescence and aging, as well as the establishment of age-related pathologies. Amidst these underlying mechanisms governing aging and related pathology metabolism assumes a pivotal role that holds promise for intervention and therapeutics. The advancements in metabolomics techniques and analysis methods have significantly propelled the study of senescence and aging, particularly with the aid of multiscale metabolomics which has facilitated the discovery of metabolic markers and therapeutic potentials. This review provides an overview of senescence and aging, emphasizing the crucial role metabolism plays in the aging process as well as age-related diseases.
Humans ; Aging/metabolism* ; Metabolomics/methods* ; Neurodegenerative Diseases/metabolism* ; Cardiovascular Diseases/metabolism*

ObjectiveTo investigate the clinical application value of a predictive model for the efficacy of third-generation cephalosporin in the treatment of community-acquired spontaneous bacterial peritonitis （CASBP）. MethodsThis prospective study was conducted among 50 patients with liver cirrhosis and CASBP who were admitted to The Ninth Hospital of Nanchang from January 2021 to June 2022， and the patients were randomly divided into optimized treatment group and traditional treatment group， with 25 patients in each group. The patients in the optimized treatment group received ceftazidime or imipenem for initial treatment based on the above predictive model， and those in the traditional treatment group received ceftazidime for initial treatment， with the subsequent use of antibiotics adjusted based on the efficacy of initial treatment. The two groups were compared in terms of the response rate of initial treatment， cure rate on day 5， and 30-day mortality rate. The independent-samples t test or the Mann-Whitney U test was used for comparison of continuous data between two groups， and the chi-square test or the Fisher’s exact test was used for comparison of categorical data between two groups. ResultsAll patients completed the study. The optimized treatment group had a significantly higher response rate of initial treatment than the traditional treatment group （88.0% vs 60.0%， χ²=5.094， P=0.024）， while there was no significant difference in the cure rate on day 5 between the two groups （80.0% vs 56.6%， χ²=3.309， P=0.069）. As for the patients who received ceftazidime for initial treatment， the optimized treatment group had a significantly higher response rate of initial treatment than the traditional treatment group （88.9% vs 60.0%， χ²=4.341， P=0.037）， while there was no significant difference in the cure rate on day 5 between the two groups （83.3% vs 56.0%， χ²=2.425， P=0.119）. There was no significant difference in 30-day mortality rate between the two groups （8.0% vs 20.0%， χ²=0.664， P=0.415）. For all patients， there was a significant association between response of initial treatment and cure on day 5 （odds ratio ［OR］=9.643， 95% confidence interval ［CI］： 2.292‍ — ‍40.564） and between cure on day 5 and 30-day mortality （OR=0.138， 95%CI： 0.023‍ — ‍0.813）. ConclusionThis predictive model for efficacy helps clinicians to identify the patients who can benefit from third-generation cephalosporin treatment and improve the efficacy of third-generation cephalosporin in the initial empirical treatment of CASBP.