ObjectiveTo investigate the effects of Chaihu Jia Longgu Mulitang（CJLM） on depression-like behaviors and neuroinflammation in rats subjected to social isolation combined with chronic unpredictable mild stress（CUMS）， and to explore the potential underlying mechanisms. MethodsSixty male SD rats were randomly divided into a normal group， a model group， and low-， medium-， and high-dose CJLM groups（2.89， 5.78， 11.56 g·kg^-1）， as well as a fluoxetine group（10 mg·kg^-1）. Except for the normal group， all other groups were subjected to social isolation combined with CUMS for 63 d. During the first 35 d， depression models were established only， and from day 36 onward， modeling and drug administration were conducted simultaneously for a total intervention period of 28 d. Depression-like behaviors were evaluated using the sucrose preference test， open-field test， and forced swimming test. Hematoxylin-eosin（HE） staining was performed to observe hippocampal histomorphology. Immunohistochemistry（IHC） was used to detect the expression levels of ionized calcium-binding adapter molecule 1（Iba1） and gasdermin D（GSDMD） proteins in the hippocampus. Western blot analysis was employed to determine the protein expression levels of adenosine 5′-monophosphate（AMP）-activated protein kinase（AMPK） and phosphorylated（p）-AMPK， silent information regulator 1（SIRT1）， nuclear factor-κB（NF-κB） and p-NF-κB， NOD-like receptor protein 3（NLRP3）， and Caspase-1 in the hippocampus. Real-time quantitative polymerase chain reaction（Real-time PCR） was used to detect the mRNA expression levels of tumor necrosis factor-α（TNF-α）， interleukin（IL）-6， and IL-1β in the hippocampus. ResultsCompared with the normal group， the model group showed a decreased sucrose preference rate（P<0.01）， reduced total movement distance（P<0.01）， prolonged immobility time（P<0.01）， and decreased central zone residence time（P<0.01） in the open-field test， and increased immobility time in the forced swimming test（P<0.01）. Hippocampal neuronal structure was damaged. The contents of Iba1 and GSDMD in the hippocampus were significantly increased（P<0.01）. The protein expression levels of p-AMPK and SIRT1 in the hippocampus were significantly decreased（P<0.01）， whereas the protein expression levels of p-NF-κB， NLRP3， and Caspase-1 were significantly increased（P<0.01）. The mRNA expression levels of IL-1β， IL-6， and TNF-α in the hippocampus were significantly upregulated（P<0.01）. Compared with the model group， the low-， medium-， and high-dose CJLM groups and the fluoxetine group all were able to reverse depression-like behavioral changes， as evidenced by increased sucrose preference rate， increased total movement distance with shortened immobility time in the open-field test， prolonged central zone residence time， and reduced immobility time in the forced swimming test（P<0.05， P<0.01）. Meanwhile， hippocampal neuronal structural damage was alleviated. In the hippocampus， the expression levels of Iba1 and GSDMD were downregulated， the expression levels of p-AMPK and SIRT1 were upregulated， and the abnormal elevations of p-NF-κB， NLRP3， Caspase-1， as well as IL-1β， IL-6， and TNF-α mRNA were suppressed（P<0.05， P<0.01）. ConclusionCJLM can ameliorate depression-like behaviors in rats subjected to social isolation combined with CUMS and attenuate hippocampal neuroinflammation and pyroptosis， suggesting that its effects may be associated with the regulation of AMPK/SIRT1/NF-κB/NLRP3 signaling pathway.

Objective: To explore the association role of anxiety and depressive symptoms with Internet gaming disorder (IGD) and emotional-behavioral problems among junior high school students, so as to provide references for preventing the occurrence and progression of emotional-behavioral problems in middle school students. Methods: From September to October 2024, a random cluster sampling method was adopted to select 38 069 students from 28 junior high schools in 14 counties (cities and districts) and functional areas of Jining City, Shandong Province. Surveys were conducted by Nine item Internet Gaming Disorder Scale-Short Form, Generalized Anxiety Disorder-7, Patient Health Questionnaire-9, and Strengths and Difficulties Questionnaire. The correlation of emotional and behavioral issues, online game addiction, anxiety and depressive symptoms among junior high school students was analyzed by Speaman correlation analysis. Mediation analysis was used to explore the role of anxiety and depressive symptoms between IGD and emotional-behavioral problems among junior high school students. Results: The detection rates of emotional-behavioral problems, anxiety symptoms, depressive symptoms, and IGD among junior high school students were 19.24 %, 9.48%, 16.69%, and 19.68%, respectively. The scores of emotional-behavioral problems, anxiety symptoms, depressive symptoms and IGD were 10.00 (6.00, 15.00), 3.00 (0.00, 7.00), and 4.00 (1.00, 8.00), 13.00 (10.00, 19.00), respectively. There were positive correlations between each pair of emotional-behavioral problem, IGD, anxiety symptoms, and depressive symptoms scores ( r =0.51-0.81, all P <0.01). Anxiety and depressive symptoms played mediating roles between IGD and emotional-behavioral problems among junior high school students, with mediating effect values of 0.16 (95% CI =0.15-0.17) and 0.12 (95% CI =0.11-0.14), respectively (both P <0.01). Conclusions Anxiety and depressive symptoms mediate the relationship between IGD and emotional-behavioral problems among junior high school students. It is necessary to identify early signs of IGD among junior high school students, strengthen intervention for anxiety and depressive symptoms, to prevent the occurrence and development of emotional and behavioral issues.

Objective To compare the pathological diagnosis rate, number of punctures, and applicability to different lesion locations between a fork-tip biopsy needle and a conventional needle. Methods A retrospective collection of data was conducted on 137 patients who underwent endoscopic ultrasound (EUS)-guided fine needle biopsy (novel needle group, n=12) or aspiration (conventional needle group, n=125) at Zhongshan Hospital, Fudan University from January to August 2023. After propensity score matching at a ratio of 1∶5, there were 12 patients in the novel needle group and 60 patients in the conventional needle group. The pathological diagnosis rate and number of punctures were compared between the two groups. The stratified analyses on the size and location of lesions were performed. Results After matching, the pathological diagnosis rate was 91.7% (11/12) in the novel needle group, with a median of 2 punctures; the rate was 61.7% (37/60) in the conventional needle group, with a median of 3 punctures. No statistically significant differences in pathological diagnosis rate and number of punctures were observed between the two groups. Subgroup analysis showed that for lesions ≥20 mm, there was no significant difference in the number of punctures between the novel and conventional needles. The novel needle achieved a significantly higher pathological diagnosis rate for subepithelial tumors compared with the conventional needle (100.0% vs 30.4%, P＝0.019), whereas no significant differences were found for pancreatic masses or lymph node lesions. Conclusions Compared with EUS-guided aspiration using the conventional needle, EUS-guided biopsy using the novel fork-tip needle shows a trend toward a higher pathological diagnosis rate, with particular advantages in subepithelial tumors.

Objectives: Hypoparathyroidism (HP) is a rare endocrine disorder caused by parathyroid hormone (PTH) defi ciency. The PTH is a candidate gene for familial isolated hypoparathyroidism (FIH). This study aimed to investigate the pathogenicity of two novel rare variants (RVs) ofPTH through in vitro functional study. Methods: Targeted next-generation sequencing was used to identify candidate gene mutations. Clinical data were retrospectively collected. Wild-type (WT) PTH was used as a template for site-directed mutagenesis to create mutant eukaryotic expression plasmids, which were transfected into cells. Treated with or without 4-phenylbu tyric acid (4-PBA), the levels of intact PTH (iPTH) and PTH (1-84) were measured by chemiluminescence, and protein expression was assessed using Western blotting. Results: Two patients carrying PTH mutations (c.154G > A: p.Val52Ile, c.270G > T: p.Leu90Phe) were identified.Patient 1, a 45-year-old male, presented with carpal and pedal numbness, muscle cramps, and low serum calcium (1.29 mmol/L). Patient 2, a 12-year-old female, had muscle twitches, convulsions, low calcium (1.50 mmol/L), and iPTH of 4 pg/mL. The iPTH or PTH (1-84) levels in the medium transfected with mutant Val52Ile and Leu90Phe PTH decreased by 31%–38%, and 51%–96% compared to WT (allP < 0.05), which were not rescued by 4-PBA. No significant changes in intracellular PTH expression were observed. Conclusions In this study, two novel RVs of PTH(Val52Ile and Leu90Phe) were identified that may impair hormone synthesis and secretion. Our study has broadened the mutation spectrum of the PTH and shed light on potential mechanisms underlying FIH.

Objective: There were associations between emotional abuse and neglect (EAN) and depression, but few studies had tested potential mechanisms underlying these relationships. We aimed to provide insights on how (the mediation role of neuroticism), and under what conditions (the moderator role of psychological resilience), led to a higher level of depression. Methods: This study was a cross-sectional study that used a random cluster sampling method. We randomly selected 3,993 participants from four junior middle schools in northern city of China. Participants were asked to complete four self-reported questionnaires, including the Childhood Trauma Questionnaire, Children Depression Inventory-Short Form, Chinese Big Five Personality Inventory Brief Version, and Chinese Resilience Scale. Results: The results showed that neuroticism mediated the associations between EAN and depression. In addition, the mediating effect of neuroticism was moderated by psychological resilience (p<0.05). Conclusion EAN and neuroticism could have an adverse impact on depression, and psychological resilience could alleviate these negative effects as a moderator. Our model suggested psychological resilience could be a particularly effective intervention point for victims of EAN.

ObjectiveTo observe ovarian microvascular damage in rats with cold coagulation and blood stasis syndrome and to explore the mechanism by which Liangfang Wenjing decoction improves this condition in rats. MethodsFifty SPF female SD rats were randomly divided into a blank group， a model group， low-dose （8.1 g·kg^-1） and high-dose groups （16.2 g·kg^-1） of Liangfang Wenjing decoction， and a 4-phenylbutyric acid （0.1 g·kg^-1） group， with 10 rats in each group. The ice-water bath method was employed to establish the rat model of cold coagulation and blood stasis syndrome. Concurrent with modeling， Liangfang Wenjing decoction was administered continuously for 21 days， once daily. The rats' syndrome manifestations and estrous cycles were recorded. The enzyme-linked immunosorbent assay （ELISA） was used to detect serum reproductive hormone levels and levels of endothelin-1 （ET-1）， nitric oxide （NO）， thrombomodulin （TM）， and von Willebrand factor （vWF） in ovarian tissue. Prothrombin time （PT）， activated partial thromboplastin time （APTT）， thrombin time （TT）， and fibrinogen （FIB） were measured. The ovarian microcirculatory blood perfusion was detected by laser speckle contrast imaging. Hematoxylin-eosin （HE） staining was performed to observe the ovarian histopathology， flow cytometry to detect ovarian apoptosis rate， and transmission electron microscopy to observe the ultrastructure of ovarian microvascular endothelial cells. Western blot was employed to detect the protein expression of endothelial nitric oxide synthase （eNOS）， phosphorylated eNOS （p-eNOS）， Caspase-3， B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X protein （Bax）， glucose-regulated protein 78 （GRP78）， C/EBP homologous protein （CHOP）， inositol-requiring enzyme1α （IRE1α）， p-IRE1α， apoptosis signal-regulating kinase 1 （ASK1）， p-ASK1， c-Jun N-terminal kinase （JNK）， and p-JNK. Immunofluorescence was used to detect ovarian Bax and Bcl-2 expression in microvascular endothelial cells. ResultsCompared with the blank group， the model group showed signs of cold coagulation and blood stasis syndrome， prolonged estrus cycles， and reproductive hormone disorders. Histopathological results revealed a decrease in follicle counts at all stages and disorganized granulosa cell arrangement. Ovarian microcirculatory perfusion was significantly decreased （P<0.01）. PT， APTT， and TT were reduced （P<0.05， P<0.01）， while FIB levels were increased （P<0.05）. In ovarian tissue， NO content was decreased， while ET-1， vWF， and TM levels were increased significantly （P<0.01）. The apoptosis rate of ovarian cells was markedly increased （P<0.01）. Furthermore， p-eNOS/eNOS and Bcl-2 were decreased （P<0.05）， whereas Bax， cleaved-Caspase-3/Caspase-3， GRP78， CHOP， p-IRE1α/IRE1α， p-ASK1/ASK1， and p-JNK/JNK expression showed significant increases （P<0.05， P<0.01）. Compared with the model group， Liangfang Wenjing decoction intervention alleviated the symptoms of cold coagulation and blood stasis， gradually restored the estrus cycle， and improved ovarian histopathology and endothelial cell ultrastructure. Microcirculatory blood perfusion was significantly elevated （P<0.05）. NO content in ovarian tissue was elevated， while ET-1， vWF， and TM levels were significantly decreased （P<0.05， P<0.01）. The p-eNOS/eNOS ratio and Bcl-2 expression were significantly elevated （P<0.05）， while the expression of Bax， cleaved-Caspase-3/Caspase-3， GRP78， CHOP， p-IRE1α/IRE1α， p-ASK1/ASK1， and p-JNK/JNK was significantly decreased （P<0.05， P<0.01）. ConclusionLiangfang Wenjing decoction may regulate the IRE1α/ASK1/JNK signaling pathway to inhibit endoplasmic reticulum stress， attenuate apoptosis， and improve microvascular endothelial injury in ovaries of rats with cold coagulation and blood stasis syndrome.

Eight butyl-phthalides, senkyunolide K(1), senkyunolide N(2), butylphthalide(3), senkyunolide I(4), senkyunolide H(5),(Z)-butylidenephthalide(6),(Z)-ligustilide(7), and 3-butylidene-7-hydroxyphthalide(8) were isolated from the aerial part of Ligusticum sinense by column chromatography on silica gel column, ODS, Sephadex LH-20 and semi-preparative HPLC. Their structures were elucidated on the basis of spectroscopic and chemical data, especially NMR and MS. Compound 1 was a new butyl-phthalide and compounds 2-8 were isolated from the aerial part of L. sinense for the first time. Furthermore, the inhibitory activities of compounds 1-8 against the nitric oxide(NO) production induced by lipopolysaccharide(LPS) in mouse RAW264.7 macrophages in vitro were evaluated. The results showed that compounds 1-8 exerted inhibitory activities on NO production with IC_(50) of 19.34-42.16 μmol·L~(-1).
Animals ; Mice ; Nitric Oxide/biosynthesis* ; Ligusticum/chemistry* ; Benzofurans/isolation & purification* ; Drugs, Chinese Herbal/isolation & purification* ; Macrophages/immunology* ; RAW 264.7 Cells ; Molecular Structure

Diterpenoid ferruginol is a key intermediate in biosynthesis of active ingredients such as tanshinone and carnosic acid.However, the traditional process of obtaining ferruginol from plants is often cumbersome and inefficient. In recent years, the increasingly developing gene editing technology has been gradually applied to the heterologous production of natural products, but the production of ferruginol in microbe is still very low, which has become an obstacle to the efficient biosynthesis of downstream chemicals, such as tanshinone. In this study, miltiradiene was produced by integrating the shortened diterpene synthase fusion protein,and the key genes in the MVA pathway were overexpressed to improve the yield of miltiradiene. Under the shake flask fermentation condition, the yield of miltiradiene reached about(113. 12±17. 4)mg·L~(-1). Subsequently, this study integrated the ferruginol synthase Sm CYP76AH1 and Sm CPR1 to reconstruct the ferruginol pathway and thereby realized the heterologous synthesis of ferruginol in Saccharomyces cerevisiae. The study selected the best ferruginol synthase(Il CYP76AH46) from different plants and optimized the expression of pathway genes through redox partner engineering to increase the yield of ferruginol. By increasing the copy number of diterpene synthase, CYP450, and CPR, the yield of ferruginol reached(370. 39± 21. 65) mg·L~(-1) in the shake flask, which was increased by 21. 57-fold compared with that when the initial ferruginol strain JMLT05 was used. Finally, 1 083. 51 mg·L~(-1) ferruginol was obtained by fed-batch fermentation, which is the highest yield of ferruginol from biosynthesis so far. This study provides not only research ideas for other metabolic engineering but also a platform for the construction of cell factories for downstream products.
Saccharomyces cerevisiae/genetics* ; Diterpenes/metabolism* ; Metabolic Engineering ; Fermentation ; Abietanes

Ginsenoside Rg_2(GRg2) is a triterpenoid compound found in Panax notoginseng. This study explored its effects and mechanisms on diabetic retinopathy and angiogenesis. The study employed endothelial cell models induced by glucose or vascular endothelial growth factor(VEGF), the chorioallantoic membrane(CAM) model, the oxygen-induced retinopathy(OIR) mouse model, and the db/db mouse model to evaluate the therapeutic effects of GRg2 on diabetic retinopathy and angiogenesis. Transwell assays and endothelial tube formation experiments were conducted to assess cell migration and tube formation, while vascular area measurements were applied to detect angiogenesis. The impact of GRg2 on the retinal structure and function of db/db mice was evaluated through retinal thickness and electroretinogram(ERG) analyses. The study investigated the mechanisms of GRg2 by analyzing the activation of Yes-associated protein(YAP) and Toll-like receptors(TLRs) pathways. The results indicated that GRg2 significantly reduced cell migration numbers and tube formation lengths in vitro. In the CAM model, GRg2 exhibited a dose-dependent decrease in the vascular area ratio. In the OIR model, GRg2 notably decreased the avascular and neovascular areas, ameliorating retinal structural disarray. In the db/db mouse model, GRg2 increased the total retinal thickness and enhanced the amplitudes of the a-wave, b-wave, and oscillatory potentials(OPs) in the ERG, improving retinal structural disarray. Transcriptomic analysis revealed that the TLR signaling pathway was significantly down-regulated following YAP knockdown, with PCR results consistent with the transcriptome sequencing findings. Concurrently, GRg2 downregulated the expression of Toll-like receptor 4(TLR4), TNF receptor-associated factor 6(TRAF6), and nuclear factor-kappaB(NF-κB) proteins in high-glucose-induced endothelial cells. Collectively, GRg2 inhibits cell migration and tube formation and significantly reduces angiogenesis in CAM and OIR models, improving retinal structure and function in db/db mice, with its pharmacological mechanism likely involving the down-regulation of YAP expression.
Animals ; Ginsenosides/pharmacology* ; Diabetic Retinopathy/physiopathology* ; Mice ; YAP-Signaling Proteins ; Humans ; Male ; Signal Transduction/drug effects* ; Cell Movement/drug effects* ; Adaptor Proteins, Signal Transducing/genetics* ; Mice, Inbred C57BL ; Neovascularization, Pathologic/metabolism* ; Drugs, Chinese Herbal/administration & dosage* ; Panax notoginseng/chemistry* ; Endothelial Cells/metabolism* ; Transcription Factors/genetics* ; Angiogenesis