Background/Aims: Natural killer (NK) cell function is generally considered dampened in chronic hepatitis B virus (HBV) infection; however, the NK cell pool exhibits phenotypic and functional heterogeneity, and the antibody--mediated effect of NK cells remains less characterized. This study evaluated the dynamic changes in antibody-mediated NK cell responses and the involvement of distinct NK subsets across disease stages and during antiviral treatment. Methods: A T-cell receptor-like antibody specific for the HBV core 18–27 peptide (cTCRL-Ab) was used to determine the antibody-mediated effect of NK cells, and an array of NK cell surface markers were analyzed in cross-sectional and longitudinal cohorts of patients with chronic HBV infection. Single-cell RNA sequencing (scRNA-seq) was performed to identify the heterogeneity of NK subsets. Results: The cTCRL-Ab enabled the detection of NK cell cytolytic activity and IFNγ production. Notably, cTCRL-Ab-mediated NK cell responses were compromised in chronically HBV-infected patients, particularly in those receiving pegylated interferon-α (Peg-IFNα), which was associated with the downregulation of CD16 expression. Correspondingly, Peg-IFNα inhibited cTCRL-Ab-mediated NK cell function by reducing CD16 expression in vitro. scRNA-seq revealed that CD16 downregulation occurred mainly within a dysfunctional CD16hi NK subset exhibiting exhaustion properties. In contrast, an activated CD16hiNK subpopulation (CX3CR1⁺KLRC2–CD16hi) with high cytotoxicity was enriched in patients who experienced favorable treatment responses. Furthermore, the intrahepatic CX3CR1+KLRC2–CD16hi subset tended to exhibit functional restoration in HBsAg-loss individuals. Conclusions Our data contribute to the understanding of antibody-mediated responses of NK cells in chronic HBV infection, and highlight a previously unappreciated functional CX3CR1+KLRC2–CD16hiNK subset as a potential therapeutic target.

ObjectiveTo investigate whether intestinal deficiency of lanosterol synthase （LSS）， a key enzyme in cholesterol synthesis， influences the progression of metabolic dysfunction-associated steatotic liver disease （MASLD） by regulating intestinal cholesterol absorption and immune response. MethodsLSS heterozygous knockout （LSS^+/-） mice and wild-type （WT） controls were generated using CRISPR/Cas9 technology and fed either a high-fat diet （HFD） or regular chow （CHOW）. The model was validated by genotyping. Hepatic steatosis was assessed by HE and oil red O staining. Immunohistochemistry was used to detect the localization and expression of NPC1L1 and CD36 proteins in the intestine. Western blot analysis was performed to measure JNK phosphorylation and TLR4 protein levels in intestinal tissues. Real-time quantitative polymerase chain reaction （qPCR） was employed to examine the mRNA expression of TLR4 and IL-6. ResultsLSS^+/- mice were successfully validated by genotyping and reduced intestinal LSS protein expression. HE and oil red O staining of liver sections showed that， compared with WT mice fed a CHOW diet， WT mice fed a HFD exhibited a marked increase in hepatic lipid vacuoles. In contrast， compared with HFD-fed WT mice， HFD-fed LSS^+/- mice displayed significantly attenuated hepatic lipid deposition and reduced serum ALT levels （P<0.05）. Immunohistochemical analysis revealed that， compared with WT mice， the expression of the cholesterol absorption protein NPC1L1 in the intestinal villi of LSS^+/- mice was downregulated under both CHOW and HFD conditions （P_HFD<0.001）. Conversely， the expression of the fatty acid transporter CD36 was upregulated in the intestines of LSS^+/- mice （P_CHOW<0.05， P_HFD<0.01）. Western blot analysis demonstrated that， compared with WT mice， TLR4 protein expression in the intestines of LSS^+/- mice significantly increased under both CHOW and HFD conditions （both P<0.05）. JNK phosphorylation level was significantly elevated in LSS^+/- mice under CHOW condition （both P<0.05）. Under HFD condition， total JNK protein expression increased， but its phosphorylation level showed no significant change. qPCR analysis showed that， compared with WT mice， the mRNA levels of TLR4 （P_CHOW<0.01， P_HFD<0.000 1） and IL-6 （P_CHOW<0.001， P_HFD<0.01） were significantly upregulated in the intestines of LSS^+/-mice. ConclusionLSS deficiency counteracts hepatic lipid deposition by orchestrating a synergistic reprogramming involving restricted intestinal cholesterol absorption， enhanced fatty acid utilization， and activation of immune pathways， suggesting intestinal LSS as a potential therapeutic target of MASLD.

ObjectiveTo investigate the effect of hypoxia on pemetrexed （PE）-mediated apoptosis in non-small cell lung cancer cells. MethodsA549 cells were cultured under 21% O₂ （normoxia） or 1% O₂ （hypoxia）， and treated with 8 μmol/L PE for 48 h. The cells were divided into normoxic control group， normoxic vehicle group， normoxic drug-treated group， hypoxic control group， hypoxic vehicle group and hypoxic drug-treated group. Trypan blue staining was used to observe cell death after PE intervention under normoxia and hypoxia. Flow cytometry was adopted to detect cell apoptosis rate. Wound healing assay was used to assess cell migration. Western blot was performed to detect the protein levels of MRP-1， Cleaved Caspase-3， E-Cadherin and N-Cadherin. ResultsTrypan blue staining results showed that the cell death rate was reduced in PE-treated cells under hypoxia compared with the normoxic PE group （P<0.001）. Flow cytometry results indicated that PE induced cell apoptosis under both normoxic and hypoxic conditions.However， the apoptosis rate of the normoxic PE group was significantly higher than that of the hypoxic PE group （P<0.000 1）. Wound healing assay showed that the migration distance of A549 cells decreased after PE treatment for 24 h and 48 h under normoxia and hypoxia when compared with the control group （P<0.05）， while no significant difference was found between the normoxic PE group and the hypoxic PE group. Western blot results revealed that MRP-1 expression was markedly higher in hypoxic PE-treated cells than in normoxic PE-treated cells （P<0.000 1）. Compared with the control group， Cleaved Caspase-3 expression was upregulated in A549 cells following PE intervention under normoxia （P<0.05）， whereas no obvious alteration in Cleaved Caspase-3 expression was detected in hypoxic PE-treated cells. Under both normoxic and hypoxic conditions， PE treatment downregulated the expression of E-Cadherin （P<0.000 1，P<0.001） and N-Cadherin （P<0.05，P<0.000 1）. In addition， the expression levels of E-Cadherin and N-Cadherin in the hypoxic PE group were significantly lower than those in the normoxic PE group （all P<0.000 1）. ConclusionHypoxia significantly attenuates the pro-apoptotic effect of PE on A549 cells and blocks Cleaved Caspase-3 activation. This mechanism may be related to hypoxia-induced MRP-1 overexpression and reduced drug accumulation. Interfering with MRP-1 or restoring the activity of the apoptosis pathway may serve as a novel strategy to overcome hypoxia-induced drug resistance.

Polycystic ovarian syndrome （PCOS） is a common endocrine and metabolic disease mainly occurring among women of childbearing age. Its main symptoms include menstrual disorders， acne， hirsutism， infertility， oily skin， acanthosis nigricans， and obesity. Currently， the etiology and pathogenesis of PCOS remain unclear. Classical formulas， which have rigorous compatibility， concise composition， precise alignment with syndromes， and definitive therapeutic effects， demonstrate unique practical and scientific value in the treatment of PCOS. These formulas exhibit significant clinical efficacy， mild adverse effects， and sustained therapeutic outcomes. To explore the current status and mechanisms of classical formulas in treating PCOS， on the basis of Zhang Zhongjing's academic thoughts on gynecological diseases， this paper reviewed the relevant literature on the treatment of PCOS with classical formulas in recent years. The findings reveal that the pathogenesis of PCOS predominantly involves a combination of internal deficiency and superficial excess， closely related to dysfunction of the liver， spleen， and kidney. The root cause lies in deficiency， and on this basis， there are also symptoms of qi stagnation， blood stasis， phlegm obstruction， and dampness encumbrance. Commonly used classical formulas for treating this disease include Guizhi Fuling pills， Danggui Shaoyao powder， Wenjing decoction， and Jingui Shenqi pills. These classical formulas have good clinical efficacy in treating PCOS. Their mechanisms of action may be related to improving serum levels of sex hormones， increasing the dominant follicle diameter and endometrial thickness， alleviating insulin resistance， lowering glucose and lipid metabolism， inhibiting oxidative and inflammatory reactions in the ovarian tissue， regulating the intestinal flora， correcting the flora disorder， protecting the intestinal barrier function， and regulating the phosphatidylinositol 3-kinase/protein kinase B signaling pathway. The above research results can help doctors use classical formulas flexibly， broaden diagnostic and therapeutic approaches for PCOS and provide ideas for improving the traditional Chinese medicine diagnosis and treatment plan for PCOS.

Objective: To investigate the effect of loss of function of lanosterol synthase( LSS) gene on intestinal function in a mouse model of non-alcoholic fatty liver disease( NAFLD) induced by a high-fat diet． Methods: LSS gene heterozygous knockout C57 mice ( LSS + / －) were established using the CRISRP / Cas9 system．After being fed a high-fat diet with 60% fat content for 6 months，the fat deposition in liver tissues was detected by HE and Oil red O staining，the morphological changes of small intestine tissue were detected by HE staining．The changes in total cholesterol content in intestinal tissue were detected by kits．The gastrointestinal motility function of mice was detected by phenol red paste．The intestinal permeability was detected by Evans blue staining，and the expression of LSS，tight junction protein ( Claudin) -1，Claudin-5，cluster of differentiation 36 ( CD36) ，and Niemann-Pick type C1-like 1 protein ( NPC1L1) proteins in small intestinal tissues were detected by Western blot． Results : The results of HE and Oil red O staining of liver tissues showed that liver fat deposition in LSS gene heterozygous knockout mice was lower than that in wild-type mice in the high-fat diet group．The total cholesterol content in intestinal tis- sue of LSS gene heterozygous knockout mice decreased ( P ＜0. 01) ，but no morphological differences were ob- served between the two groups of mice by HE staining of intestinal tissues．The gastrointestinal motility function of LSS gene heterozygous knockout mice did not show significant changes．The intestinal permeability of LSS gene het- erozygous knockout mice in the high-fat diet group decreased as detected by Evans blue ( P＜0. 05) ．The expres- sion levels of Claudin-5 protein in the intestinal tissue of LSS gene heterozygous knockout mice in the high-fat diet group increased ( P ＜0. 05 ) ，while the expression of LSS protein in the intestinal tissues of LSS heterozygous knockout mice decreased ( P ＜0. 05) ． Conclusion In the NAFLD model induced by a high-fat diet，LSS gene heterozygous knockout reduces liver fat deposition induced by a high-fat diet and improves intestinal barrier function by regulating cholesterol metabolism in intestinal tissues and up-regulating the expression of Claudin-5．

Objective : To explore the changes in behavior and spatial memory of C57BL/6J female mice of different ages (youth , middle-aged , and elderly) . Methods: C57BL/6J female mice were divided into female youth group (YG group) , female middle-aged group ( MG group) and female elderly group ( OG group) according to age. The Morris water maze test measured spatial memory ability , and the open field and elevated cross maze test observed activity level and anxiety level. Western blot was used to determine the protein expressions of CREB , CaMKⅡ(pan) and CaMKⅡ(p) in the hippocampus of the brain tissues of female mice in each group. Results: Compared with the YG group , the weight of the MG group and the OG group significantly increased (P < 0. 01 , P < 0. 001) . Compared with the OG group , the third quadrant escape latency and the number of crossings in the YG group and MG group were shortened , and the difference was not statistically significant. Compared with the OG group , there was a statistically significant difference in the exercise speed in the open field of the YG group (P < 0. 01) , there was no significant difference in the movement speed in the open field of the MG group , the number of entries into the central zone significantly increased in the MG group ( P < 0. 05 ) , and there was no significant difference in the number of entries in the YG group (P > 0. 05) . Compared with the OG group , the YG group had a statistically significant difference in the elevated cross maze (P < 0. 05) , the MG group had no statistically signif- icant difference in the elevated cross maze , and the number of closed arm entries in the YG group and MG group significantly increased (P < 0. 001 , P < 0. 01) . Compared with the YG group , the relative expression level of CaMKⅡ(pan) in the OG group was statistically significant ( P < 0. 05 ) , while the relative expression level of CaMKⅡ(pan) in the MG group was not statistically significant ( P > 0. 05) . Conclusion With the increase of age , the weight of C57BL/6J female mice gradually increased , the activity level and desire to explore gradually de- creased , the spatial memory ability also declined , and the anxiety level and anxiety-like behavior increased. This study helps to reveal the effect of age on the activity level and cognitive function of females , and provides a refer- ence for studying cognitive and memory decline in older females.

Polycystic ovarian syndrome （PCOS） is a common endocrine and metabolic disease mainly occurring among women of childbearing age. Its main symptoms include menstrual disorders， acne， hirsutism， infertility， oily skin， acanthosis nigricans， and obesity. Currently， the etiology and pathogenesis of PCOS remain unclear. Classical formulas， which have rigorous compatibility， concise composition， precise alignment with syndromes， and definitive therapeutic effects， demonstrate unique practical and scientific value in the treatment of PCOS. These formulas exhibit significant clinical efficacy， mild adverse effects， and sustained therapeutic outcomes. To explore the current status and mechanisms of classical formulas in treating PCOS， on the basis of Zhang Zhongjing's academic thoughts on gynecological diseases， this paper reviewed the relevant literature on the treatment of PCOS with classical formulas in recent years. The findings reveal that the pathogenesis of PCOS predominantly involves a combination of internal deficiency and superficial excess， closely related to dysfunction of the liver， spleen， and kidney. The root cause lies in deficiency， and on this basis， there are also symptoms of qi stagnation， blood stasis， phlegm obstruction， and dampness encumbrance. Commonly used classical formulas for treating this disease include Guizhi Fuling pills， Danggui Shaoyao powder， Wenjing decoction， and Jingui Shenqi pills. These classical formulas have good clinical efficacy in treating PCOS. Their mechanisms of action may be related to improving serum levels of sex hormones， increasing the dominant follicle diameter and endometrial thickness， alleviating insulin resistance， lowering glucose and lipid metabolism， inhibiting oxidative and inflammatory reactions in the ovarian tissue， regulating the intestinal flora， correcting the flora disorder， protecting the intestinal barrier function， and regulating the phosphatidylinositol 3-kinase/protein kinase B signaling pathway. The above research results can help doctors use classical formulas flexibly， broaden diagnostic and therapeutic approaches for PCOS and provide ideas for improving the traditional Chinese medicine diagnosis and treatment plan for PCOS.

[Objective]To analyze the Yunqi characteristics and periodicity of drought disasters in He'nan Province during 2000 years.[Methods]On the foundation of the drought data of He'nan Province from 221 BC to 1840 AD,a hierarchical classification was conducted,using statistical tools and wavelet analysis to explore the frequency and periodicity of drought in different Yunqi periods.[Results]The most frequent dominant Qi period of drought in He'nan Province was 4th Qi(Taiyin Shitu)(P<0.001),the most of Sitian period was Jueyin Fengmu and Shaoyin Junhuo(P<0.05),the most of Zaiquan period was Shaoyang Xianghuo(P<0.05).At multiple time scales,drought disasters in He'nan Province had 4 quasi-cycles,the 1st to the 4th quasi-cycles were 510,690,300 and 150 years,respectively.[Conclusion]There was a significant difference in the frequency of drought levels in He'nan Province in different periods of dominant Qi,Sitian period and Zaiquan period,which was partly consistent with the Yunqi theory.Besides,the drought disaster in He'nan Province had certain periodic characteristics.

Objective Neurological disorders,constitute the majority of acupuncture-treated ailments.By critically examining a comprehensive selection of high-quality clinical randomized controlled trials(RCTs)from both domestic and international sources,we aimed to uncover the prevailing hot research topic and potential future directions in acupuncture therapy for these conditions.Methods Four databases,including PubMed,Web of science,Cochrane Library and EMBASE were searched.The search period is set from the establishment of each database to November 30,2023.And the search content was RCTs of acupuncture for neurological diseases classified as Ⅰ and Ⅱ by the Chinese Academy of Sciences were extracted.Key aspects extracted included annual publication volume,journals,disease types,outcomes,conclusions,acupuncture methods and control groups.Results A total of 81 high-quality RCTs were identified,revealing a consistent annual publication trend of acupuncture for neurological disorders.Key journals contributing to this research were Cephalalgia,Clinical Rehabilitation and Clinical Journal Of Pain,etc.The studies predominantly focused on migraine and sequela of apoplexy with migraine days/frequency and Ashworth scale and its modifications as commonly used primary outcome indicators,respectively.Overall,there were 73 RCTs that concluded that acupuncture for neurologic disorders were effective,8 were ineffective.The acupuncture methods were diverse,encompassing acupuncture,individualized acupuncture and transcutaneous electrical nerve stimulation,etc.The control measures were also varied,featuring sham acupuncture,sham-acupoint and micro-needle,etc.Conclusion With the development of acupuncture RCTs,their international influence has expanded.Research has shifted from validation of effectiveness to exploration of mechanisms,and despite the expansion of the disease spectrum of acupuncture,acupuncture trials have yet to be regulated and standardized,and the assessment of acupuncture efficacy remains controversial and challenging for the discipline.It is recommended to promote multi-center cooperation and strengthen the training of trialists etc.to provide more high-quality clinical evidence.

Objective To explore methods for point estimation in adaptive enrichment design,providing a reference for effect estimation inn this context.Methods We simulated efficacy data under adaptive enrichment design and subsequentially estimated the effects using five methods:maximum likelihood method,stage 2 method,Lindley's method,parametric Bootstrap method,and the uniformly minimum variance conditionally unbiased estimated(UMVCUE)method.The performance of these methods was further evaluated.Results The simulation study showed that the maximum likelihood method has a large bias and is very sensitive to some parameters,while Lindley's method tends to underestimate and the parametric bootstrap method sometimes overestimates.The stage 2 method and UMVCUE are almost unbiased.In terms of mean squared error(MSE),all methods perform well except stage 2 estimator.Conclusion In adaptive enrichment design,the UMVCUE performs well in terms of bias and MSE.Therefore,we recommend it as the point estimator for the treatment effect in adaptive enrichment design.