ObjectiveTo investigate the mechanisms by which Mahuang Lianqiao Chixiaodoutang （MLC） improves obesity-type polycystic ovary syndrome （PCOS） through the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） signaling pathway. MethodsThirty-six female Sprague-Dawley （SD） rats were randomly divided into a blank control group （Con） and an obesity-type PCOS model preparation group. The model was induced by gavage with letrozole （1 mg·kg^-1） combined with a high-fat diet （HFD）. After model establishment， the obesity-type PCOS model preparation group was further divided into the model group （Mod， normal saline）， metformin group （Met， 0.3 g·kg^-1）， low-dose MLC group （MLC-L， 4.3 g·kg^-1）， medium-dose MLC group （MLC-M， 8.6 g·kg^-1）， and high-dose MLC group （MLC-H， 17.2 g·kg^-1）. Active components of MLC and targets of obesity-type PCOS were screened from databases， a protein-protein interaction （PPI） network was constructed， and gene ontology（GO）and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis was performed. The gut microbiota structure was analyzed based on 16S rRNA sequencing and correlated with network pharmacology pathways. Body weight and estrous cycle were dynamically monitored. Ovarian morphology was observed by hematoxylin-eosin （HE） staining. Cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling （TUNEL）. Enzyme-linked immunosorbent assay （ELISA） was used to detect levels of follicle-stimulating hormone （FSH）， luteinizing hormone （LH）， anti-Müllerian hormone （AMH）， testosterone （T）， and estradiol （E₂）. Western blot was used to detect the protein expression levels of phosphorylated PI3K/PI3K （p-PI3K/PI3K）， phosphorylated Akt/Akt （p-Akt/Akt）， B-cell lymphoma-2 （Bcl-2）， and Bcl-2-associated X protein （Bax）. ResultsNetwork pharmacology screening identified 124 active components of MLC and 408 overlapping targets between the herbal formula and the disease. Core targets such as Akt1 and Bcl-2 were revealed. As indicated by 16S rRNA sequencing， the abundances of Lachnospiraceae， Lachnoclostridium， and Dorea were increased in the MLC groups （P<0.05）， while the abundance of Veillonella was decreased （P<0.05）. KEGG correlation analysis integrating network pharmacology and gut microbiota data showed significant enrichment of the PI3K/Akt signaling pathway. Animal experiments showed that， compared with the Mod group， body weight decreased to normal levels in the Met， MLC-M， and MLC-H groups. The estrous cycle became regular. The number of corpora lutea increased and cystic follicles decreased. Serum levels of T， FSH， and LH/FSH were reduced （P<0.05， P<0.01）， while the E₂ level was increased （P<0.01）. Ovarian cell apoptosis was reduced （P<0.01）， and the protein expression levels of p-PI3K/PI3K， p-Akt/Akt， and Bcl-2 in ovarian tissue were significantly increased， whereas Bax protein expression was significantly decreased （P<0.05， P<0.01）. ConclusionMLC can regulate gut microbiota structure， effectively improve ovarian pathology in rats with obesity-type PCOS， and inhibit ovarian granulosa cell apoptosis. The mechanism may be associated with upregulation of the PI3K/Akt signaling pathway.

ObjectiveTo investigate the mechanisms by which Mahuang Lianqiao Chixiaodoutang （MLC） improves obesity-type polycystic ovary syndrome （PCOS） through the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） signaling pathway. MethodsThirty-six female Sprague-Dawley （SD） rats were randomly divided into a blank control group （Con） and an obesity-type PCOS model preparation group. The model was induced by gavage with letrozole （1 mg·kg^-1） combined with a high-fat diet （HFD）. After model establishment， the obesity-type PCOS model preparation group was further divided into the model group （Mod， normal saline）， metformin group （Met， 0.3 g·kg^-1）， low-dose MLC group （MLC-L， 4.3 g·kg^-1）， medium-dose MLC group （MLC-M， 8.6 g·kg^-1）， and high-dose MLC group （MLC-H， 17.2 g·kg^-1）. Active components of MLC and targets of obesity-type PCOS were screened from databases， a protein-protein interaction （PPI） network was constructed， and gene ontology（GO）and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis was performed. The gut microbiota structure was analyzed based on 16S rRNA sequencing and correlated with network pharmacology pathways. Body weight and estrous cycle were dynamically monitored. Ovarian morphology was observed by hematoxylin-eosin （HE） staining. Cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling （TUNEL）. Enzyme-linked immunosorbent assay （ELISA） was used to detect levels of follicle-stimulating hormone （FSH）， luteinizing hormone （LH）， anti-Müllerian hormone （AMH）， testosterone （T）， and estradiol （E₂）. Western blot was used to detect the protein expression levels of phosphorylated PI3K/PI3K （p-PI3K/PI3K）， phosphorylated Akt/Akt （p-Akt/Akt）， B-cell lymphoma-2 （Bcl-2）， and Bcl-2-associated X protein （Bax）. ResultsNetwork pharmacology screening identified 124 active components of MLC and 408 overlapping targets between the herbal formula and the disease. Core targets such as Akt1 and Bcl-2 were revealed. As indicated by 16S rRNA sequencing， the abundances of Lachnospiraceae， Lachnoclostridium， and Dorea were increased in the MLC groups （P<0.05）， while the abundance of Veillonella was decreased （P<0.05）. KEGG correlation analysis integrating network pharmacology and gut microbiota data showed significant enrichment of the PI3K/Akt signaling pathway. Animal experiments showed that， compared with the Mod group， body weight decreased to normal levels in the Met， MLC-M， and MLC-H groups. The estrous cycle became regular. The number of corpora lutea increased and cystic follicles decreased. Serum levels of T， FSH， and LH/FSH were reduced （P<0.05， P<0.01）， while the E₂ level was increased （P<0.01）. Ovarian cell apoptosis was reduced （P<0.01）， and the protein expression levels of p-PI3K/PI3K， p-Akt/Akt， and Bcl-2 in ovarian tissue were significantly increased， whereas Bax protein expression was significantly decreased （P<0.05， P<0.01）. ConclusionMLC can regulate gut microbiota structure， effectively improve ovarian pathology in rats with obesity-type PCOS， and inhibit ovarian granulosa cell apoptosis. The mechanism may be associated with upregulation of the PI3K/Akt signaling pathway.

Increasing evidence shows that the early lesions of Parkinson's disease (PD) originate from gut, and correction of microbiota dysbiosis is a promising therapy for PD. FLZ is a neuroprotective agent on PD, which has been validated capable of alleviating microbiota dysbiosis in PD mice. However, the detailed mechanisms still need elucidated. Through metabolomics and 16S rRNA analysis, we identified glycoursodeoxycholic acid (GUDCA) was the most affected differential microbial metabolite by FLZ treatment, which was specially and negatively regulated by Clostridium innocuum, a differential microbiota with the strongest correlation to GUDCA production, through inhibiting bile salt hydrolase (BSH) enzyme. The protection of GUDCA on colon and brain were also clarified in PD models, showing that it could activate Nrf2 pathway, further validating that FLZ protected dopaminergic neurons through promoting GUDCA production. Our study uncovered that FLZ improved PD through microbiota-gut-brain axis, and also gave insights into modulation of microbial metabolites may serve as an important strategy for treating PD.

Although enteric glial cell (EGC) abnormal activation is reported to be involved in the pathogenesis of Parkinson's disease (PD), and inhibition of EGC gliosis alleviated gut and dopaminergic neuronal dysfunction was verified in our previous study, the potential role of gut microbiota on EGC function in PD still need to be addressed. In the present study, fecal microbiota transplantation revealed that EGC function was regulated by gut microbiota. By employing 16S rRNA and metabolomic analysis, we identified that 3-indolepropionic acid (IPA) was the most affected differential microbial metabolite that regulated EGC gliosis. The protective effects of IPA on PD were validated in rotenone-stimulated EGCs and rotenone (30 mg/kg i.g. for 4 weeks)-induced PD mice, as indicated by decreased inflammation, improved intestinal and brain barrier as well as dopaminergic neuronal function. Mechanistic study showed that IPA targeted pregnane X receptor (PXR) in EGCs, and inhibition of IL-13Rα1 involved cytokine-cytokine receptor interaction pathway, leading to inactivation of downstream JAK1-STAT6 pathway. Our data not only provided evidence that EGC gliosis was critical in spreading intestinal damage to brain, but also highlighted the potential role of microbial metabolite IPA in alleviating PD pathological damages through gut-brain axis.

[This corrects the article DOI: 10.1016/j.apsb.2025.02.029.].

Osteoarthritis (OA) causes chronic pain that significantly impairs quality of life, with current treatments often proving insufficient and accompanied by adverse effects. Recent research has identified the dorsal root ganglion (DRG) and its resident macrophages as crucial mediators of chronic OA pain through neuroinflammation driven by macrophage polarization. We present a novel injectable thermo-sensitive hydrogel system, KAF@PLEL, designed to deliver an anti-inflammatory peptide (KAF) specifically to the DRG. This biodegradable hydrogel enables sustained KAF release, promoting the reprogramming of DRG macrophages from pro-inflammatory to anti-inflammatory phenotypes. Through comprehensive in vitro and in vivo studies, we evaluated the hydrogel's biocompatibility, effects on macrophage polarization, and therapeutic efficacy in chronic OA pain management. The system demonstrated significant capabilities in preserving macrophage mitochondrial function, suppressing neuroinflammation, alleviating chronic OA pain, reducing cartilage degradation, and improving motor function in OA rat models. The sustained-release properties of KAF@PLEL enabled prolonged therapeutic effects while minimizing systemic exposure and side effects. These findings suggest that KAF@PLEL represents a promising therapeutic approach for improving outcomes in OA patients through targeted, sustained treatment.

Naringenin (4,5,7-trihydroxyflavonoid) is a naturally occurring bioflavonoid found in citrus fruits, which plays an important role in metabolic syndrome, neurological disorders, and cardiovascular diseases. However, the pharmacological mechanism and biological function of naringenin on anti-angiogenesis and anti-tumor immunity have not yet been elucidated. Our study firstly demonstrates that naringenin inhibits the growth of hepatocellular carcinoma (HCC) cells both in vivo and in vitro. Naringenin diminishes the ability of HCC cells to induce tube formation and migration of human umbilical vein endothelial cells (HUVECs) and suppresses neovascularization in chicken chorioallantoic membrane (CAM) assays. Meanwhile, in vivo results demonstrate that naringenin can significantly upregulate level of CD8⁺ T cells, subsequently increasing the level of immune-related cytokines in the tumor immune microenvironment. Mechanistically, we found that naringenin facilitate the K48-linked ubiquitination and subsequent protein degradation of vascular endothelial growth factor A (VEGFA) and mesenchymal-epithelial transition factor (c-Met), which reduces the expression of programmed death ligand 1 (PD-L1). Importantly, combination therapy naringenin with PD-L1 antibody or bevacizumab provided better therapeutic effects in liver cancer. Our study reveals that naringenin can effectively inhibit angiogenesis and anti-tumor immunity in liver cancer by degradation of VEGFA and c-Met in a K48-linked ubiquitination manner. This work enlightens the potential effect of naringenin as a promising therapeutic strategy against anti-angiogenesis and anti-tumor immunity in HCC.

Objective:To screen the Alzheimer's disease(AD)-related genes and construct its diagnostic model using bioinformatics technology and machine learning(ML)algorithms,to discuss the immunological characteristics of AD patients,and to provide novel biomarkers for AD diagnosis.Methods:The AD-related gene expression dataset GSE125583 was downloaded from the Gene Expression Omnibus(GEO)database.Differentially expressed genes(DEGs)were identified through differential analysis.Gene Ontology(GO)functional enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG)signaling pathway enrichment analyses were performed to explore the biological functions and signaling pathways of DEGs.A protein-protein interaction(PPI)network was constructed,and hub genes were screened using Cytoscape software combined with three ML algorithms:Least Absolute Shrinkage and Selection Operator(LASSO),eXtreme Gradient Boosting(XGBoost),and Random Forest(RF).The screened hub genes were utilized to build an AD diagnostic model via RF,followed by feature importance ranking.The model's efficacy and key genes were evaluated using a test set.Single-sample gene set enrichment analysis(ssGSEA)was used for immune cell infiltration analysis between AD group and control group.Results:Differential analysis identified 1 287 DEGs.The GO functional enrichment analysis results revealed that DEGs were primarily involved in biological functions related to neural signaling,synapses,and vesicles.KEGG signaling pathway enrichment analysis indicated significant enrichment of DEGs in ion transport,neurotransmitter,and ligand-gated channel pathways.Nine overlapping hub genes were screened by the three ML algorithms.In the AD diagnostic model,the top four key genes with highest diagnostic performance were adenylate cyclase-activating polypeptide 1(ADCYAP1),brain-derived neurotrophic factor(BDNF),platelet-derived growth factor receptor β(PDGFRB),and C-X-C motif chemokine receptor 4(CXCR4),with corresponding area under the curve(AUC)values of 0.852,0.795,0.820,and 0.756,respectively.The model achieved an AUC of 0.828,accuracy of 81.25%,sensitivity of 84.40%,and specificity of 71.43%.The immune cell infiltration analysis results demonstrated higher infiltration of macrophages,monocytes,natural killer(NK)cells,and lymphocytes in AD tissue.Among these,NK/natural killer T(NKT)cells and plasmacytoid dendritic cells showed significant correlations with the four key genes(P<0.05).Conclusion:The feature genes screened based on bioinformatics and ML exhibit diagnostic potential for AD.Genes such as ADCYAP1 may serve as potential biomarkers for AD diagnosis,offering significant implications for early prevention and treatment.

ObjectiveTo investigate the effect of Zuoguiwan on the ovarian function in the rat model of cyclophosphamide-induced premature ovarian failure （POF） based on the changes of ferroptosis pathway. MethodForty SD rats were randomized into blank， model， and low- and high-dose （2， 8 g·kg^-1， respectively） Zuoguiwan groups， with 10 rats in each group. The rats in the other groups except the normal group were intraperitoneally injected with CTX at a dose of 50 mg·kg^-1 on the first day and 8 mg·kg^-1 from the second day to the fifteenth day for the modeling of POF. After modeling， the rats were administrated with corresponding drugs or normal saline by gavage for four weeks. Hematoxylin-eosin staining was performed to observe the pathological changes in the ovarian tissue. The mitochondria of the ovarian tissue was observed by electron microscopy. The serum levels of follicle-stimulating hormone （FSH）， estradiol （E₂）， luteinizing hormone （LH）， anti-Mullerian hormone （AMH）， malondialdehyde （MDA）， catalase （CAT）， superoxide dismutase （SOD）， and iron ion were measured by biochemical methods and enzyme-linked immunosorbent assay. Western blot was employed to determine the protein levels of glutathione peroxidase 4 （GPX4）， solute carrier family 7 member 11 （SLC7A11）， ferritin heavy chain （FTH1）， and acyl-CoA synthetase long chain family member 4 （ACSL4）. ResultCompared with the blank group， the model group showcased significantly increased atretic follicles， atrophied， fragmented， and vacuolated mitochondria， and reduced， loose， and disordered cristae in mitochondria. Compared with the model group， high-dose Zuoguiwan increased mature follicles， the volume of mitochondria in the ovary， alleviated the vacuolation， and improved the number and arrangement of mitochondrial cristae. Compared with the blank group， the modeling elevated the levels of iron， MDA， FSH， and LH， up-regulated the expression of GPX4， SLC7A11， and FTH1 （P<0.05， P<0.01）， decreased the activities of SOD and CAT， lowered the levels of E₂ and AMH， and down-regulated the expression of ACSL4 （P<0.05， P<0.01）. Compared with the model group， drug interventions lowered the levels of iron， MDA， FSH， and LH， down-regulated the expression of GPX4， SLC7A11， and FTH1 （P<0.05， P<0.01）， increased the activity of CAT， elevated the levels of E₂ and AMH， and up-regulated the expression of ACSL4 （P<0.05， P<0.01）. ConclusionZuoguiwan may inhibit the occurrence of ferroptosis by regulating the SLC7A11/GPX4 axis， thereby improving the ovarian function of POF rats.

ObjectiveTo explore the potential mechanism of Zuoguiwan in ameliorating polycystic ovary syndrome （PCOS） by network pharmacology and liquid chromatography-mass spectrometry （LC-MS）-based metabolomics. MethodThe active ingredients and potential targets of Zuoguiwan for treating PCOS were predicted by bioinformatics. SD rats were assigned into a control group and a modeling group. The rat model of PCOS was established by gavage with letrozole （1 mg·kg^-1） combined with feeding with a high-fat diet. At the end of modeling， the modeled rats were assigned into model （normal saline）， metformin （300 mg·kg^-1）， and Zuoguiwan （concentrate 1.62 g·kg^-1） groups. The body weight and oestrous cycle of each rat were recorded， and the ovary was stained with hematoxylin and eosin for observation of ovarian morphology. Enzyme-linked immunosorbent assay was employed to determine the serum levels of follicle-stimulating hormone （FSH）， luteinizing hormone （LH）， anti-mullerian hormone （AMH）， testosterone （T）， and estradiol （E₂）， and the LH/FSH ratio was calculated. Serum metabolomics of rats was conducted by orthogonal partial least squares-discriminant analysis （OPLS-DA） to screen the metabolite-enriched pathways. Furthermore， network pharmacology and association analysis were employed construct the compound-response-enzyme-gene network. ResultA total of 503 potential targets of Zuoguiwan and 5 843 targets of PCOS were screened out， with 271 common targets. The Gene Ontology enrichment analysis revealed that the common targets were involved in the response to lipopolysaccharide， etc.， and the Kyoto Encyclopedia of Genes and Genomes （KEGG） enrichment yielded 119 pathways. Animal experiments showed that compared with the control group， the model group presented increased body weight （P<0.01）， elevated LH and AMH levels （P<0.01）， increased LH/FSH ratio （P<0.01）， lowered E₂ level （P<0.01）， and increased cystic follicles. Compared with the model group， Zuoguiwan and metformin decreased the body weight （P<0.01）， reduced atretic follicles and cystic follicles， increased mature follicles and corpus luteum， and thickened the granulosa layer. Moreover， Zuoguiwan lowered the T， FSH， LH， and AMH， and LH/FSH levels （P<0.01） and elevated the E₂ level （P<0.01）. The principal component analysis and OPLS-DA in metabolomics showed that the differential metabolites between Zuoguiwan and model groups included 26 up-regulated metabolites in the Zuoguiwan group. There were 8 common pathways predicted by the KEGG enrichment analysis in network pharmacology and the metabolite enrichment in metabolomics. The results of topological analysis revealed the pathways of steroid hormone biosynthesis and glycerol-phospholipid metabolism， and the constructed compound-response-enzyme-gene network revealed that the key targets were protein kinase B1 （Akt1）， epithelial growth factor receptor （EGFR）， prostaglandin-endoperoxide synthase 2 （PTGS2）， and mitogen-activated protein kinase 1 （MAPK1）. ConclusionZuoguiwan regulated the steroid hormone biosynthesis pathway to recover hormone levels， promote follicle production and development， and improve ovarian function， which may be the potential mechanism of this medicine in treating PCOS.