OBJECTIVE To investigate the mechanism of pachymic acid on renal injury in pregnancy induced hypertension （PIH） rats by regulating the silent information regulator transcript 1/peroxisome proliferator-activated receptor γ coactivator-1α （Sirt1/PGC-1α） pathway. METHODS Pregnant SD rats were prepared by co-caging and PIH model was induced using N-nitro-L- arginine methyl ester （L-NAME） method. PIH rats were randomly divided into model group， L-pachymic acid （low-dose pachymic acid， 10 mg/kg） group， H-pachymic acid （high-dose pachymic acid， 20 mg/kg） group， and H-pachymic acid+EX527 （20 mg/kg pachymic acid+10 mg/kg EX527） group， with 6 rats in each group. Another 6 normal pregnant rats were selected as blank group. Each group was given relevant medicine or solvent intragastrically or intraperitoneally daily， once a day， for 28 consecutive days. After the last administration， 24 h urinary protein and tail artery systolic blood pressure （SBP） were measured in pregnant rats from each group， along with the levels of serum creatinine （Scr）， blood urea nitrogen （BUN），uric acid （UA）， and cystatin C （Cys-C）. The contents of superoxide dismutase （SOD）， malondialdehyde （MDA）， glutathione peroxidase （GSH-Px）， and 8-hydroxy-2′-deoxyguanosine （8-OHdG） in renal tissue， as well as the mRNA and protein expression levels of Sirt1 and PGC-1α， were also determined. Meanwhile， renal histopathological changes in rats from each group were evaluated using hematoxylin-eosin （HE） staining and periodic acid-Schiff （PAS） staining. RESULTS Compared with model group， L-pachymic acid group and H-pachymic acid group exhibited significant decreases in 24 h urine protein quantification， tail artery SBP， Scr， BUN， UA， Cys-C levels， glomerulosclerosis index score of renal tissue， renal tubular injury score， the percentage of PAS positive area， MDA and 8-OHdG （P＜0.05）. Conversely， the contents of SOD and GSH-Px， along with the mRNA and protein expression levels of Sirt1 and PGC-1α， were significantly increased （P＜0.05）. Moreover， these improvements were more pronounced in H-pachymic acid group （P＜0.05）. Compared with H-pachymic acid group， the aforementioned indicators in pregnant rats from the H-pachymic acid+EX527 group showed significant reversal （P＜0.05）. CONCLUSIONS Pachymic acid significantly ameliorates renal injury induced by PIH in rats， potentially through activation of the Sirt1/PGC-1α pathway.

ObjectiveTo elucidate the underlying mechanism through which Zhuluan decoction suppresses excessive autophagy in human ovarian granulosa cells （KGN） and ameliorates premature ovarian insufficiency （POI） via the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）/mammalian target of rapamycin （mTOR） signaling pathway. MethodsThe optimal concentration of cyclophosphamide for inducing a POI model in KGN cells was identified via the cell counting kit-8 （CCK-8） assay. Subsequently， the impacts of varying concentrations of Zhuluan decoction-containing serum on the viability of the KGN cell model were assessed. After the optimal drug concentration was determined， KGN cells were categorized into the following groups： blank control （20% blank serum）， model （20% blank serum + 5 μmol·L^-1 cyclophosphamide）， Zhuluan decoction-containing serum （20% Zhuluan decoction-containing serum + 5 μmol·L^-1 cyclophosphamide）， autophagy inhibitor （20% blank serum + 5 μmol·L^-1 cyclophosphamide + 20 μmol·L^-1 chloroquine phosphate）， autophagy inhibitor + Zhuluan decoction-containing serum （20% Zhuluan decoction-containing serum + 5 μmol·L^-1 cyclophosphamide + 20 μmol·L^-1 chloroquine phosphate）， and estradiol valerate （20% estradiol valerate-containing serum + 5 μmol·L^-1 cyclophosphamide）. Following 48 hours of incubation， flow cytometry was utilized to measure the apoptosis rate of KGN cells in each group. Western blotting was employed to quantify the protein levels of PI3K， phosphorylated （p）-Akt， Akt， p-mTOR， and mTOR， along with the expression levels of autophagy-related proteins such as Beclin1， autophagy-related 5 homolog （ATG5）， and microtubule-associated protein 1 light chain 3 （LC3）， in each group. Additionally， monodansylcadaverine （MDC） staining was performed to evaluate the extent of autophagy in each group. ResultsIncubation of KGN cells with 5 μmol·L^-1 cyclophosphamide for 48 h successfully established a POI model， marked by a significant inhibition of KGN cell proliferation. Notably， the inhibitory effect of cyclophosphamide on KGN cell proliferation exhibited a positive correlation with its concentration. Zhuluan decoction-containing serum at 20% and 30% promoted cell proliferation and mitigated the inhibitory effect of cyclophosphamide on KGN cell proliferation， with comparable therapeutic efficacy observed at both concentrations. Compared with the blank control group， the model group displayed an elevated apoptosis rate （P<0.01）， reduced protein levels of PI3K， p-Akt， and p-mTOR （P<0.01）， increased protein levels of Beclin1， LC3， and ATG5 （P<0.01）， no significant alterations in the protein levels of Akt and mTOR， and an enhanced MDC autophagy fluorescence intensity （P<0.01）. In comparison to that the model group， the apoptosis rates in the blank control group， model group， Zhuluan decoction-containing serum group， autophagy inhibitor group， autophagy inhibitor + Zhuluan decoction-containing serum group， and estradiol valerate group all reduced （P<0.05， P<0.01）， with the most pronounced reduction observed in the autophagy inhibitor + Zhuluan decoction-containing serum group. The protein levels of PI3K， p-Akt， and p-mTOR were higher in other groups than in the model group （P<0.05， P<0.01）， being the highest in the autophagy inhibitor + Zhuluan decoctio-containing serum group （P<0.01）. The protein levels of Beclin1 and ATG5 were lower in other groups than in the model group （P<0.05， P<0.01）. The expression level of LC3 declined in the Zhuluan decoction-containing serum group and the estradiol valerate group （P<0.05， P<0.01）， while it decreased without statistical significance in the autophagy inhibitor group and the autophagy inhibitor + Zhuluan decoction-containing serum group. ConclusionZhuluan decoction may activate the PI3K/Akt/mTOR pathway to inhibit excessive autophagy and counteract the detrimental effects of cyclophosphamide on the KGN cell model， thus managing POI.

ObjectiveTo elucidate the underlying mechanism through which Zhuluan decoction suppresses excessive autophagy in human ovarian granulosa cells （KGN） and ameliorates premature ovarian insufficiency （POI） via the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）/mammalian target of rapamycin （mTOR） signaling pathway. MethodsThe optimal concentration of cyclophosphamide for inducing a POI model in KGN cells was identified via the cell counting kit-8 （CCK-8） assay. Subsequently， the impacts of varying concentrations of Zhuluan decoction-containing serum on the viability of the KGN cell model were assessed. After the optimal drug concentration was determined， KGN cells were categorized into the following groups： blank control （20% blank serum）， model （20% blank serum + 5 μmol·L^-1 cyclophosphamide）， Zhuluan decoction-containing serum （20% Zhuluan decoction-containing serum + 5 μmol·L^-1 cyclophosphamide）， autophagy inhibitor （20% blank serum + 5 μmol·L^-1 cyclophosphamide + 20 μmol·L^-1 chloroquine phosphate）， autophagy inhibitor + Zhuluan decoction-containing serum （20% Zhuluan decoction-containing serum + 5 μmol·L^-1 cyclophosphamide + 20 μmol·L^-1 chloroquine phosphate）， and estradiol valerate （20% estradiol valerate-containing serum + 5 μmol·L^-1 cyclophosphamide）. Following 48 hours of incubation， flow cytometry was utilized to measure the apoptosis rate of KGN cells in each group. Western blotting was employed to quantify the protein levels of PI3K， phosphorylated （p）-Akt， Akt， p-mTOR， and mTOR， along with the expression levels of autophagy-related proteins such as Beclin1， autophagy-related 5 homolog （ATG5）， and microtubule-associated protein 1 light chain 3 （LC3）， in each group. Additionally， monodansylcadaverine （MDC） staining was performed to evaluate the extent of autophagy in each group. ResultsIncubation of KGN cells with 5 μmol·L^-1 cyclophosphamide for 48 h successfully established a POI model， marked by a significant inhibition of KGN cell proliferation. Notably， the inhibitory effect of cyclophosphamide on KGN cell proliferation exhibited a positive correlation with its concentration. Zhuluan decoction-containing serum at 20% and 30% promoted cell proliferation and mitigated the inhibitory effect of cyclophosphamide on KGN cell proliferation， with comparable therapeutic efficacy observed at both concentrations. Compared with the blank control group， the model group displayed an elevated apoptosis rate （P<0.01）， reduced protein levels of PI3K， p-Akt， and p-mTOR （P<0.01）， increased protein levels of Beclin1， LC3， and ATG5 （P<0.01）， no significant alterations in the protein levels of Akt and mTOR， and an enhanced MDC autophagy fluorescence intensity （P<0.01）. In comparison to that the model group， the apoptosis rates in the blank control group， model group， Zhuluan decoction-containing serum group， autophagy inhibitor group， autophagy inhibitor + Zhuluan decoction-containing serum group， and estradiol valerate group all reduced （P<0.05， P<0.01）， with the most pronounced reduction observed in the autophagy inhibitor + Zhuluan decoction-containing serum group. The protein levels of PI3K， p-Akt， and p-mTOR were higher in other groups than in the model group （P<0.05， P<0.01）， being the highest in the autophagy inhibitor + Zhuluan decoctio-containing serum group （P<0.01）. The protein levels of Beclin1 and ATG5 were lower in other groups than in the model group （P<0.05， P<0.01）. The expression level of LC3 declined in the Zhuluan decoction-containing serum group and the estradiol valerate group （P<0.05， P<0.01）， while it decreased without statistical significance in the autophagy inhibitor group and the autophagy inhibitor + Zhuluan decoction-containing serum group. ConclusionZhuluan decoction may activate the PI3K/Akt/mTOR pathway to inhibit excessive autophagy and counteract the detrimental effects of cyclophosphamide on the KGN cell model， thus managing POI.

Background/Aims: Lenvatinib resistance remains a critical barrier in advanced hepatocellular carcinoma (HCC) therapy. However, the underlying mechanisms and strategies for reversing resistance remain incompletely understood. Methods: Integrated transcriptomics of lenvatinib-resistant patient tumors and an acquired-resistance murine model identified a novel macrophage subpopulation. Functional validation employed CRISPR-SAM screening, conditioned medium (CM) assays, subcutaneous/orthotopic xenografts, patient-derived organoids (PDOs), and patient-derived xenografts (PDXs). Mechanistic studies included ChIP-qPCR, co-immunoprecipitation, and pharmacologic targeting. Clinical relevance was assessed in a retrospective cohort. Results: Resistant HCC exhibited significant enrichment of a COLEC12high TAM subset , which correlated with poor survival and treatment response. These TAMs secreted neuregulin-1 (NRG1) , activating HER2/HER3-AKT signaling in tumor cells to drive cancer stemness and lenvatinib resistance. Mechanistically, in TAMs COLEC12 sequestered STAT1 in the cytoplasm, preventing its phosphorylation, and thereby derepressing STAT3-mediated NRG1 transcription. Depletion of NRG1 reversed the stemness phenotypes and resensitized tumors to lenvatinib both in vitro and in vivo. Clinically, high NRG1 expression predicted an inferior lenvatinib response and shorter survival. Crucially, the bispecific anti-HER2/HER3 antibody zenocutuzumab restored lenvatinib efficacy in PDOs, PDXs, and murine models. Conclusions Our work establishes the COLEC12high TAM/NRG1 axis as a master regulator of therapeutic resistance and identifies NRG1 as a predictive biomarker, providing a clinically actionable strategy to overcome lenvatinib resistance in HCC.

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 the protective effect of Huayu Jiedu prescription against PC12 cell damage induced by a combination of neutrophil extracellular traps （NETs） and oxygen-glucose deprivation （OGD）. MethodsFirst， dimethyl sulfoxide （DMSO） was used to induce the differentiation of the human promyelocytic leukemia cell line （HL-60） into neutrophil-like cells （dHL-60）， and cell differentiation was assessed through Giemsa staining and flow cytometry. Next， phorbol myristate （PMA） was used to induce the formation of NETs in dHL-60 cells. The formation of NETs was assessed via SYTOX Green staining， and NETs were extracted by low-speed centrifugation. Finally， the NETs prepared through the conditioned medium were combined with OGD to induce a high-differentiation PC12 neuronal cell damage model through the co-culture method. This model was treated with the Huayu Jiedu prescription-containing serum. Cells were randomly allocated into a normal control group， a model （NETs + OGD） group， a traditional Chinese medicine （NETs + OGD + 5% Huayu Jiedu prescription-containing serum） group， and a NETosis inhibitor （100 mg·L^-1 DNase I + NETs + OGD） group. The cell counting kit-8 （CCK-8） assay was used to evaluate cell viability and screen the optimal intervention concentrations of NETs and Huayu Jiedu Formula-containing serum， as well as the optimal intervention time for OGD. Enzyme-linked immunosorbent assay （ELISA） was adopted to measure the levels of myeloperoxidase-DNA complexes （MPO-DNA）， citrullinated histone H3 （CitH3）， and neutrophil elastase （NE）. Western blot was employed to quantify the protein levels of CitH3 and cleaved Caspase-3. Terminal-deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay （TUNEL） staining and flow cytometry were employed to assess neuronal apoptosis. ResultsCompared with those in the normal control group， cells in the model group successfully differentiated into dHL60 cells after DMSO induction. Wright-Giemsa staining revealed that the cells in the model group had disappeared nucleoli， distinct reniform and lobulated changes of nuclei， highly condensed chromatin， and the cytoplasm containing pale purple fine granules. Flow cytometry showed that the mean fluorescence intensity （MFI） of CD11b in the model group was enhanced （P<0.01）. Compared with those in the normal control group， dHL60 cells in the model group successfully generated NETs after PMA induction. SYTOX Green staining revealed loss of cell membrane integrity， with chromatin released from the cytoplasm to form abundant extracellular， cloud-like fibrillar structures， and a marked increase in fluorescence intensity （P<0.01）. Through the CCK-8 assay for assessing the viability of highly differentiated PC12 cells， the optimal concentration for NETs was determined as 1 000 ng·L^-1， the optimal concentration of Huayu Jiedu formula-containing serum as 5%， and the optimal duration for OGD as 2 h. Compared with the normal control group， the model group showed elevated levels of MPO-DNA， CitH3， and NE （P<0.01） and upregulated protein levels of CitH3 and cleaved Caspase-3 （P<0.01）. Furthermore， TUNEL staining revealed an increased proportion of apoptotic cells （P<0.01） and flow cytometry showed an increased apoptosis rate （P<0.01） in the model group. Compared with the model group， the Huayu Jiedu formula-containing serum group and the NETosis inhibitor group exhibited declined levels of MPO-DNA， CitH3， and NE （P<0.05，P<0.01） and downregulated protein levels of CitH3 and cleaved Caspase-3 （P<0.01）. In addition， TUNEL staining showed a decrease in the proportion of apoptotic cells （P<0.01）， and flow cytometry revealed a decrease in the apoptosis rate （P<0.01）. ConclusionHuayu Jiedu prescription may exert a protective effect against ischemic neuronal damage by inhibiting NET formation or alleviating NET-induced neurotoxicity

ObjectiveTo investigate the protective effect of Huayu Jiedu prescription against PC12 cell damage induced by a combination of neutrophil extracellular traps （NETs） and oxygen-glucose deprivation （OGD）. MethodsFirst， dimethyl sulfoxide （DMSO） was used to induce the differentiation of the human promyelocytic leukemia cell line （HL-60） into neutrophil-like cells （dHL-60）， and cell differentiation was assessed through Giemsa staining and flow cytometry. Next， phorbol myristate （PMA） was used to induce the formation of NETs in dHL-60 cells. The formation of NETs was assessed via SYTOX Green staining， and NETs were extracted by low-speed centrifugation. Finally， the NETs prepared through the conditioned medium were combined with OGD to induce a high-differentiation PC12 neuronal cell damage model through the co-culture method. This model was treated with the Huayu Jiedu prescription-containing serum. Cells were randomly allocated into a normal control group， a model （NETs + OGD） group， a traditional Chinese medicine （NETs + OGD + 5% Huayu Jiedu prescription-containing serum） group， and a NETosis inhibitor （100 mg·L^-1 DNase I + NETs + OGD） group. The cell counting kit-8 （CCK-8） assay was used to evaluate cell viability and screen the optimal intervention concentrations of NETs and Huayu Jiedu Formula-containing serum， as well as the optimal intervention time for OGD. Enzyme-linked immunosorbent assay （ELISA） was adopted to measure the levels of myeloperoxidase-DNA complexes （MPO-DNA）， citrullinated histone H3 （CitH3）， and neutrophil elastase （NE）. Western blot was employed to quantify the protein levels of CitH3 and cleaved Caspase-3. Terminal-deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay （TUNEL） staining and flow cytometry were employed to assess neuronal apoptosis. ResultsCompared with those in the normal control group， cells in the model group successfully differentiated into dHL60 cells after DMSO induction. Wright-Giemsa staining revealed that the cells in the model group had disappeared nucleoli， distinct reniform and lobulated changes of nuclei， highly condensed chromatin， and the cytoplasm containing pale purple fine granules. Flow cytometry showed that the mean fluorescence intensity （MFI） of CD11b in the model group was enhanced （P<0.01）. Compared with those in the normal control group， dHL60 cells in the model group successfully generated NETs after PMA induction. SYTOX Green staining revealed loss of cell membrane integrity， with chromatin released from the cytoplasm to form abundant extracellular， cloud-like fibrillar structures， and a marked increase in fluorescence intensity （P<0.01）. Through the CCK-8 assay for assessing the viability of highly differentiated PC12 cells， the optimal concentration for NETs was determined as 1 000 ng·L^-1， the optimal concentration of Huayu Jiedu formula-containing serum as 5%， and the optimal duration for OGD as 2 h. Compared with the normal control group， the model group showed elevated levels of MPO-DNA， CitH3， and NE （P<0.01） and upregulated protein levels of CitH3 and cleaved Caspase-3 （P<0.01）. Furthermore， TUNEL staining revealed an increased proportion of apoptotic cells （P<0.01） and flow cytometry showed an increased apoptosis rate （P<0.01） in the model group. Compared with the model group， the Huayu Jiedu formula-containing serum group and the NETosis inhibitor group exhibited declined levels of MPO-DNA， CitH3， and NE （P<0.05，P<0.01） and downregulated protein levels of CitH3 and cleaved Caspase-3 （P<0.01）. In addition， TUNEL staining showed a decrease in the proportion of apoptotic cells （P<0.01）， and flow cytometry revealed a decrease in the apoptosis rate （P<0.01）. ConclusionHuayu Jiedu prescription may exert a protective effect against ischemic neuronal damage by inhibiting NET formation or alleviating NET-induced neurotoxicity

OBJECTIVE To investigate the potential mechanism of berberine improving diabetes mellitus type 2 （T2DM） combined with metabolic-associated fatty liver disease （MAFLD） by regulating ceramide. METHODS Thirty-two db/db mice with blood glucose levels＞11.1 mmol/L （T2DM model） were divided into four groups： model group， berberine low- and high-dose groups [100， 200 mg/（kg·d）] and metformin group [300 mg/（kg·d）]， with 8 mice in each group. Additionally， 8 wt/wt mice were selected as the normal control group. Mice in each group were administered the corresponding drug solution or water by gavage once daily for a continuous period of 6 weeks. During the experiment， the body weight of the mice was monitored， and the differences in final body weight were analyzed. After the last administration， the body shape of the mice in each group was observed， and their fasting blood glucose （FBG） and the lipid indicators [total cholesterol （TC）， triglyceride （TG）， low-density lipoprotein cholesterol （LDL-C）， high-density lipoprotein cholesterol （HDL-C）] were measured. Fasting serum insulin （FINS） levels were also measured， and the insulin resistance index HOMA-IR） and insulin sensitivity index （ISI） were calculated. Liver weight， liver index and serum liver function indicators [alanine transaminase （ALT）， aspartate transaminase（AST）] were assessed， and hepatic histopathological changes were observed. Additionally， the expression of fatty acid synthesis-related proteins [sterol regulatory element-binding protein 1 （SREBP1）， fatty acid synthase （FASN）， acetyl-CoA carboxylase 1 （ACC1）] in liver tissue was examined. Serum samples from the normal control group， model group， and berberine high-dose group were collected for non-targeted lipidomics analysis and validation. RESULTS Compared with the model group， the pathological changes， including disordered liver tissue cell arrangement and lipid vacuoles， were significantly improved in the berberine low- and high-dose groups. The significant decreases or down-regulations were observed in body weight in the last week， as well as FBG， TC， TG， and LDL-C levels， HOMA-IR （except for the berberine low-dose group）， liver weight， liver index， AST and ALT levels， and protein expressions of SREBP1， FASN and ACC1. Additionally， HDL-C levels， FINS （except for the berberine high-dose group）， and ISI （except for the berberine low-dose group） were significantly increased （P＜0.05）. A total of 21 potential differential metabolites， including multiple types of ceramides， were identified； these metabolites were primarily enriched in sphingolipid metabolism and glycerophospholipid metabolism pathways. Verification experiments confirmed that high-dose berberine significantly reduced the serum content of ceramide in model mice （P＜0.05）. CONCLUSIONS Berberine reduces insulin resistance， improves liver damage and lipid accumulation in the T2DM combined with MAFLD mice， and these effects may be related to the reduction of ceramide content.

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive cognitive decline, functional impairment, and neuropsychiatric symptoms. It represents the most prevalent form of dementia among the elderly population. Accumulating evidence indicates that oxidative stress plays a pivotal role in the pathogenesis of AD. Notably, elevated levels of oxidative stress have been observed in the brains of AD patients, where excessive reactive oxygen species (ROS) can cause extensive damage to lipids, proteins, and DNA, ultimately compromising neuronal structure and function. Amyloid β‑protein (Aβ) has been shown to induce mitochondrial dysfunction and calcium overload, thereby promoting the generation of ROS. This, in turn, exacerbates Aβ aggregation and enhances tau phosphorylation, leading to the formation of two pathological features of AD: extracellular Aβ plaque deposition and intracellular neurofibrillary tangles (NFTs). These events ultimately culminate in neuronal death, forming a vicious cycle. The interplay between oxidative stress and these pathological processes constitutes a core link in the pathogenesis of AD. The signaling pathways mediating oxidative stress in AD include Nrf2, RCAN1, PP2A, CREB, Notch1, NF‑κB, ApoE, and ferroptosis. Nrf2 signaling pathway serves as a key regulator of cellular redox homeostasis, exerts important antioxidant capacity and protective effects in AD. RCAN1 signaling pathway, as a calcineurin inhibitor, and modulates AD progression through multiple mechanisms. PP2A signaling pathway is involved in regulating tau phosphorylation and neuroinflammation processes. CREB signaling pathway contributes to neuroplasticity and memory formation; activation of CREB improves cognitive function and reduce oxidative stress. Notch1 signaling pathway regulates neuronal development and memory, participates in modulation of Aβ production, and interacts with Nrf2 toco-regulate antioxidant activity. NF‑κB signaling pathway governs immune and inflammatory responses; sustained activation of this pathway forms “inflammatory memory”, thereby exacerbating AD pathology. ApoE signaling pathway is associated with lipid metabolism; among its isoforms, ApoE-ε4 significantly increases the risk of AD, leading to elevated oxidative stress, abnormal lipid metabolism, and neuroinflammation. The ferroptosis signaling pathway is driven by iron-dependent lipid peroxidation, and the subsequent release of lipid peroxidation products and ROS exacerbate oxidative stress and neuronal damage. These interconnected pathways form a complex regulatory network that regulates the progression of AD through oxidative stress and related pathological cascades. In terms of therapeutic strategies targeting oxidative stress, among the drugs currently used in clinical practice for AD treatment, memantine and donepezil demonstrate significant therapeutic efficacy and can improve the level of oxidative stress in AD patients. Some compounds with antioxidant effects (such asα-lipoic acid and melatonin) have shown certain potential in AD treatment research and can be used as dietary supplements to ameliorate AD symptoms. In addition, non-drug interventions such as calorie restriction and exercise have been proven to exerted neuroprotective effects and have a positive effect on the treatment of AD. By comprehensively utilizing the therapeutic characteristics of different signaling pathways, it is expected that more comprehensive multi-target combination therapy regimens and combined nanomolecular delivery systems will be developed in the future to bypass the blood-brain barrier, providing more effective therapeutic strategies for AD.

Osteoporosis(OP) is a senile bone disease characterized by an imbalance between bone remodeling and bone formation. Targeting pathogenesis of kidney deficiency, spleen deficiency, and blood stasis, Bushen Jianpi Huoxue Decoction has a significant effect on the treatment of OP by tonifying kidney, invigorating spleen, and activating blood circulation. MicroRNA(miRNA) and the anti-apoptotic protein B-cell lymphoma-2-like protein 1(BCL2L1) are closely related to bone cell metabolism. Therefore, in this study, the binding of miR-140-5p to BCL2L1 was detected by dual luciferase assay and polymerase chain reaction(PCR). After silencing or overexpressing miR-140-5p, the apoptosis, autophagy, and osteogenic function of human fetal osteoblast cell line 1.19(HFOB1.19) were observed by flow cytometry and Western blot. Bushen Jianpi Huoxue Decoction-containing serum was prepared by intragastric administration of Bushen Jianpi Huoxue Decoction in rats. Different concentrations of Bushen Jianpi Huoxue Decoction-containing serum were used to treat HFOB1.19 with or without miR-140-5p mimic. The expression of osteogenic proteins in each group was observed, and the role of miR-140-5p/BCL2L1 in apoptosis and autophagy of HFOB1.19 was studied, along with the effect of Bushen Jianpi Huoxue Decoction on these processes. As indicated by the dual luciferase assay, miR-140-5p bound to BCL2L1. Flow cytometry and Western blot showed that miR-140-5p promoted apoptosis and inhibited autophagy in HFOB1.19. After intervention with high, medium, and low doses of Bushen Jianpi Huoxue Decoction-medicated serum, compared with the miR-140-5p NC group, the expression of osteocalcin(OCN), osteopontin(OPN), Runt-related transcription factor 2(RUNX2), and transforming growth factor beta 1(TGF-β1) decreased in the miR-140-5p mimic group, while the expression of bone morphogenetic protein 2(BMP2) showed no significant difference under high-dose intervention. Therefore, miR-140-5p/BCL2L1 can promote apoptosis and inhibit autophagy in HFOB1.19. Bushen Jianpi Huoxue Decoction can affect the osteogenic effect of miR-140-5p through BMP2.
MicroRNAs/metabolism* ; Autophagy/drug effects* ; Apoptosis/drug effects* ; Humans ; Drugs, Chinese Herbal/administration & dosage* ; Animals ; Cell Line ; bcl-X Protein/metabolism* ; Osteoblasts/metabolism* ; Rats ; Osteoporosis/physiopathology* ; Male ; Rats, Sprague-Dawley ; Osteogenesis/drug effects*