OBJECTIVE: To investigate the therapeutic potential of octaarginine (R8)-modified essential oil from Fructus Alpiniae zerumbet (EOFAZ) lipid microspheres (EOFAZ@^R8LM) for cardiovascular therapy. METHODS: EOFAZ@^R8LM was developed by leveraging the volatilization of EOFAZ and integrating it with the oil phase of LM, followed by surface modification with cell-penetrating peptide R8 to target the site of vascular endothelial injury. The therapeutic effects of this formulation in alleviating lipopolysaccharide-induced vascular endothelial inflammation were evaluated by assessing mitochondrial membrane potential (MMP), intracellular reactive oxygen species (ROS) levels, as well as inflammatory factors interleukin-6 (IL-6) and interleukin-1β (IL-1β) levels. RESULTS: EOFAZ@^R8LM effectively delivered EOFAZ to the site of injury and specifically targeted the mitochondria in vascular endothelial cells, thereby ameliorating mitochondrial dysfunction through regulation of MMP and reduction of intracellular ROS levels. Moreover, it attenuated the expression levels of IL-6 and IL-1β, exerting protective effects on the vascular endothelium. CONCLUSION Our findings highlight the significant therapeutic potential of EOFAZ@^R8LM in cardiovascular therapy, providing valuable insights for developing novel dosage forms utilizing EOFAZ for effective treatment against cardiovascular diseases.

Cholesterol (CH) plays a crucial role in enhancing the membrane stability of drug delivery systems (DDS). However, its association with conditions such as hyperlipidemia often leads to criticism, overshadowing its influence on the biological effects of formulations. In this study, we reevaluated the delivery effect of CH using widely applied lipid microspheres (LM) as a model DDS. We conducted comprehensive investigations into the impact of CH on the distribution, cell uptake, and protein corona (PC) of LM at sites of cardiovascular inflammatory injury. The results demonstrated that moderate CH promoted the accumulation of LM at inflamed cardiac and vascular sites without exacerbating damage while partially mitigating pathological damage. Then, the slow cellular uptake rate observed for CH@LM contributed to a prolonged duration of drug efficacy. Network pharmacology and molecular docking analyses revealed that CH depended on LM and exerted its biological effects by modulating peroxisome proliferator-activated receptor gamma (PPAR-γ) expression in vascular endothelial cells and estrogen receptor alpha (ERα) protein levels in myocardial cells, thereby enhancing LM uptake at cardiovascular inflammation sites. Proteomics analysis unveiled a serum adsorption pattern for CH@LM under inflammatory conditions showing significant adsorption with CH metabolism-related apolipoprotein family members such as apolipoprotein A-V (Apoa5); this may be a major contributing factor to their prolonged circulation in vivo and explains why CH enhances the distribution of LM at cardiovascular inflammatory injury sites. It should be noted that changes in cell types and physiological environments can also influence the biological behavior of formulations. The findings enhance the conceptualization of CH and LM delivery, providing novel strategies for investigating prescription factors' bioactivity.

Cholesterol(CH)plays a crucial role in enhancing the membrane stability of drug delivery systems(DDS).However,its association with conditions such as hyperlipidemia often leads to criticism,overshadowing its influence on the biological effects of formulations.In this study,we reevaluated the delivery effect of CH using widely applied lipid microspheres(LM)as a model DDS.We conducted comprehensive in-vestigations into the impact of CH on the distribution,cell uptake,and protein corona(PC)of LM at sites of cardiovascular inflammatory injury.The results demonstrated that moderate CH promoted the accumulation of LM at inflamed cardiac and vascular sites without exacerbating damage while partially mitigating pathological damage.Then,the slow cellular uptake rate observed for CH@LM contributed to a prolonged duration of drug efficacy.Network pharmacology and molecular docking analyses revealed that CH depended on LM and exerted its biological effects by modulating peroxisome proliferator-activated receptor gamma(PPAR-γ)expression in vascular endothelial cells and estrogen receptor alpha(ERα)protein levels in myocardial cells,thereby enhancing LM uptake at cardiovascular inflam-mation sites.Proteomics analysis unveiled a serum adsorption pattern for CH@LM under inflammatory conditions showing significant adsorption with CH metabolism-related apolipoprotein family members such as apolipoprotein A-V(Apoa5);this may be a major contributing factor to their prolonged circu-lation in vivo and explains why CH enhances the distribution of LM at cardiovascular inflammatory injury sites.It should be noted that changes in cell types and physiological environments can also influence the biological behavior of formulations.The findings enhance the conceptualization of CH and LM delivery,providing novel strategies for investigating prescription factors' bioactivity.

Objective To investigate the causal relationship between frailty and epilepsy based on the traditional Chinese medicine(TCM)theory of"epilepsy caused by frailty"utilizing a two-sample Mendelian randomization approach,to enrich the modern connota-tion of the"epilepsy caused by frailty"theory and provide a theoretical basis for treating epilepsy from a frailty perspective in TCM.Methods Data on frailty and epilepsy were extracted from publicly available genome-wide association study summary datasets.Single nucleotide polymorphism(SNP)were selected as instrumental variable under the conditions of P＜5 x 10-8,a linkage disequilibrium co-efficient of 0.001,and a linkage disequilibrium region width of 10000kb.Mendelian randomization analysis was performed using five methods:MR-Egger regression,weighted median,inverse variance weighting,simple mode and weighted mode,with inverse variance weighting as the primary analysis method.The causal relationship between frailty and epilepsy was interpreted using OR and 95％CI.Pleiotropy was assessed using the MR-Egger intercept and MR-PRESSO analysis methods,while heterogeneity was analyzed using MR-Egger Cochran's Q test,and sensitivity analysis was conducted using the leave-one-out method.Results The results of inverse variance weighting and weighted median analysis indicated that there was a positive causal relationship between frailty and epilepsy.This result passed pleiotropy tests,confirming its robustness.Conclusion Frailty is associated with an increased risk of epilepsy,which is consistent with the TCM understanding of"epilepsy caused by frailty".It is suggested that in clinical treatment,besides conventional ap-proaches such as resolving phlegm,eliminating blood stasis,calming wind,and opening orifices,there should be a greater emphasis on tonifying frailty and nurturing the body's vital energy.

Objective To investigate the causal relationship between frailty and epilepsy based on the traditional Chinese medicine(TCM)theory of"epilepsy caused by frailty"utilizing a two-sample Mendelian randomization approach,to enrich the modern connota-tion of the"epilepsy caused by frailty"theory and provide a theoretical basis for treating epilepsy from a frailty perspective in TCM.Methods Data on frailty and epilepsy were extracted from publicly available genome-wide association study summary datasets.Single nucleotide polymorphism(SNP)were selected as instrumental variable under the conditions of P＜5 x 10-8,a linkage disequilibrium co-efficient of 0.001,and a linkage disequilibrium region width of 10000kb.Mendelian randomization analysis was performed using five methods:MR-Egger regression,weighted median,inverse variance weighting,simple mode and weighted mode,with inverse variance weighting as the primary analysis method.The causal relationship between frailty and epilepsy was interpreted using OR and 95％CI.Pleiotropy was assessed using the MR-Egger intercept and MR-PRESSO analysis methods,while heterogeneity was analyzed using MR-Egger Cochran's Q test,and sensitivity analysis was conducted using the leave-one-out method.Results The results of inverse variance weighting and weighted median analysis indicated that there was a positive causal relationship between frailty and epilepsy.This result passed pleiotropy tests,confirming its robustness.Conclusion Frailty is associated with an increased risk of epilepsy,which is consistent with the TCM understanding of"epilepsy caused by frailty".It is suggested that in clinical treatment,besides conventional ap-proaches such as resolving phlegm,eliminating blood stasis,calming wind,and opening orifices,there should be a greater emphasis on tonifying frailty and nurturing the body's vital energy.

ObjectiveTo prepare oral nanoemulsions encapsulating essential oil from Alpinia zerumbet fructus（EOFAZ） and to investigate its pro-absorption effect in vitro and distribution in vivo. MethodThe proteoglycan conjugate polysaccharides of vinegar-processed Bupleuri Radix-bovine serum albumin（VBCP-BSA） was prepared by Maillard reaction of VBCP and BSA. Taking VBCP-BSA as emulsifier， vitamin B₁₂（VB₁₂） as absorption enhancer， and medium chain triglycerides mixed with EOFAZ as oil phase， the nanoemulsions loaded with EOFAZ was prepared by high energy emulsification method. The particle size， particle size distribution， surface Zeta potential， EOFAZ content and appearance and morphology of the nanoemulsions were characterized， and fluorescein tracer method was used to investigate the absorption effect of fluorescein-labeled EOFAZ nanoemulsions in vitro and their distribution in vivo. ResultVBCP-BSA was formed by Maillard reaction for 48 h with high grafting rate. Using VBCP-BSA as emulsifier， the homogeneous pink nanoemulsions was prepared and denoted as EOFAZ@VBCP-BSA/VB₁₂. The particle size of the nanoemulsions was less than 100 nm and the particle size distribution was uniform. The surface of the nanoemulsions was a weak negative charge， and the shape was spherical. The encapsulation rate of the nanoemulsions for EOFAZ was greater than 80%， which had a good absorption effect in vitro and could enhance liver accumulation after oral administration. ConclusionThe designed proteoglycan nanoemulsions can effectively load EOFAZ， promote oral absorption and enhance liver distribution， which can provide experimental basis for the development of oral EOFAZ liver protection preparations.

OBJECTIVE To study the interventional effect and mechanism of 1，8-cineole on pancreatic β cell ferroptosis induced by type 2 diabetes. METHODS In vitro ferroptosis model was established in pancreatic β cells of mice by using high glucose. The effects of low-dose and high-dose 1，8-cineole （0.25， 0.5 μmol/L） on the level of Fe2+ in pancreatic β cells were investigated. The effects of 1，8-cineole （0.5 μmol/L） combined with ferroptosis inducer Erastin （20 μmol/L） and ferroptosis inhibitor Ferrostatin-1 （20 μmol/L） on the protein expressions of glutathione peroxidase-4 （GPX4） and cyclooxygenase-2 （COX2） were also detected. The type 2 diabetes model mice were established by feeding high-sugar and high-fat diet combined with intraperitoneal injection of streptozotocin. The effects of low-dose and high-dose 1，8-cineole （50， 200 mg/kg） on the pathological morphology of pancreatic tissue， the content of iron as well as the protein expressions of GPX4 and COX2 were investigated. RESULTS The results of the cell experiment showed that compared with the model group， pretreatment with 1，8-cineole significantly reduced intracellular Fe2+ levels and upregulated GPX4 protein expression， while downregulated COX2 protein expression in pancreatic β cells （P＜0.05）. After combining with Ferrostatin-1， the expression trends of the above two proteins were the same， while there was no statistically significant difference after combining with Erastin. The results of animal experiments showed that compared with the model group， after intervention with 1，8-cineole， the structure of the pancreatic islets in mice recovered intact and their morphology improved； the iron content of pancreatic tissue and protein expression of COX2 were decreased significantly （P＜0.05）， while protein expression of GPX4 was increased significantly （P＜0.05）. CONCLUSIONS 1，8-cineole could ameliorate pancreatic β cell injury induced by diabetes， the mechanism of which may be related to reducing intracellular iron deposition and regulating ferroptosis-related proteins.

Objective To investigate the effects of zalcitabine(ddC),a mitochondrial DNA polymerase γ(POLG)inhibitor,on the migration,invasion,and to preliminarily explore mitochondrial biogenesis of human tri-ple-negative breast cancer MDA-MB-231 cells.Methods The effect of ddC on cell viability was detected using the MTT assay.The migration and invasion abilities of the cells were evaluated using the cell scratch and Transwell in-vasion assays.Cell apoptosis was determined using flow cytometry and a V-FITC/PI cell apoptosis detection kit.The protein expression of POLG,NADH dehydrogenase subunit Ⅰ(NADH1),NADH dehydrogenase subunit Ⅱ(NADH2),ATP synthase subunit 6(ATPase6),cytochrome c oxidase subunit Ⅰ(COX-1)and cytochrome c ox-idase subunit Ⅲ(COX-3)were determined using Western blot.The POLG mRNA level and mtDNA copy number were determined using qPCR.The mitochondrial content and ATP levels were determined using MitoTracker Green fluorescent probe staining and an ATP determination kit.MDA-MB-231 cells were transfected with pcDNA3.1-EG-FP-POLG plasmids to overexpress POLG.The inhibitory effects of ddC on cell migration and invasion were detected in POLG-overexpressed MDA-MB-231 cells.Results POLG expression was higher in MDA-MB-231 cells than in normal mammary epithelial cells(MCF-10A)(P＜0.01).ddC inhibited cell viability in a dose-dependent man-ner.ddC inhibited the migration(P＜0.01)and invasion(P＜0.01)of MDA-MB-231 cells;however,it dis-played no significant inhibitory effects on cell viability in normal mammary epithelial cells(MCF-10A)at the same concentration.ddC downregulated the protein(P＜0.01)and mRNA(P＜0.01)levels of POLG,reduced mtD-NA copy number(P＜0.01)and downregulated mtDNA-coded NADH1,NADH2,ATPase6,COX-1 and COX-3 protein expression(P＜0.01)in MDA-MB-231 cells.Furthermore ddC inhibited mitochondrial content(P＜0.01)and ATP(P＜0.01)levels in MDA-MB-231 cells.POLG overexpression increased the migration(P＜0.05)and invasion(P＜0.05)abilities of MDA-MB-231 cells,while ddC did not significantly inhibit the migra-tion and invasion abilities of MDA-MB-231 cells overexpressing POLG.Conclusion ddC downregulates POLG ex-pression in MDA-MB-231 cells and inhibits mitochondrial biogenesis and ATP levels,thereby inhibiting the migra-tion and invasion of MDA-MB-231 cells.

Objective We aimed to compared matrix metalloproteinase-13 (MMP-13) and transforming growth factor-β1 (TGF-β1) in synovial fluid,the phosphorylation level of Smad3 in articular cartilage (P-Smad3),and their correlation with traditional Chinese medicine (TCM) patterns in patients with knee osteoarthritis (KOA) of liver-kidney deficiency pattern and pattern of intermingled phlegm and blood stasis.Methods Using a cross-sectional field investigation method,KOA patients hospitalized in the Orthopedics Department of Dongzhimen Hospital,Beijing University of Chinese Medicine from September 2019 to February 2023 were collected. A total of 112 KOA patients were included,among which 63 cases were diagnosed with liver-kidney deficiency pattern,and 49 cases were diagnosed with pattern of intermingled phlegm and blood stasis. The intensity of knee pain,function,and X-ray imaging result were quantified using the Visual Analogue Scale (VAS),Lysholm Knee Scoring Scale,and Kellgren-Lawrence (K-L) Grading Scale,respectively. The TCM pattern was identified and quantified using a TCM Pattern Scoring Scale. Immunohistochemistry was used to determine the phosphorylation characteristics of Smad3 in articular cartilage,and ELISA was used to measure the contents of MMP-13 and TGF-β1 in synovial fluid. The level characteristics and their correlation with the degree of syndrome were analyzed.Results (i) There was no statistically significant difference in VAS scores,Lysholm scores,and K-L grades between KOA patients with different TCM patterns. (ii) Compared with KOA patients with pattern of intermingled phlegm and blood stasis,patients with pattern of liver-kidney deficiency had higher levels of MMP-13 in synovial fluid and lower levels of TGF-β1 in synovial fluid (P<0.05). (iii) In KOA patients with liver-kidney deficiency pattern,there was a positive correlation between the level of MMP-13 in synovial fluid and the score of TCM pattern (r=0.292,P=0.020),while there was a negative correlation between the level of TGF-β1 in synovial fluid and the score of TCM pattern (r=-0.781,P<0.001). In KOA patients with pattern of intermingled phlegm and blood stasis,there was also a positive correlation between the level of MMP-13 in synovial fluid and the score of TCM pattern (r=0.936,P<0.001). (iv) The mean optical density value of P-Smad3 in articular cartilage was lower in KOA patients with liver-kidney deficiency pattern than in pattern of intermingled phlegm and blood stasis (P<0.05).Conclusion KOA patients with liver-kidney deficiency pattern or pattern of intermingled phlegm and blood stasis have different levels of TGF-β1 and MMP-13 in synovial fluid,as well as varying degrees of Smad3 phosphorylation in articular cartilage,which is consistent with the analysis of etiology and pathogenesis under different patterns. The levels of TGF-β1 and MMP-13 in synovial fluid of patients with liver-kidney deficiency pattern can reflect the severity of the pattern to a certain extent,and the mechanism may be related to the inhibition of the activation level of the TGF-β/Smad signaling pathway. This study enriches the research content of the material basis of TCM patterns.

Objective: To explore the effect of hypoxia inducible factor 1α(HIF-1α)on tamoxifen resistance in breast cancer MCF-7,and to established a human breast cancer cell line(MCF-7/TR)with 4-hydroxy tamoxifen(4-OHT)resistance. Methods: 4-OHT was an active form of tamoxifenin vivo, resistant breast cancer cells MCF-7/TR were establishedin vitrousing 4-OHT. MTT assay was used to detect the effect of 4-OHT on the MCF-7 and MCF-7/TR cells, and the drug resistance multiple was detected. Western blot was used to detect the expression level of HIF-1α protein in MCF-7 and MCF-7/TR cells. The effects of HIF-1α inhibitor(LW6) or siRNA HIF-1α on MCF-7/TR cells and 4-OHT on MCF-7 cells treated with HIF-1α stabilizer(FG-4592) were detected by MTT and flow cytometry AV/PI staining. Results: The results showed that the MCF-7/TR was successfully constructed, and the drug resistance ratio was(5.56±0.80). Compared with MCF-7 cells, MCF-7/TR cells had higher expression of HIF-1α protein and it was up-regulated after tamoxifen treatment. After giving LW6 or silencing the expression of HIF-1α,the down-regulation of HIF-1α expression enhanced the inhibitory effect of 4-OHT on MCF-7/TR cells. After treatment of FG-4592, the expression level of HIF-1α in breast cancer cells MCF-7 was up-regulated, and hindered the inhibition effect of tamoxifen on MCF-7 cells. Conclusion The above results indicate that HIF-1α plays an important role in 4-OHT resistant breast cancer, and targeting HIF-1α may be an effective way to increase the sensitivity of MCF7/TR cells to tamoxifen.