For the treatment of medial compartment knee osteoarthritis, unicompartmental knee arthroplasty was chosen on the basis of the clinical effects and the rate of prosthesis survival. A retrospective analysis was performed on 500 patients with osteoarthritis of the medial compartment knee treated by unicompartmental knee arthroplasty between April 2007 and April 2017. The cohort was comprised of 176 males and 324 females, aged (61.12 ± 8.0) years old. The clinical treatment was evaluated in terms of the range of movement (ROM), the Knee Society score (KSS), and the Oxford knee score.The follow-up lasted 1.59–11.60 years. Grouped in terms of age, 133 cases were in the middle-age, 295 were in the old-age group, and 72 in the advanced-age group. Against the Iwano classification of the patellofemoral joint, 104 cases were graded 0–1; 179 were graded 2; 182 were graded 3 and 35 were graded 4. The KSS score improved from (59 ± 5.6) points before the operation to (93 ± 3.5) points after the operation. The OKS score increased from (24 ± 2.4) points before the operation to (45 ± 3.8) points after the operation. The ROM of knee joint was (111 ± 6.8)° before the operation and was (117 ± 9.7)° after the operation. The 10-year survival rate of the prosthesis was 96%.UKA is one of the treatments for medial compartmental knee osteoarthritis. The issues, such as age and patellofemoral joint degeneration, can be addressed by careful selection of patients and precise operational manipulation. Osteoarthritis (OA) of the knee represents one of the most common diseases among middle-aged and elderly people, and tends to involve the medial compartment of the knee joint . Since MThe OUKA only involves the single compartment of the knee and retains the bone mass and ligament function of the joint. It goes well with minimally invasive incision and restores the knee joint function early and quickly . Some clHowever, the indications, complications, and prosthesis revision rate of the OUKA remain controversial –23. The Inclusion criteria: (1) The patients were diagnosed as having osteoarthritis of knee; (2) Preoperative imaging confirmed the onset of medial compartmental osteoarthritis of the knee , with tExclusion criteria: (1) Concurrently having other joint diseases, such as rheumatoid arthritis, joint infection, joint tuberculosis, bone tumor, and acute phase of intra-articular fracture; (2) Having degeneration of the lateral compartment of the knee joint or with concurrent severe deformity of the knee joint; (3) Patients had received ipsilateral knee osteotomy or joint replacement; (4) Patients could not tolerate surgery.2 . The unilateral replacement was performed in 400 cases (80.0%), including 205 cases of the right knee (41.0%) and 195 cases of the left knee (39.0%). The bilateral replacement was done in 88 cases (17.6%). One side received OUKA and the other side received TKA in 12 cases (2.4%). Iwano et al. [From April 2007 to April 2017, a total of 522 patients with knee osteoarthritis were treated with OUKA. According to the above-mentioned criteria, 500 cases of UKA surgery were included in this study. The third generation of Oxford mobile-bearing prosthesis system  was selected for all patients. The cohort was comprised of 176 (35.2%) males and 324 (64.8%) females, with the average age being (61.12 ± 8.0) years . The patients were divided into three groups according to the age: a middle age group (< 55 years old), an old-age group (55–70 years old), and an advanced-age group (> 70 years-old). The body mass index (BMI) was (23.7 ± 4.2) kg/mo et al.  proposedIn all patients, operation was performed by the same group of surgeons. These patients were reviewed by a non-surgical medical team 1 month, 3 months, and 1 year after operation. The ROM, the Knee Society score (KSS) , and Oxft-test was utilized to compare the pre- or postoperative ROM, KSS, OKS. One-way analysis of variance (ANOVA) was used to compare postoperative ROM, KSS, and OKS between different age groups and patellofemoral joint degeneration groups. A p < 0.05 indicated a statistically significant difference. Statistical analysis was performed using SPSS statistical software .Descriptive statistics were used for data analysis. Kaplan-Meier survival analysis was employed for the evaluation of the prosthesis survival rate at different ages and the degree of patellofemoral joint degeneration. The revision of prosthesis for any reason was used as the endpoint, including replacement or removal of any component of the prosthesis. Two-sided independent sample t=–2.586, p < 0.05). The OKS score increased from (24 ± 2.4) preoperatively to (45 ± 3.8) postoperatively . The average preoperative ROM of the knee joint was (111 ± 6.8) °, and the postoperative ROM was (117 ± 9.7)° .The 500 patients recruited in this study were followed up for 1.59–11.60 years (average: 5.27 years). The last follow-up was in November 2018. In terms of age, 133 (26.6%) cases were in the middle-age group, 295 (59.0%) in the old-age group, and 72 (14.4%) in the advanced-age group. Against the Iwano imaging classification, 104 cases (20.8%) were rated 0–1; 179 cases (35.8%) were rated 2, 182 cases (36.4%) were rated 3 and 35 (7.0%) were rated 4. The KSS score was improved from (59 ± 5.6) preoperatively to (93 ± 3.5) postoperatively  and the infection developed 2 years after surgery and was treated by cement spacer and staged total knee arthroplasty. Moreover, 4 cases (0.8%) had loosening of the femoral component of the prosthesis (including 2 cases of bearing dislocation), of which 3 underwent revision of femoral component and 1 received total knee arthroplasty. In 2 cases, prosthesis loosening resulted from spontaneous osteonecrosis of the knee (SONK). Two patients (0.4%) developed repeated aseptic hydrops in the joint and recovered after knee puncture and physical therapy. 2 cases (0.4%) had a bony free body from the osteophyte in the joint, which was removed by arthroscopic surgery, and 1 patient (0.2%) suffered from postoperative knee joint pain of unknown origin, which subsided after conservative treatment and rehabilitation exercise.n = 133), 99.0% in the old-age group (n = 295), and 97.2% in the advanced-age group (n = 72). The long-term survival rate of the prosthesis was significantly reduced in patients < 55-year old  .The 10-year prosthesis survival rate was 89.8% in the middle-age group  , but was significantly reduced in the grade 4 group.According to the Iwano imaging classification, the 10-year prosthesis survival rate was 98.9% in the grade 0–1 group, 92.7% in the grade 2 group, 97.8% in the grade 3 group, and 96% in the grade 4 group. Interestingly, the degree of patellofemoral joint degeneration did not affect the survival rate of the UKA (This study reviewed 500 OUKAs for the treatment of medial compartment osteoarthritis of the knee in a single institution. The results showed that OUKA significantly improved knee joint mobility, and could achieve reliable clinical effect. Thus, OUKA is the best choice for the treatment of anteromedial osteoarthritis. Murray et al. first reported that the 10-year survival rate of the phase III Oxford UKA was 98% . In thisThe complications of OUKA include: polyethylene bearing dislocation, bearing wear, aseptic loosening, medial collateral ligament or cruciate ligament injury, arthritis progression, deep infection, articular cavity recurrent hematoma, bone cement residual, and unexplained pain. The complications varied with different periods of follow-up , 36. TheThe complications can be treated with revision surgery or non-revision surgery , 42. NonFor advanced-age patients, especially those > 75-year-old, the OUKA has the apparent advantages of small trauma, fast postoperative recovery, reliable clinical efficacy, and high long-term survival rate of the prosthesis, as compared with TKA . AlthougPatellofemoral joint degeneration in the past has been regarded as a contraindication of UKA . HoweverIn conclusion, OUKA is one of the treatments for medial compartmental osteoarthritis and has reliable clinical efficacy, high long-term survival rate, and low incidence of related complications. Some issues, such as age-related problems and patellofemoral joint degeneration, can be addressed by careful patient selection and skillful operation.


Daphnia magna sublethal indices: behavioral  and physiological endpoints . The results showed that FIP induced reduction of swimming speed and distance traveled in a concentration- and time-dependent manner at all the concentrations used. The lowest concentration of the insecticide temporarily stimulated post-abdominal claw activity after 24 h and thoracic limb activity after 48 h; however, the highest concentrations reduced all the studied physiological endpoints. IC50 values showed that thoracic limb activity, swimming speed, and distance traveled were most sensitive to FIP after 24-h exposure. The most sensitive parameter after 48 h and 72 h was swimming speed and post-abdominal claw activity, respectively. The study indicated that (i) behavioral and physiological endpoints of Daphnia magna are reliable and valuable sublethal indicators of toxic alterations induced by FIP; however, they respond with different sensitivity at various times of exposure, (ii) FIP may alter cladoceran behavior and physiological processes at concentrations detected in the aquatic environment; therefore, it should be considered as an ecotoxicological hazard to freshwater cladocerans.Fipronil (FIP) is an organic pesticide with many practical uses. Although some results indicated toxic effects in some terrestrial and aquatic animal species, little is known on its influence on behavioral and physiological endpoints of cladocerans. The aim of our study was to determine the short-term effects of FIP at concentrations of 0.1 μg/L, 1 μg/L, 10 μg/L, and 100 μg/L on  Fipronil (FIP), a derivative of phenylpyrazole, is one of the most widely used organic pesticides in agriculture, veterinary medicine, and urban households for selective elimination of insect pests such as ants, mosquitos, termites, cockroaches, spiders, fleas, and mites Tomlin . The mecMassive use of FIP for veterinary purposes led to hazardous incidents. Eradication of red mites in poultry farms in European countries and Japan   per tank and supplemented with a few drops of baker’s yeast (10 mg/L per tank).FIP  was purchased from Merck, and the following series of its nominal concentrations was prepared: 0.1 μg/L, 1 μg/L, 10 μg/L, and 100 μg/L. Initially, the stock solution (100 mg/L) was prepared by diluting 10 mg of FIP in the 100 mL of filtered culture medium containing 0.1% of methanol. After mixing the flask with the stock solution, the highest FIP working concentration of 100 μg/L was prepared by transferring 100 μL of the stock solution to a volumetric flask containing 99.9 mL of clean culture medium. The experimental concentration of 10 μg/L was done by transferring 10 mL from the flask with 100 μg/L of FIP to another volumetric flask containing 90 mL of clean medium. A concentration of 1 μg/L was prepared by transferring 10 mL from the flask containing 10 μg/mL of FIP to another flask with 90 mL of clean medium. A concentration of 0.1 μg/L was prepared by transferring 10 mL from the flask containing FIP at 1 μg/mL to another flask with 90 mL of clean medium. The appropriate solutions of the insecticide prepared in the above-mentioned flasks were transferred at a volume of 8 mL to glass Petri dishes (75 mm of diameter). Each studied endpoint was examined at 24 h, 48 h, and 72 h of the exposure. Animals were considered as dead when no heart activity was noted under microscopic examination.Each experimental group consisted of 15 neonates grouped into 5 individuals placed in three glass Petri dishes (for three independent treatments) with a diameter of 75 mm containing one of FIP concentrations of 0.1 μg/L, 1 μg/L, 10 μg/L, and 100 μg/L. Additionally, 15 animals grouped into 5 individuals placed in three experimental dishes with medium only were treated as the control. Daphnids were left 15 min for acclimation  concentration (EC50) for immobilization and inhibition  concentration (IC50) for swimming speed, distance traveled, heart rate, thoracic limb activity, and post-abdominal claw activity after 24 h, 48 h, and 72 h of exposure were calculated using probit regression analysis with MedCalc 19.3.1 statistical software . Maximum log likelihood estimation  was used as an overall fit model, and logarithmic curve was plotted as a result.p < 0.05. The results are presented as means ± standard deviation (SD).Data were analyzed with statistical software Statistica® 13.1. Normality and homogeneity of variances were estimated by the Shapiro-Wilk and Levene’s tests, respectively. The tests were parametric with homogenous data. The comparisons of means among the experimental groups were done by two-way ANOVA  followed by Dunnett’s post hoc test to calculate significant differences between the experimental groups and the control. Results were significant when Calculation of nominal IC50 of the studied endpoints revealed that the most sensitive endpoints after 24-h exposure to FIP were thoracic limb activity (IC50 = 2.8 ± 0.7 μg/L) Table ; Fig. 1,Daphnia magna exposed to FIP showed concentration- and time-dependent immobilization  when compared to the control (5.32 ± 0.22 mm/s). More reduced parameter was found in the treated animals after 48 h, and complete cessation of motility was noted in the crustaceans exposed to 100 μg/L after 48 h and 10 μg/L after 72 h.Effects of FIP on daphnids swimming speed are presented in Fig. p < 0.01) when compared to the control (319 ± 15 mm/min). The distance traveled was decreasing with the increasing time of exposure to FIP. No distance traveled was noted in daphnids at 100 μg/L after 48 h and at 10 μg/L after 72 h.Daphnids exposed to FIP showed a concentration- and time-dependent reduction of distance traveled Fig. . A distip < 0.01, and 124 ± 26.6 bpm, p < 0.01, respectively), when compared to the control (197.5 ± 25.8 bpm) .The results show that FIP at concentrations of 1 μg/L, 10 μg/L, and 100 μg/L reduced daphnid heart rate after 24-h exposure  and 100 μg/L  after 24-h exposure when compared to the control (11.5 ± 2.5 bpm). The highest reduction of this physiological activity showed daphnids at 72-h exposure to the pesticide  at concentrations of 0.1 μg /L, 1 μg/L, 10 μg/L, respectively).The results presented in Fig. p < 0.01) both after 48 h  and after 72 h of exposure.Figure Daphnia pulex was estimated to be 0.0156 mg/L (Stark and Vargas Farfantepenaeus aztecus) (96-h LC50 = 0.12 μg/L)   exposed to FIP  (Jinguji et al. Thoracic limbs play an important role in daphnid feeding and ventilation (Pirow et al. The present study showed that daphnid behavioral and physiological endpoints are sensitive indicators of toxic changes induced by FIP; however, sensitivity of these responses was different at various times of exposure. Since FIP induced alterations of daphnid endpoints at environmentally relevant concentrations, it may be expected that vital behavioral and physiological processes may be impaired by this pesticide in the real scenario. More studies in daphnids are required that would provide results useful in explanation of the neurological mechanisms responsible for the behavioral and physiological alterations.


Cysticercus cellulosae (C. cellulosae) excretes and secretes antigens during the parasitic process to regulate the host immune response; however, resulting immune response and cytokine production in the host during infection still remains unclear. We used C. cellulosae crude antigens (CAs) as controls to explore the effect of excretory secretory antigens (ESAs) on T-cell immune responses in piglets. C. cellulosae ESAs induced imbalanced CD4+/CD8+ T-cell proportions, increased the CD4+Foxp3+ and CD8+Foxp3+ T-cell frequencies, and induced lymphocytes to produce interleukin-10, which was mainly attributed to CD4+ and CD4−CD8− T cells. The ESAs also induced Th2-type immune responses. The results showed that the ability of C. cellulosae to escape the host immune attacks and establish a persistent infection may be related to host immune response regulation by the ESAs. Taenia solium (T. solium) is a cestode parasite that has zoonotic importance and is harmful to human health. Its developmental stages include four life cycles: eggs, oncospheres, larvae, and adults  is the most serious form of the disease , including Th1, Th2, and Th17. Th1-type immune responses inhibit infections, and Th2-type immune responses aggravate infections -γ and IL-2 production, but not IL-4 or IL-10 production, in CD4+ and CD8+ T cells of rat mesenteric lymph nodes. It shows that Th2 immune responses are closely related to persistent parasitic infections  enables evaluating the treatment responses of these patients. C. cellulosae survival is directly related to the existence of anti-ESA antibodies, and these antibodies can be used in new diagnostic tests and subsequent treatments , then cultivated at 37°C and 5% CO2 in RPMI 1640 medium  containing penicillin G (100 U/ml) and streptomycin (100 μg/ml). Then the culture supernatants were collected aseptically after 72 h. The supernatants were filtered through 0.22 μm sterile filters, then concentrated by transferring them into a 3 kDa ultrafiltration tube . The liquid after concentration was C. cellulosae ESAs, and its protein concentration was determined using a Bradford protein assay kit  per the manufacturer’s instructions and stored at −80°C until used.To prepare the ESAs and CAs of the C. cellulosae CAs were prepared as previously described with some modifications  at 4°C until completely lysed. The supernatants were collected via centrifugation as C. cellulosae CAs, and its protein concentration was measured using a Bradford protein assay kit per the manufacturer’s instructions and stored at −80°C until further use.The ications . The C. via jugular venipuncture and diluted 1:1 with sample diluent according to the lymphocyte separation solution kit  as per manufacturer’s instructions. The diluted blood was added to the surface of an equal volume of separation liquid. The solutions were centrifuged at 2,500 rpm for 20 min according to the density gradient centrifugation to obtain the PBMCs layer. PBMCs were collected and washed twice with cleaning solution. Erythrocyte lysis buffer  was used to eliminate erythrocyte contamination. PBMCs were suspended in RPMI 1640 medium with 10% inactivated fetal calf serum  after ensuring that the cell viability exceeded 95% via trypan blue staining.Peripheral blood mononuclear cells (PBMCs) were isolated as previously described with minor modifications . Briefly6 cells/well at 37°C and 5% CO2 and separately incubated with control medium, concanavalin A  , ESAs (40 μg/ml), or CAs (40 μg/ml). Phytohemagglutinin  was added to a final concentration of 2.5 μg/ml after cultivation for 3 h. Cells were harvested on day 2, stained with CD4 and CD8, and analyzed via flow cytometry.To analyze the effects of ESAs and CAs on mitogen-induced lymphocyte proliferation, PBMCs were cultured in 96-well plates at 1×106 cells/well and treated with control medium, ConA, ESAs, or CAs for 48 h under standard conditions. The cells were then collected and analyzed for Foxp3, CD4, and CD8 expression by flow cytometry. Foxp3 and Helios mRNA expression levels were calculated via quantitative real-time PCR (qRT-PCR).To further analyze whether ESAs and CAs can induce Treg production, PBMCs were incubated in 96-well plates at 1×106/well and added with ESAs, CAs, 2 μg/ml lipopolysaccharide , ESA+LPS, CA+LPS, or control medium. The culture supernatants were harvested after 24 h, and IL-10 expression was measured via ELISA.To investigate whether ESAs and CAs can induce IL-10 production, PBMCs were cultured in 96-well plates at 1×10via intracellular cytokine staining. PBMCs were cultured in 96-well plates at 1×106/well and stimulated with ESAs, CAs, LPS, ESA+LPS, CA+LPS, or control medium. During the last 12 h of culturing, 5 μg/ml of Brefeldin A  was added to block intracellular cytokine secretion into the extracellular area. After 24 h, the cells were stained with IL-10, CD4, CD8, IL-10, and Foxp3 and analyzed via flow cytometry.The lymphocyte subsets expressing IL-10 were evaluated C. cellulosae ESAs and CAs on T-cell polarization, CD4+ T cells were enriched from the spleens of the healthy piglets via positive selection using anti-CD4 magnetic beads  and a lymphocyte separation solution kit  as per manufacturer’s instructions. CD4+ T cells at 1×107 were co-cultivated with 1×106 immature DCs in 96-well pates and added with ESAs (40 μg/ml), CAs (40 μg/ml), ConA (10 μg/ml), or control medium stimulation. The medium was removed via centrifugation, and 10 ng/ml pig recombinant IL-2  was added to promote polarization and maintain T-lymphocyte activity when 24 h of culture. After 48 h, the culture supernatants were collected, and the expression levels of IFN-γ (Th1), IL-4 (Th2), and IL-17 (Th17) were analyzed via ELISA.Healthy piglets were anesthetized, 20 ml bone marrow was extracted, and the bone marrow precursor cells were isolated under sterile conditions. The cells were cultured under standard conditions, and the immature DCs were induced. The purity of immature DCs was more than 80% by flow cytometry. To determine the influence of For cell surface staining, cells were washed twice in precooling staining buffer, then resuspended in staining buffer and incubated with primary antibodies for 30 min on ice in the dark. Cells were then washed once with staining buffer and resuspended in it. The antibodies were used as CD4a:PE-Cy7  and CD8a:AF647 . To detect the intracellular antigens, cells were first incubated for 30 min with surface-labeled antibodies, then washed and resuspended in staining buffer. Cells were then fixed and permeabilized for 45 min with transcription factor buffer  as per manufacturer’s instructions, then incubated with intracellular antigens fluorescent antibodies for 45 min on ice in the dark. The antibodies were used as CD4a:PE-Cy7, CD8a:AF647, Foxp3:FITC , and IL-10:PE .Cytokine secretion was detected using pig IFN-γ, IL-4, and IL-17 ELISA kits . The culture supernatants were collected, and then the cytokine production levels were analyzed as per manufacturer’s instructions.via qRT-PCR. The primer sequences used were pig GAPDH , pig Foxp3 , and pig Helios . TRIzol universal total RNA extraction reagent  was used to extract total RNA from the stimulated cells per the manufacturer’s instructions. Reverse transcription and qRT-PCR were performed using RevertAid First Strand cDNA synthesis kit  and FastStart Universal SYBR Green Master (Rox) . The cycle conditions were one cycle at 95°C for 15 s, 56°C for 30 s, and 72°C for 20 s for 44 cycles. The relative Foxp3 and Helios mRNA expression levels were determined via the 2−ΔΔCT method.The relative changes of Foxp3 and Helios mRNA expression levels in the PBMCs were measured P<0.05 was considered the lowest statistically significant; P<0.01 was considered moderately statistically significant; P<0.001 was considered the highest statistically significant.One-way analysis of variance was used for multiple-group comparisons, and the least significant differences test was used for pairwise comparisons between groups. + and CD8+ T-cell subset expressions were detected via flow cytometry. The ESAs-induced CD8+ T cells increased to levels near those of ConA. Compared with the normal controls, the CAs-induced CD8+ T cells were elevated, but CD4+ T cells were decreased, and both levels were lower than those of ConA .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: Gouty arthritis (GA) is a common metabolic disease caused by a long-term disorder of purine metabolism and increased serum levels of uric acid. The processed product of dried root of Aconitum carmichaeli Debeaux  is used often in traditional Chinese medicine (TCM) to treat GA, but its specific active components and mechanism of action are not clear.Methods: First, we used ultra-performance liquid chromatography-quadrupole/time-of-flight tandem mass spectrometry to identify the chemical spectrum of ARC. Based on this result, we explored the active components of ARC in GA treatment and their potential targets and pathways. Simultaneously, we used computer simulations, in vitro cell experiments and animal experiments to verify the prediction results of systems pharmacology. In vitro, we used aurantiamide acetate (AA) to treat monosodium urate (MSU)-stimulated THP-1 cells and demonstrated the reliability of the prediction by western blotting and real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR). ELISAs kit were used to measure changes in levels of proinflammatory factors in rats with GA induced by MSU to demonstrate the efficacy of ARC in GA treatment.Results: Forty-three chemical constituents in ARC were identified. ARC could regulate 65 targets through 29 active components, and then treat GA, which involved 1427 Gene Ontology (GO) terms and 146 signaling pathways. Signaling pathways such as proteoglycans in cancer, C-type lectin receptor signaling pathway, and TNF signaling pathway may have an important role in GA treatment with ARC. In silico results showed that the active components songoramine and ignavine had high binding to mitogen-activated protein kinase p38 alpha (MAPK14) and matrix metallopeptidase (MMP)9, indicating that ARC treatment of GA was through multiple components and multiple targets. In vitro experiments showed that AA in ARC could effectively reduce expression of MAPK14, MMP9, and cyclooxygenase2 (PTGS2) in THP-1 cells stimulated by MSU, whereas it could significantly inhibit the mRNA expression of Caspase-1, spleen tyrosine kinase (SYK), and PTGS2. Animal experiments showed that a ARC aqueous extract could significantly reduce expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and intereleukin (IL)-18 in the serum of GA rats stimulated by MSU. Hence, ARC may inhibit inflammation by regulating the proteoglycans in cancer-associated signaling pathways.Conclusion: ARC treatment of GA may have the following mechanisms, ARC can reduce MSU crystal-induced joint swelling, reduce synovial tissue damage, and reduce the expression of inflammatory factors in serum. AA in ARC may inhibit inflammation by regulating the protein expression of MAPK14, MMP9, and PTGS2 and the mRNA expression of caspase-1, SYK, and PTGS2. Gouty arthritis (GA) is a rheumatic disease that often causes lesions and inflammatory reactions due to deposition of urate crystals in tissues  . In acutTraditional Chinese medicine (TCM) has been practiced in East Asia for centuries . IncreasAconitum carmichaeli Debeaux  can be used to eliminate wind, remove dampness, disperse cold, relieve pain, and warm meridians. Originally in the Eastern Han Dynasty of China (24-220 AD), ARC was recorded by “Shennong Materia Medica” (Shengnong Ben Cao Jing), the earliest Pharmacopeia of China, and was described for its medicinal effect against chronic arthritic diseases by “Treatise on Febrile Diseases” (Shang Han Lun), a classical TCM book   values of 1.8, 1.9, and 5.8 mg/kg, respectively, for oral administration in mice. Through this heat-processing, aconitine, mesaconitine, and hypaconitine are hydrolyzed at the carbon 8-position to become benzoylaconine, benzoylmesaconine, and benzoylhypaconine, respectively. It is reported that the LD50 values of benzoylaconine, benzoylmesaconine, and benzoylhypaconine are 1.5, 0.81, and 0.83 g/kg, respectively (ARC is highly toxic before processing and should not be eaten . Patientycardia) . The rawectively . The toxectively .Ultra-performance liquid chromatography-quadrupole/time-of-flight tandem mass spectrometry (UPLC-QTOF-MS/MS) has the advantages of fast separation, high sensitivity, and can provide accurate relative molecular mass. UPLC-QTOF-MS/MS is used commonly for analyses of the chemical constituents of complex TCM formulations . Therefo.In recent years, systems pharmacology has been used widely in TCM research. This approach enables systematic determination of the effects and mechanisms of drugs used to treat complex diseases at molecular, cellular, tissue, and biological levels . Several.The animal study was reviewed and approved by the Research Ethics Committee of the Jiangxi University of Traditional Chinese Medicine . NO potentially identifiable human images or data is presented in this study.Aconitum carmichaeli Debeaux , Zingiber officinale Roscoe , dried root of Glycyrrhiza glabra L.  and Saponaria officinalis L.  were purchased from Xujiang Ecological Technology . UHPLC-MS-grade methanol and formic acid were purchased from Anaqua Chemicals Supply , UHPLC-MS-grade acetonitrile was obtained from Merck Drugs and Biotechnology . Ultrapure water was provided by Watson's Food and Beverage .Dried root of As reference standards, four pure compounds were used (purity≥98%). Among these compounds, benzoylmesaconine (25), benzoylaconine (26), Benzoylhypacoitine (28) were purchased from Chendu Chroma Biotechnology Co., Ltd. . Aurantiamide acetate (41) was purchased from Must Biotechnology .THP-1 cells were purchased from Beina Chuanglian Biotechnology . A Bicinchoninic Acid (BCA) protein quantification kit, Dulbecco’s modified Eagle’s medium and Cell Counting Kit (CCK)-8 kit were obtained from Beijing Solaibao Technology . Phorbol-12-myristate-13-acetate (PMA) was purchased from Abcam . Fetal bovine serum was from Zhejiang Tianhang Biotechnology . Monosodium urate (MSU) and colchicine were purchased from Sigma–Aldrich . Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody and anti-matrix metallopeptidase (MMP)9 antibody were obtained from Abcam. Rabbit anti-cyclooxygenase-2 antibody was purchased from Beijing Boosen Biotechnology . An ECL chemiluminescence kit was from Millipore . A tumor necrosis factor (TNF)-α kit, interleukin (IL)-1β kit, and IL-18 kit were purchased from Jiangxi Boyinda Biotechnology Co., Ltd. .CW was soaked in water (which was changed twice a day) for 2 days until its core was completely moist. CW was removed, and SJ, GC, and ZJ added. The water was changed and CW boiled for 4 h until its core was cooked completely. It was boiled for 2 h, removed, dried to 60% and cut into thin slices, followed by further drying . Per 100The dried sample was pulverized into powder and passed through a size-3 sieve. We weighed 1 g of powder accurately, added 30 ml of methanol, and undertook ultrasonic extraction for 20 min. After cooling, the weight loss was compensated, followed by filtering. The filtrate was diluted 3-fold, and passed through 0.22-μm microporous filter membrane .A Triple TOF™ 5600 liquid chromatography high-resolution tandem mass spectrometer  equipped with a DuoSpray™ ion source, 30-A liquid chromatography system, and Acquity UPLC BEH C18 column  was used. The mobile phase was 0.1% formic acid aqueous solution (A) and acetonitrile (B). The gradient elution was: 0.01–5 min, 5%–10% B; 5–12 min, 10%–35% B; 12–20 min, 35%–80% B; 20–30 min, 80%–85% B; 30–35 min, 85%–95% B; 35–37.1 min, 95%–5% B. The flow rate was 0.25 ml/min, and 2 μL of filtrate was injected into the UPLC-QTOF-MS/MS system for analyses.m/z) range from 50 to 1,000. The collision energy spread was 15 V. Data were collected in information-dependent acquisition mode and analyzed by PeakView®1.2 (AB Sciex).The instrument settings for the QTOF-MS/MS system were chosen carefully. The electrospray ionization (ESI) source was positive and negative modes. The positive and negative ionization spray voltages were 5,500 and 4,500V, respectively. The ion source temperature was 500°C. The declustering potential was 100 V. The collision energy was 40 eV. Ion source gas 1 (GS1) and GS2 were both set to 50 psi. The curtain gas was set to 40psi. Samples were analyzed in positive and negative ionization modes with a scanning mass-to-charge (https://pubchem.ncbi.nlm.nih.gov/) into the Molinspiration website (www.molinspiration.com/cgi-bin/properties) to calculate the prediction parameters for drug absorption  and SwissTargetPrediction (http://www.swisstargetprediction.ch/) databases for target collection.Established biological/chemoinformatics methods can aid prediction of the targets of chemical constituents in TCM formulations . We obtawww.genecards.org/) and the Comparative Toxicogenomics Database  to identify disease targets associated with GA.We used “gouty arthritis” as the keyword in the GeneCards database (www.cytoscape.org/) to undertake visual analyses of the C–T network   v11.0 (db.org/) . We seleWe used “clusterProfiler” within R   for funchttp://biogps.org/). The species we selected was “human”. We obtained the protein-expression datasets of related targets in different organs, and exported them to Heml 1.0 to generate organ-expression heatmaps of targets.TCM theory holds that the human body is a whole, and that the way to treat diseases is to regulate multiple organs to achieve a “peaceful” state . We wishwww.rcsb.org/). The X-ray crystal structures of MAPK14 and MMP9 were obtained. The PDB entry code of these two proteins was 5ET1 and 1L6J, respectively. For molecular docking, we employed a semi-empirical free-energy calculation to evaluate the energy matching between the compound and target. The more negative the docking score, the more favorable was the interaction of the complex   for 24 h to induce their differentiation into resting M0 macrophages. Fresh complete culture medium was added and cultured allowed for 24 h after washing cells with phosphate-buffered saline. Then, differentiated THP-1 cells were stimulated with MSU  and treated with aurantiamide acetate (AA) at a final concentration of 0, 20, 60, 80 or 100 µM as well as colchicine  simultaneously for 24 h at 37 C . The protein concentration was determined using the BCA Protein Quantification Kit according to manufacturer instructions. Cell lysates containing equal amounts of protein were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 10% gels and then transferred to polyvinylidene fluoride (PVDF) membranes. After blockade with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20 for 0.5 h at room temperature, the PVDF membranes were incubated with primary antibodies overnight at 4 C and horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature, respectively. Protein bands were probed with ECL Plus chemiluminescent reagent and exposed to the ChemiScope Mini 3,300 chemiluminescent imaging system .® Reagent , and reverse-transcribed with oligo-DT using HiScript™ Reverse Transcriptase  according to manufacturer instructions. The primers used were synthesized by Beijing Ruixingke Biotechnology . The sequences were : 5”-CATCCCACAATGGGCTCTGT-3” and 5”-CTCTTCACTTCCTGCCCACA-3” for Caspase-1; 5”-CCTGGCGCAGGTGGAC-3” and 5”-GTAGTGGTGTGCCTTCCTCC-3” for SYK; 5”-GATCCCCAGGGCTCAAACAT-3” and 5”-GAAAAGGCGCAGTTTACGCT-3” for PTGS2; 5”-AATGGGCAGCCGTTAGGAAA-3” and 5”-GCGCCCAATACGACCAAATC-3” for the internal control (GAPDH).Total RNA was extracted with TRIzol−△△CT method was the basis for measuring relative expression of genes.The PCR conditions were: 95°C for 3 min, 94 C for 30 s, 60°C for 30 s, 72°C for 30 s, 35 cycles, and extension at 72°C for 10 min. Then, 6 μL of the PCR-amplification products were electrophoresed in 1.5% agarose gel, imaged and photographed under a gel imager . GAPDH was used as an internal reference to determine relative mRNA expression of the genes to be measured. The 2Thirty-two specific pathogen-free male Sprague−Dawley rats (180–220 g) were purchased from Nanjing Junke Bioengineering . After 1 week of adaptive feeding, 32 rats were divided randomly into four groups of eight: control; GA model (MSU); positive control (colchicine); ARC. The positive-control group was given colchicine (0.8 mg/kg i.g.). The ARC group was given an aqueous extract of ARC (0.3 g/kg i.g.). The control and MSU groups were given an equal volume of physiologic (0.9%) saline (i.g.) once a day for 7 days. On day-5, 200 μL of MSU (25 mg/ml) was injected into the knees of rats in the MSU group, colchicine group, and ARC group . In the Next, the circumference of the knee joint in rats was measured by using the tie line method for 0, 4, 6, and 24 h after MSU injection, respectively. The change in knee joint edema was calculated as follows: joint swelling = b–a. Here, a, b are the initial circumference of rat knee joint and the circumference after the injection of MSU crystal at each time-point, respectively.On day-7, all rats in each group were anesthetized using 3% sodium pentobarbital. Blood was drawn from the abdominal aorta into a vacuum collection tube. Serum was obtained by centrifugation at 3,500 rpm/min for 10 min at 4°C. Levels of TNF-α, IL-1β, and IL-18 were measured by enzyme-linked immunosorbent assay (ELISA) kits. Synovium tissue samples were obtained from the joints of rats for histological analysis.p < 0.05 was considered significant. All experiments were repeated at least thrice.Data are the mean ± SD. The significance of results was determined based on one-way analysis of variance using Prism 8.0.1 . The response values of different chemical compositions differ in different modes. Hence, we undertook UPLC-QTOF-MS/MS in positive and negative ionization modes. Forty-three compounds were identified  and theivia inhibition of activation of mitogen-activated protein kinase (MAPK) , c-Jun N-terminal kinase (JNK), p38), protein kinase B (Akt) and nuclear factor-kappa B (NF-κB) pathways in SW982 human synovial sarcoma cells , songoramine (degree = 18), cytochalasin B (degree = 16), and ignavine (degree = 17). Hence, the same active component in ARC could act on multiple targets. Simultaneously, the same target could be affected by multiple components, such as AKT1 (degree = 44), caspase 1 (degree = 18), IL-18 (degree = 17) and SYK (degree = 15). This finding also illustrated, from another perspective, that TCM theory involves is a mode of action with multiple components and multiple targets. Details on the active ingredients and targets are provided in We constructed a PPI network consisting of 65 nodes and 443 edges . This PPThe light-blue edges denote known interactions from curated databases. The pink edges show that the known interactions were determined by experimental methods. The green edges demonstrate that the predicted interactions arose from a neighboring gene. The red edges show that the predicted interactions arose from gene fusions. The dark-blue edges denote that the predicted interactions arose from gene co-occurrence. The yellow edges demonstrate that the predicted interactions arose from text-mining. The black edges denote that the predicted interactions arose from co-expression. The lavender edges show that the predicted interactions arose from protein homology. The details of the PPI network are described in We took the top-20 proteins in the PPI network. AKT1 may be associated with 40 other proteins . TNF andFirst, we undertook pathway-enrichment analyses using the GO database to elucidate the relevant biological processes . The y-aDetails of pathway-enrichment analyses using the GO database are provided in Pathway enrichment analyses using the KEGG database are shown in Pathway enrichment analyses using the KEGG database indicated that ARC treatment of GA could affect the modules of “tumor”, “immune response”, and “inflammation”. Sixty-five overlapping targets were closely related to the relevant pathways of prostate cancer (hsa05215), C-type lectin receptor signaling pathway (hsa04625), TNF signaling pathway (hsa04668) and endocrine resistance (hsa01522) . These pExpression of the top-20 targets in the PPI-network node degree in different organs is shown in The liver is the main site of catabolism of endogenous free purine bases. The small intestine is responsible for catabolism of purine bases from excessive dietary sources. The excretion of uric acid occurs primarily at three sites: kidney (urine), intestine (feces) and liver (bile). About 90% of uric acid is reabsorbed as it passes through renal glomeruli. When its production (and reabsorption) rate exceeds the excretion rate, accumulation occurs. In turn, this causes hyperuricemia and increases the number of uric-acid crystals, and leads ultimately to GA .We showed that therapeutic targets could exist in two or more organs simultaneously, and that multiple targets could be distributed in the same organ. This finding suggests that human organs are interconnected during the occurrence and progression of diseases, and that ARC may have a therapeutic role in GA through multiple targets and multiple pathways.in silico simulations.In general, the smaller the binding energy, the more stable the ligand binds to the receptor. Binding energy <0 indicates that the ligand molecule can bind spontaneously to the receptor molecule . TherefoPrevious analyses of C–T networks have revealed a close association of the component songoramine to GA targets. Therefore, we first undertook molecular docking of songoramine with MAPK14. The binding energy between songoramine and MAPK14 was −7.83 kcal/mol, which indicated the possibility of strong binding between them. Songoramine and MAPK14 formed a hydrogen bond at glutamate (GLU)-3 . The molAs another active component of treatable GA in ARC, ignavine was docked with MAPK14 and MMP9, respectively. The binding energy between ignavine and MAPK14 and MMP9 was −5.71 and −5.81 kcal/mol, respectively. This result indicated a good possibility of binding between ignavine and two GA targets. Ignavine could form hydrogen bonds with MAPK14 and MMP9 at lysine (LYS)-152, aspartate (ASP)-177, threonine (THR)-331, and arginine (ARG)-134, respectively .Based on these data, we suggest that the interactions between these active ingredients and targets underlie their biological activities. It also means that ARC has multiple components and multiple targets.Cells were treated with MSU  to evaluate the activity of THP-1 cells . FinallyWe chose AA to test our predictions. Western blotting was done to measure expression of MAPK14, MMP9, and PTGS2. RT-qPCR was used to measure expression of caspase-1, SYK, and PTGS2 to confirm the previously predicted effect. Compared with that in the control group, expression of MAPK14, MMP9 and PTGS2 in THP-1 cells was increased significantly by MSU stimulation, and expression of MAPK14 and MMP9 was decreased significantly after treatment with AA (80 μmol/L); for PTGS2, it was decreased significantly after treatment with only a high dose of AA .RT-qPCR results showed that mRNA expression of caspase-1, SYK, and PTGS2 was increased significantly in the model compared with that in the control group, and expression of PTGS2 mRNA was decreased significantly in all dose groups. Expression of caspase-1 mRNA and SYK mRNA showed a significant decreasing trend upon treatment with AA at 80 μmol/L .in vitro experiments showed that AA had a therapeutic effect on GA possibly by inhibiting inflammation thanks to regulation of the protein expression of MAPK14, MMP9, and PTGS2 and mRNA expression of caspase-1, SYK, and PTGS2.The results of The MSU crystal-induced rats had a significant increase in knee joint circumference . The intFurthermore, synovium tissue were stained with H&E and visualized via using a microscope . In contThe proinflammatory cytokines IL-1β, IL-18, and TNF-α are the main cytokines involved in acute GA. Their expression can be used to characterize objectively inflammation severity in the body . To demoAconitum carmichaeli Debeaux toxicity is the heart. However, we have previously conducted cardiotoxicity studies of ARC. The ARC group was given an aqueous extract of ARC (0.3 g/kg i.g.). The control group were given an equal volume of physiologic (0.9%) saline (i.g.) once a day for 28 days. It was found that there was no statistically significant difference in the intensity of cardiotoxicity between ARC group and blank group, indicating that ARC was weakly or even non-toxic at reasonable doses , especially in developing countries . The etile doses . Colchicle doses . Therefoin vitro cell experiments. Therefore, we suggest that one of the mechanisms of ARC in GA treatment may be through the inhibition of inflammation by AA.The aconitine found in ARC is used widely to treat pain and inflammation . IgnavinHelicobacter pylori infection. Caspase-1 and SYK were relatively posterior in the PPI network, but several studies have shown that they are closely related to the occurrence and treatment of GA  is a proinflammatory cytokine that can lead to acute GA . MMP9 rent of GA . TherefoIn vivo experimental studies revealed that MSU crystal-induced knee joint swelling was significantly reduced in rats after treatment with ARC. At the same time, synovial tissue injury was mild, synovial hyperplasia was inhibited, and the inflammatory response was attenuated. IL-1β, IL-18, and TNF-α expression was significantly lower in serum.in vitro experiments showed that AA had a therapeutic effect on GA possibly by inhibiting inflammation thanks to regulation of the protein expression of MAPK14, MMP9, and PTGS2 and mRNA expression of caspase-1, SYK, and PTGS2.The results of By analyzing the enrichment of pathways using GO and KEGG databases, 1427 GO terms and 146 pathways were obtained. Among them, functional-enrichment results using the GO database were related mostly to peptidyl-serine modification, the cellular response to chemical stress, and protein-tyrosine-kinase activity in transmembrane receptors. Pathway-enrichment analyses showed that the active components of ARC could treat GA by regulating signaling pathways such as prostate cancer (hsa05215), C-type lectin receptor signaling pathway (hsa04625), TNF signaling pathway (hsa04668) and endocrine resistance (hsa01522). To a certain extent, these data also suggest that ARC has multiple components, multiple targets, and multiple pathways.We propose that the following mechanisms may exist for ARC treatment of GA, and ARC can reduce joint swelling caused by MSU crystals, reduce synovial tissue damage, and decrease the expression of inflammatory factors in serum. AA in ARC may inhibit inflammation by regulating the protein expression of MAPK14, MMP9, and PTGS2 and the mRNA expression of caspase-1, SYK, and PTGS2.In China, ARC is used commonly to treat rheumatic fever, joint pain, inflammation, and edema . However


Here we survey 28 H3K27ac-marked active enhancer profiles and 50 transcriptomes in primary EC, metastatic lymph node cancer (LNC), and adjacent normal (Nor) esophageal tissues. Thousands of gained or lost enhancers and hundreds of altered putative super-enhancers are identified in EC and LNC samples respectively relative to Nor, with a large number of common gained or lost enhancers. Moreover, these differential enhancers contribute to the transcriptomic aberrations in ECs and LNCs. We also reveal putative driver onco-transcription factors, depletion of which diminishes cell proliferation and migration. The administration of chemical inhibitors to suppress the predicted targets of gained super-enhances reveals HSP90AA1 and PDE4B as potential therapeutic targets for ESCC. Thus, our epigenomic profiling reveals a compendium of reprogrammed cis-regulatory elements during ESCC carcinogenesis and metastasis for uncovering promising targets for cancer treatment.The role of  The role of regulatory cis-elements in carcinogenesis and metastasis in esophageal squamous cell carcinoma remains crucial. Here the authors investigate H3K27ac-marked active enhancer profiles and transcriptomes in different types of esophageal tissues and identify oncogenic events and potential therapeutic targets. It is associated with a poor prognosis and a 5‐year overall survival rate of 18% only2. ESCC accounts for ~90% of esophageal cancer cases worldwide and predominates in eastern Asia and Africa3. Although the 5-year survival rate of patients with esophageal cancer has improved in the past decade, this cancer still has a very poor prognosis because patients generally do not show clinical features before an advanced stage unless via screening. Stage III tumors invade through the muscular layer and involve lymph nodes or other adjacent structures3. The 5-year survival rate after esophagectomy can benefit from lymph node removal, and the depth of invasion and presence of node metastasis are important predictors4. Thus, a better understanding of molecular dependencies and vulnerabilities during esophageal carcinogenesis and its metastasis to lymph nodes is urgently needed to develop promising diagnostic and therapeutic strategies.Esophageal cancer has two main subtypes, esophageal squamous cell carcinoma  and esophageal adenocarcinoma (EAC)5. The leading causes of aberrant transcriptional patterns are genetic alterations and the deregulated signaling pathways and epigenomic traits, including DNA methylation, histone modification, and other cis-regulatory elements6. Multiple studies have highlighted the importance of histone modifications in the establishment and maintenance of disease and cancer statuses8, and super-enhancers (SEs), comprising clustered enhancers, are considered to be the key controllers of different cell types and disease conditions9, associated with disease-specific genetic variants and mutations10. We and others recently proposed that key transcription factor (TF)-driven reprogramming of enhancers, which are marked by histone H3 lysine 27 acetylation (H3K27ac) and localized distal to promoters and transcription start sites (TSSs), promotes carcinogenesis and metastasis13.An aberrant and reprogrammed transcriptome is a universal hallmark of human cancers that is associated with deregulated cell proliferation, invasion, and metastasisMSR1, ASCC1, CTHRC1, CRTC1, BARX1, FOXP1, TP53, CDKN2A, SMAD4, and NFE2L2, have been identified in Barrett’s esophagus and adenocarcinoma patients18. Meanwhile, some familial form of ESCC and a number of susceptibility loci in ESCC patients as well some genetic drivers  have been reported22, while the complexity of carcinogenesis might be more than just genetic alterations. In recent years, the epigenomic profiling of tumor-specific promoter, enhancer, and super-enhancer landscapes during carcinogenesis has been widely used to elucidate the molecular mechanisms underlying the dysregulated local and regional expression of oncogenic genes in several cancers, such as colon cancer, gastric cancer, renal cell carcinoma (RCC), and ependymoma27. Although global histone modifications have been linked to the evolution of distant metastasis during pancreatic and prostate cancer progression29, the genome-wide epigenomic landscape of ESCC tumorigenesis and metastasis is poorly understood.Several familial genetic risk mutations in trans-elements within altered enhancers, we discover several key TFs regulating esophageal cancer cell proliferation and migration. By performing an integrated analysis of SE-associated genes with drug interaction databases, we identify some promising oncotargets that are responsive to pharmacologic inhibition. These findings indicate that the identification of variant enhancers and SEs is of great help for illustrating core transcriptional regulatory circuitries and discovering therapeutic targets for esophageal and LNCs.In this work, to address the role of enhancer dynamics during ESCC progression and metastasis to lymph nodes, we characterize the enhancer and SE landscapes in primary ESCC and lymph node cancer (LNC) tissues as well as normal esophageal epithelium. We identify a large set of commonly altered enhancers and LNC-specific enhancers associated with regional gene expression that can be used for precise subclassification. By analyzing the cis-regulatory elements in esophageal carcinogenesis and metastasis, we generated 28 active histone mark (H3K27ac) profiles from ten paired ESCC patients by chromatin immunoprecipitation and sequencing (ChIP-seq) with freshly dissected samples of adjacent normal esophageal (Nor or Adj) tissues, primary ESCC (further abbreviated EC herein), and LNC tissues. In addition, a total of 50 transcriptomes  were identified with DESeq2  with the Epigenomics Roadmap dataset31 and found an overlap rate of ~90%  in H3K27ac reads per kilobase per million reads (RPKM) of ≥2 and an absolute difference of ≥0.5. Considering the large variation between patients and samples, we evaluated the distribution of altered elements in tumor or LNC/normal pairs. Approximately 80% of the altered enhancer regions were significantly different  at the threshold of ≥6/10 patients (EC vs. Nor) , G2 , G3 , G4 , G5 , and G6   in the promoter analysis and genes with negative PC1 scores  in the enhancer analysis were considered potential oncogenes promoting carcinogenesis  Fig. . Unsuper15) Fig. . Similar15) Fig. . Among tConsidering that the epigenetic discrepancy among the three groups in distal regions, the epigenetic features of which were more dynamic among multiple patients, was much more stable than that in proximal promoter regions  or repression (EHF)  from other cancers34 in our unbiased profiling of H3K27ac enrichment further supports the reliability of our data.Relative to the normal esophageal epithelium, 392 (46.3%) and 494 (52.1%) cancer-specific SEs were recovered in the EC and LNC H3K27ac profiles, respectively, and these SEs were enriched with cancer-associated genes or oncogenes, such as HF) Fig. , 7. Our 9, we assigned predicted enhancers to nearby genes with the nearest TSSs, and then analyzed the expression of differential enhancer-linked genes in the six groups  and G6  groups, although the expression of some genes was not consistent with the patterns of their nearby H3K27ac marks  without considering the pairing of patients and the threshold of ≥6/10 patients used for differential enhancer identification ; for example, the H3K27ac level in an EHD3 enhancer was highly correlated with its expression  , g2 , g3 , and g6  with region-specific enhancer and transcript enrichment , EC (g1+ g2− g3+ g6−), and LNC (g1− g2+ g3+ g6−) tissues  or pre-silenced  before transcriptional alterations in EC samples, and considered that these enhancers or their associated genes might be the priming factors for the initiation of cancer metastasis-related processes  and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that gained enhancers in EC and LNC were enriched in essential cancer processes, such as cell proliferation, motility, and migration, and were associated with genes involved in proteoglycans, chemokine signaling, transcriptional misregulation, Rap1 signaling, and other pathways in cancer  among multiple patients  may predominantly mediate cellular responses to cytokines, such as TGFβ signaling, to promote tumourigenesis and metastasis.GO analysis showed that the SEs in the EC and LNC cohorts were enriched in genes involved in the regulation of metabolic processes, signal transduction, responses to cytokines, cell proliferation, cell migration, and cell mobility, and the enrichment was more significant than that of the GO terms predicted from the top 2000 typical enhancers with the strongest average H3K27ac signals Fig. . Because26 to map gained or lost SEs , as well as many lost SE-linked genes  involved in epithelial development, morphogenesis, and developmental processes  or lost  SEs overlapped with the results shown in Fig. n = 456) (Supplementary Data MEIS1 and CXCR4)  that were activated in metastatic cancers in two of the three cancer types  , ZEB1, TCF3, and NFE2L2 led to a modest decrease in cell growth , SOX12 (weak but significant), and IRF2 resulted in impaired cell migration  for each target were used to investigate the functional roles of altered enhancer-predicted top-ranked TFs by efficiently knocking down the corresponding TFs , with individual low efficient shRNAs  Fig. . Then, cwth Fig. . Wound hion Fig. . ConsistZXDC, CTNNB1, CXCR5, CCND2, STAT3, and SNAI1, were bound by IRF2, RXRA, RELA, or ZEB1  was twofold higher than that of HSP90AA1-high patients  , which were analyzed from a set of Hi-C data in fetal lung fibroblasts (IMR90)11, with some predicted TFs binding sites  upon TF depletion, demonstrating that the knockdown of some key TFs, which may mediate SE gain, resulted in the downregulation of ANO1, HSP90AA1, or PMEPA1 expression  or weak  suppressive effects on cell migration Fig. . Furtheroup Fig. , validat/9) Fig. . Althougcer Fig. . Considecer Fig. . These d4. Therefore, for this kind of clinically heterogeneous disease, understanding its epigenomic deregulation signatures has emerged as a pivotal strategy to identify biomarkers, vulnerabilities, and therapeutic targets for this clinically heterogeneous disease23. By profiling histone H3K27ac modifications in paired normal, primary, and lymph node tumor pairs, we generated a comprehensive compendium of promoter and enhancer alterations in ESCC.Esophageal cancer is a severe disease with high mortality, accompanied by poor prognosis and frequent metastasis to lymph nodes; in particular, its survival rate is inversely correlated with its metastasis and recurrence. In addition to surgery and chemotherapy, very few chemical drugs targeting biological molecules are clinically approved17 and provides a valuable resource of epigenomic information for investigating cancer metastasis. Similar to a recent report regarding DNA methylation alterations in ESCC43, the significance of our work was partially limited by the limited sample size; a larger number of patients may help to dissect more clinical and therapeutic highlights, such as clinical subgrouping and personalized treatment, as reported previously in EAC44. Second, we show that the epigenomic and transcriptomic profiles of LNC are quite similar to those of primary tumors, with some LNC-specific characteristics. It suggests that targeting the common gained enhancer-linked oncogenic targets in primary and metastatic tumors, such as HSP90AA1, is a potential strategy for ESCC therapy. In addition, the blockade of LNC-specific enhancer-linked genes may specifically inhibit cancer cell metastasis. It demonstrates that epigenomic landscapes, which might be more stable than transcriptional differences35, are able to dissect the molecular differences between similar tumor entities. Third, we demonstrate that variant enhancer and super-enhancer reprogramming contributes to transcriptional remodeling, which is further emphasized by the reverse engineering of core transcriptional regulatory circuitries functionally involved in tumor cell proliferation and migration  profiling will be quite helpful for precisely separating active promoter- and enhancer-enriched H3K27ac signals in promoter regions26. Therefore, we focused mainly on the distal enhancers outside promoter regions, which account for the majority of enhancer-linked cis-regulatory elements47. The identification of promoters herein using H3K27ac alone was not accurate, so we defined TSS ±2 kb regions with significant H3K27ac occupancy as active promoter elements  but not as active promoters. Considering the neighboring occupancy between enhancer-marked H3K27ac and H3K4me3 (tri-methylation) around TSSs, the number of our identified active promoter elements was comparable to that of H3K27ac + /H3K4me3 + -defined active promoters within TSS ±2 kb regions reported in another work26, indicating similarities between active promoter elements and active promoters.In recent years, epigenomic studies regarding promoter alterationsles Fig. , similar48. To build a perfect distal interaction model capable of identifying enhancers and paired target genes within a TAD, it will be meaningful to generate ESCC-specific Hi-C data; alternatively, integrating the altered enhancers or SEs with data from interaction datasets, such as PreSTIGE30, GREAT31, and RNAPII ChIA-PET, is another feasible strategy23 to accurately map the functional distal enhancers. Additionally, we identified a subgroup of genes, with delayed expression changes after H3K27ac alterations ues Fig. . We alsoues Fig. . Common ues Fig. , our surtrans-element prediction using gained enhancers . KYSE30 and KYSE150 cells were gifts from Dr. Zhihua Liu’s lab . All cells were tested without mycoplasma contamination. TE1 cells were authenticated by Cell Bank  using STR profiling. KYSE30 and KYSE150 cells were authenticated by Dr. Zhihua Liu’s lab using STR profiling. HEK293T cells were maintained in DMEM supplemented with 10% FBS (Gibco), and TE1, KYSE30, and KYSE150 cells were maintained in RPMI 1640 medium supplemented with 10% FBS (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified incubator with 5% CO2. Chemical inhibitors, including AH, palbociclib, endovion, SB297006, navitoclax, ML-030, hydroxyfasudil, and IT1t dihydrochloride (IT1t), were purchased from MCE, and the powders were dissolved according to the manufacturer’s instructions.https://www.sigmaaldrich.com/life-science/functional-genomics-andrnai/sirna/mission-predesigned-sirna.html), and two shRNAs were constructed for each TF. The shRNA sequences are provided in Supplementary Table shRNAs targeting TFs predicted from gained enhancers were constructed into the lentiviral vector pLVX-shRNA2 (Clontech), and an shRNA targeting luciferase coding genes was used as a control. shRNAs were designed with an online tool  every day until day 6. For chemical treatment, inhibitors were added to the medium 24 h after cells were seeded, and the cell number was determined at 72 h. Three wells were counted, and the average number of live cells was designated as a single replicate for each sample; each experiment was repeated three times.TE1 cells were plated into 24-well plates at 50,000 cells per well and treated with the indicated chemical drugs or DMSO after attachment. Cell apoptosis was detected using a Cell Cycle and Apoptosis Detection Kit . After cells were treated for three days, they were trypsinized, centrifuged, washed, and fixed with cold 70% ethanol at 4 °C overnight. Then, the fixation solution was removed, and the cells were incubated at 37 °C for 30 min in a solution containing propidium iodide and RNase A. Then the treated cells were analyzed using a FACScan flow cytometer  and 10,000 cells were collected per sample. The data were analyzed using FlowJo 10.0 software  with two or three replicates per sample, and each sample was analyzed independently three times.Mock TE1 cells or cells expressing indicated shRNAs were cultured in 12-well plates, and a wound was made in the confluent cell monolayer with 1000 µl pipette tip. Then, the wound width was monitored every 24 h using an inverted phase microscope. The relative cell migration rates were analyzed relative to the wound width at the time of initial wounding, with three replicates per sample.6 TE1 cells. The tumor volume was monitored every 2–3 days. The tumor volume was calculated as the length x width x width x 0.52; tumors were weighed after the mice were sacrificed on day 21. For evaluating the effects of AH and ML-030 on tumor growth, a total of 1 × 106 cells were injected subcutaneously into nude mice (n = 5 for each group). Mice injected with TE1 cells were treated with AH (2.5 mg/kg every time) or ML-030 (2.5 mg/kg every time) every 3 days starting on day 2 after cell implantation. Tumor volumes were determined on days 4, 8, 14, and 21. On day 21, the tumors were collected from sacrificed mice for image acquisition and tumor weight determination. For detecting the metastasis of tumors in vivo, KYSE150 cells stably expressing firefly luciferase (Luc) cDNA was established by using lentiviruses. A total of, 1 × 105 KYSE150/Luc cells were intramuscularly injected into the neck region to mimic esophagus cancer. Four weeks postinjection, in vivo luciferase imaging was conducted upon intraperitoneal injection of beetle luciferin  (0.2 ml of 15 mg/ml) using an IVIS Spectrum equipment . For administration of chemical inhibitor, ML-030 (2.5 mg/kg) or was intraperitoneally injected every 3 days starting from the day after tumor cell implantation.Animal studies were conducted according to animal protocols approved by the local Ethics Committee of Shanghai Chest Hospital Affiliated with Shanghai Jiaotong University. We complied with all relevant ethical regulations for animal experiments. Male BALB/c nude mice (6 weeks old) were implanted with 1 × 10PPL, SIM2, TRIOBP, VPS37B, THSD4, SLC7A1, AMOTL2, ZBTB7A, AHDC1, ZSWIM4, PHYHD1, PSCA, CCDC85C, PAQR7, ST3GAL4, EHF, TFAP2B, EPHA2, MINK1, RREB1, SMURF1, ESYT2, ZBTB7C, ATP8B1, RFX2, TMEM105, TMEM211, HOXA2, ATP5D, and WASF2; loss: RELT, IL4R, CCR3, MMP25, ADAM8, ADGRE5, THEMIS2, EPPK1, SLC7A5, NCF2, CD44, LDHA, CTSD, TMSB10, IER5L, FCGR3A, FCGR2A, SOCS3, NNMT, SPHK1, ITGA5, SIRPA, LMNA, DAD1, RPL35, ZC3HAV1, AHSA2, TBCD, IRX5, and HRAT92) were used for survival analysis. Log-rank tests were applied to examine the significance of differences between Kaplan–Meier curves (Log-rank P < 0.05 was considered significant). GraphPad Prism 8 was used for analyzing the 2-year survival rate using our clinical data from 18 donors. For analysis of the correlation between ESCC and HSP90AA1, GEPIA (http://gepia.cancer-pku.cn/) was used for disease-free survival (DFS) analysis.Kaplan–Meier survival analysis was employed to analyze the overall survival probability with default parameters. To analyse survival related to altered SE-linked genes, because the maximum number of genes is limited with this tool, only the top altered SE-associated genes (gain: 2O (1:10) and amplified using SYBRGreen PCR Master Mix . Relative gene expression levels were normalized to those of GAPDH. All relative quantification results were obtained from three independent experiments, and the primer sequences are listed in Supplementary Table Total RNA was isolated using TRIzol reagents (Invitrogen), and 1 µg of RNA was reverse transcribed using a Superscript III First-Strand Synthesis Kit . Then complementary DNA was diluted with ddHTotal RNA was extracted from fresh samples using TRIzol reagent. RNA-seq libraries were constructed using Stranded Total RNA Sample Prep Kit  according to the manufacturer’s instructions, and 500 ng of total RNA was used for library construction using a TruSeq™ RNA Sample Prep Kit v2  according to the manufacturer’s protocol. Sequencing was performed using the 150 bp pair-end read protocol .63. Fresh tissues obtained during surgery were collected and digested into single cells or small cell aggregates, and ~1 × 105 cells were fixed with 1% formaldehyde for 10 min in PBS at room temperature. Then, fixation was terminated with 125 nM glycine. After washing with cold PBS three times, the cells were centrifuged for storage at –80 °C. When a batch of samples was collected, the stored samples were subjected to ChIP experiments. Generally, cells were lysed in 100 µl of lysis buffer and sonicated for 15 cycles . Then, 5% of the lysate was resolved for genomic DNA purification as input DNA, and 95 µl of the lysate was diluted to a volume of 1 mL. Through the ChIP experiments, samples were supplemented with a protease inhibitor. Then, 2 µg of an anti-H3K27ac antibody  was added to the diluted lysis buffer and mixed at 4 °C for 2 h. Protein G Dynabeads  were precleaned with wash buffer three times, and then a 20 µl volume of beads was added to the mixture and incubated overnight at 4 °C. Subsequently, the beads were washed with wash buffer five times and ChIP DNA was recovered for quantification. ChIP and input DNAs were subjected to library preparation with NEBNext ChIP-seq Library Pre Reagent . Each library was sequenced to an average of 30 million raw reads on the X10 sequencing platform .ChIP-seq was performed as previously described64 with the following parameters:–twopassMode Basic–sjdbOverhang 149–outFilterMultimapNmax 20–alignSJoverhangMin 8–alignSJDBoverhangMin 1–outFilterMismatchNmax 999–outFilterMismatchNoverLmax 0.1–alignIntronMin 20–alignIntronMax 500000–alignMatesGapMax 1000000–outFilterScoreMinOverLread 0.33–outFilterMatchNminOverLread 0.33–outSAMstrandField intronMotif–chimSegmentMin 15–chimJunctionOverhangMin 15–chimOutType WithinBAM SoftClip–chimMainSegmentMultNmax 1 and other default parameters. Transcript abundances at the gene level were calculated as TPM values using RSEM65. Transcripts with a count per million (cpm) of >1 in at least 18 samples were retained for subsequent analysis, and 16,173 genes passed this filtering criterion. The TPM values were further normalized through Z-score transformation when presented in heatmaps.RNA-seq reads were aligned to the human genome (hg19) using STAR-2.6.1n = 340 samples; GSE71729) and CA  were downloaded from the NCBI Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/)67. ChIP-seq data for eight crucial TFs were downloaded from ENCODE, and detailed information about these datasets is provided in Supplementary Data Independent public RNA-seq datasets of PDA  for subsequent analyses. Significant H3K27ac peaks were called by using MACS2 (2.1.1.20160309) with all default parameters except -p 1e-9 and -f BAMPE70. Bigwig files were generated from BAM files using deepTools71 with the following parameters: -binSize 50 -extendReads 200 and–normalizeUsing RPKM. The signal intensities of each ChIP library were scored against the corresponding input library, respectively. The input-subtracted peak signal within a region was measured as the RPKM value using bigWigAverageOverBed. The RPKM values were further normalized through Z-score transformation when presented in heatmaps.ChIP-seq raw reads were mapped to the human reference genome (hg19) using Bowtie2 (version 2.3.4)https://www.ncbi.nlm.nih.gov/geo/roadmap/epigenomics/) under accession numbers GSM906393 and GSM1013127. Similarities between the epigenome roadmap and our H3K27ac ChIP-seq data were assessed by determining the percentage of overlapping peaks relative to our H3K27ac peaks using BEDtools72.Two publicly available H3K27ac peaks from normal esophageal tissues were downloaded from the NIH Roadmap Epigenomics Project Data -adjusted P value of <0.05 were considered to be significantly differentially expressed.Differential gene expression analysis between groups was performed with the DESeq2 R package75.PCA was performed on the altered promoter elements or enhancers or all SEs using the first two PCs of the H3K27ac signals, and the cumulative variance and the proportion of variance accounted for each PC were computed using the FactorMineR R packagehttps://github.com/bmbolstad/preprocessCore) to reduce potential batch effects. SE regions were identified using Rank Ordering of Super-Enhancers (ROSE)33 from merged enhancers or individual enhancers from each sample with default parameters.We defined active promoter elements as peaks located completely within ±2.0 kb of TSSs, which were excluded from enhancers. Furthermore, the H3K27ac peaks were merged into integrated but nonoverlapping peak sets across all samples . Quantile-normalization was performed using the R package preprocessCore , were applied to identify gained or lost regions. The track of differential regions were visualized using Integrative Genomics Viewer (IGV)n = 1–28) was stated, and as the number of samples increased, the number of identified regions tended to be stable. The same methods were used to analyze altered enhancers and promoter elements.Saturation analysis was performed for all enhancers and promoter elements. Specifically, the possible frequency of enriched regions in all samples  previously obtained in IMR90 cells [ENCFF307RGV]. The Hi-C maps of regions around the TADs covering the http://geneontology.org/ and performed KEGG pathway analysis using DAVID (http://david.abcc.ncifcrf.gov/home.jsp). The terms and pathways with an adjusted P value < 0.05 were considered significantly enriched.We performed GO enrichment analysis to identify key terms enriched in genes associated with recurrent enhancers or predicted SEs http://dgidb.org/search_interactions); the interactions between genes and drugs are presented in Supplementary Data 25.Common gained and LNC-specific gained super-enhancer-associated genes were used to query the Washington University Drug Gene Interaction database (t-test. Differences were considered significant when *P < 0.05, **P < 0.01, or ***P < 0.001 without statements. For analyzing differences between gained and lost enhancers or linked genes, the cut-off values are presented within the figures. For cell growth, wound healing rate, and apoptosis analysis, two or three independent experiments were performed, and similar results were obtained.The results were obtained from three independent experiments and are presented as the mean ± s.d. values. For analyzing relative expression levels as FPKM values, the FPKM values from RNA-seq data were used and are presented as the mean ± s.d. values. All original data presented in the main figures or supplemental figures are provided in the Source Data file. Each treatment was performed in triplicate, and the values for the individual treatments were averaged and normalized to those for the control group. Statistical analyses and plotting were carried out using GraphPad Prism 8.0. Comparisons of mean values were performed with Student’s unpaired Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Supplementary Data 9Supplementary Data 10Supplementary Data 11Supplementary Data 12Supplementary Data 13Supplementary Data 14Supplementary Data 15Supplementary Data 16Supplementary Data 17Supplementary Data 18Supplementary Data 19Supplementary Data 20Supplementary Data 21Reporting Summary


Kaempferia parviflora extracellular vesicles (KPEVs) were isolated by differential and sucrose density gradient centrifugation, and their size, morphology, and surface charge were characterized using transmission electron microscopy and dynamic light scattering. The biological properties of KPEVs, including their bioactive compound composition, gastric uptake, cytotoxicity, acid tolerance, and storage stability, were also examined. In addition, KPEVs had an average and uniform size of 200–300 nm and a negative surface charge of 14.7 ± 3.61 mV. Moreover, 5,7-dimethoxyflavone, the major bioactive compound of KP, was packaged into KPEVs. Meanwhile, KPEVs were resistant to gastric digestion and stably maintained at −20°C and −80°C for 8 weeks with no freeze-thaw cycle. The lipid hydrolysis during EVs storage at room temperature and 4°C were also demonstrated for the first time. Furthermore, the labeled KPEVs were internalized into adenocarcinoma gastric cells, and the cell viability was reduced in a dose-dependent manner, according to the results of the thiazolyl blue tetrazolium assay. Our study supports the potential application of KPEVs as a vehicle for anticancer or oral drugs.Plant-derived extracellular vesicles (EVs) are a promising candidate for nanomedicine delivery due to their bioactive cargos, high biocompatibility to human cells, biodegradability, low cytotoxicity, and potential for large-scale production. However, the research on EVs derived from medicinal plants is very limited. In this study,  Oral drug administration is the main route for administering drugs for various infectious and noninfectious diseases due to its noninvasiveness, convenience, and high patient compliance. However, drug bioavailability depends on many factors, such as stability in a highly acid environment pH 1.2–2.2), the solubility of a drug in the gastrointestinal tract, absorption efficiency, and permeability through the mucus membrane barrier [.2–2.2, tvia the blebbing, budding, and shedding of plasma membranes or exocytosis of multivesicular bodies [In recent years, it has been found that plant-derived extracellular vesicles (PDEVs) can potentially help overcome such limitations. PDEV biogenesis naturally occurs r bodies . Thus, tr bodies .in vitro and in vivo [via the oral route, than other synthetic nanoparticle-based therapy [Moreover, PDEVs offer health benefits. For example, sulforaphane-containing EVs from broccoli can prevent intestinal inflammation by activating dendritic cell AMP-activated protein kinase . In addi in vivo . Further in vivo . Therefo in vivo . Additio therapy .Therefore, PDEVs represent a novel therapeutic strategy acting as nanodelivery vehicles. For example, grape- and grapefruit-derived EVs can migrate through the intestinal barrier and be absorbed by intestinal stem cells in mice. In addition, the results of treatment with these PDEVs showed a lower mortality rate and prevention of dextran sulfate sodium (DSS)-induced colitis progression in mice , 9. MoreKaempferia parviflora (KP) has been used in traditional medicine since ancient times. A crude KP extract exerts various biological and pharmacological effects, including antiinflammatory, antioxidant, anticancer, and antibacterial activities [H. pylori, the causative agent of gastric cancer. Moreover, KP extracts also exerted anti-internalization activity on H. pylori in Hep-2 cells [H. pylori-infected gastric cells [While most PDEVs studies have been documented in fruit and vegetable sources, such as ginger, grape, grapefruit, and broccoli , only a tivities –15. The tivities . In our -2 cells , 18 and ic cells . Lastly,ic cells .This study aimed to characterize the physical and biochemical properties and bioactive compounds of KPEVs. In addition, the factors affecting the potential application of KPEVs for oral drug delivery, including stability in acidic pH, cellular uptake, and cytotoxicity, were examined in gastric cells. The insights from this study will be useful for applying KPEVs in drug encapsulation for therapeutic purposes.™)  were cultured in RPMI 1640 supplement with 10% (v/v) fetal bovine serum  at 37°C in 5% CO2 and 80% humidity. After the cells reached 80%–90% confluency, they were harvested using 0.25% EDTA-trypsin  and subcultured.Adenocarcinoma gastric AGS (CRL-1739K. parviflora (KP) rhizomes were purchased from a local farm in Khao Kho, Phetchabun, Thailand (voucher number ES280306). The KPEVs were isolated according to the method of Sung et al. [™ Legend™ XT/XF Centrifuge, Fiberlite™ F14-6x250 rotor) to remove fiber and cell debris. The resultant supernatant was then pelleted by ultracentrifugation at 150,000 × g for 2 h . Afterward, sucrose gradient centrifugation (8%/30%/45% sucrose) was performed at 150,000 × g for 1.5 h at 4°C . Finally, the KPEV pellets were resuspended in 1X PBS  through mild ultrasonic dispersion, pooled, filtered with a 0.45-μm filter, and stored at −80°C until use.g et al.  with modTotal protein content was measured and used to indicate the amount of KPEVs. Protein concentration (μg/ml) was determined using the Bradford method with a Bio-Rad protein quantification assay kit  and measured the absorbance at 595 nm using a microplate reader . The KPEV samples and bovine serum albumin (BSA) standards were measured at least in triplicates. The concentration of the KPEV protein was calculated using the BSA standard curve. The final yield was calculated and reported as KPEV total proteins per fresh KP rhizome weight (mg/kg).The morphology of the KPEVs was observed using TEM. Ten microliters of KPEVs were placed on a carbon-coated copper grid and incubated for 2 min. Then, the KPEVs were negatively stained with 1% uranyl acetate for 3 min. After the grid was air-dried for 10 min, it was imaged using a JEM-1400 electron microscope  at an accelerating voltage of 100 kV.The KPEVs were diluted in 1X PBS buffer and analyzed using dynamic light scattering with a Zetasizer Nano ZS  and a He–Ne laser light source at 633 nm at 25°C. The scattered light was detected at 173° and 13° angles to measure dynamic light scattering (size) and electrophoretic mobility (surface charge), respectively. Each sample was measured 3 times with 30 runs per measurement.® reverse phase column C18 columns (4.0 × 250 mm) , with a methanol:water (70:30 v/v) buffer used as the mobile phase at 40°C and a flow rate of 1.0 ml/min. The DMF was detected by UV-VIS at 210 nm. The HPLC fingerprint of KPEVs lysate was compared with that of the standard DMF and quantified by plotting peak areas against DMF standard concentrations.The KPEVs were lysed, dissolved in 100% methanol at 4°C overnight, and subsequently identified and quantified under conditions according to those reported by Malakul et al. . BrieflyThe shelf life and storage temperature of the KPEVs were assessed with different storage durations at 0, 1, 2, 4, and 8 weeks and different temperatures at 4°C, −20°C, and −80°C. Then, the physical properties, including size and zeta potential, of the KPEVs were examined. In addition, lipid hydrolysis in the PDEV membranes was investigated by measuring free fatty acid content, octanoate (C-8) and longer FFA, with a free fatty acid quantification assay fluorometric kit . The free fatty acid contents were calculated based on the palmitic acid standards.The freeze-thaw stability of the KPEVs was examined after freezing them at −80°C and thawing them at room temperature for 1 to 3 cycles. The physical stability of the KPEVs was analyzed by measuring their size and zeta potential.KPEVs were incubated in an HCl/pepsin solution, pH 1.2 , a simulated gastric buffer, in slow rotation at 95 rpm at 37°C for 1 h . Then, t100 μg/ml were incubated with 3 μg/ml FM4-64 at 4°C in the dark for 1 h. Then, the labeled KPEVs were washed with 1X PBS, transferred to 10-kDa MWCO spin column , and centrifuged at 10,000 × g for 15 min at 4°C to remove the unbounded FM4-64.The conditions of KPEV uptake by gastric cells was modified from those reported by Yang et al.  and asse4 cells/well and cultured in a complete medium for 24 h. First, each coverslip was washed with 1X PBS with Ca2+, Mg2+  before 10 μg/ml labeled KPEVs or the RPMI 1640 control medium were added to the test and control wells, respectively, and incubated for 6, 12, and 24 h at 37°C. Next, the cells on the coverslips were washed with RPMI 1640, fixed with 1% paraformaldehyde, and nuclear stained with DAPI . The uptake of the labeled KPEVs by the gastric cells was visualized using a confocal laser scanning microscope  at 515/640 nm (FM 46–4) and 358/461 nm (DAPI). In addition, the localization of the KPEVs in the cells was observed by 2D imaging in the z-stack mode.Before labeling, AGS cells were seeded on sterile cover slips placed in a 24-well plate at 8 × 104 cells/well and incubated in a complete medium (RPMI with 10% FBS) without antibiotics for 24 h. The cells were then washed with 1X PBS with Ca2+, Mg2+  and incubated at 37°C with 5, 10, 30, 50, or 100 μg/ml KPEVs for 24 h. Then, 5 mg/ml thiazolyl blue tetrazolium bromide (MTT) was added to each well for a 4-h incubation at 37°C in the dark. Next, the cells were permeabilized in 100 μl of 10% (w/v) SDS overnight to solubilize the intracellular formazan crystals. Lastly, the absorbance of the cells was read at 570 nm, and the percentage of viable cells was calculated compared to the untreated control cells.AGS cells were seeded in a 96-well plate at 1 × 10The experiments were performed in at least triplicates, and the data are presented as a mean ± standard deviation (SD). Graph plotting and statistical analysis were performed using GraphPad Prism 9 . The difference between the control and test groups was analyzed using one-way analysis of variance (ANOVA) followed by the Bonferroni test. The effects of the storage temperatures on the KPEVs over time were analyzed using two-way ANOVA followed by the Bonferroni test. In addition, p-values of less than 0.05 were considered statistically significant.The KPEVs were isolated from KP juice by ultracentrifugation and sucrose density gradient. Two EV populations were observed . Most EVCorresponding to the TEM imaging data, the dynamic light scattering data showed the average size of the KPEVs was 256.8 ± 14.81 and 346.8 ± 37.43 nm was for the lower and upper bands, respectively . In addiAs the quantity of bioactive compounds in KPEV nanoparticles is significantly lower than that of KP crude extracts, only 5,7-dimethoxyflavone (DMF) the most crucial KP rhizome active medicinal compound, was identified and quantified compared to a reference standard. According to the HPLC results , the KPEThe size, PDI, and zeta potential of the KPEVs were not significantly changed until after 8 weeks of storage at 4°C, −20°C, and −80°C . PDI of The KPEV acid resistance mimics stomach conditions, assessed at 37°C for 1 h by suspending and rotating the KPEVs in a stimulated gastric buffer, pH 1.2 compared to 1X PBS, pH 7.0. After acid digestion, the size of the KPEVs slightly enlarged as their diameter shifted from 250.00 ± 10.00 nm (pH 7.0) to 268.40 ± 37.73 nm (pH 1.2), and their PDI value increased from 0.25 ± 0.02 (pH 7.0) to 0.45 ± 0.08 (pH 1.2) . In addiFM4-64, a lipophilic fluorescence dye that stained KPEV membranes red , was use50 of 68.29 ± 11.44 μg/ml.The cytotoxicity of the KPEVs to the gastric cancer cells was examined by coculturing AGS cells with various concentrations of KPEVs for 24 h. At the lowest concentration of 5 μg/ml, the KPEVs did not affect cell viability (84.90% ± 5.10%). In contrast, higher KPEV concentrations at 10, 30, 50, and 100 μg/ml significantly decreased cells viability to 78.76% ± 7.76%, 64.84% ± 6.66%, 52.90% ± 9.41%, and 41.00% ± 8.30%, respectively . TherefoPlant-derived extracellular vesicles have promising therapeutic applications, especially in drug nanodelivery systems . EVs froKaempferia parviflora (KP) rhizome, or black ginger, is a traditional remedy for gastrointestinal disorders. KP potions are prepared by decoction and consumed to relieve excessive flatulence and gastric ulcer [ic ulcer . KP rhizic ulcer –15, 27. ic ulcer , 28. Thiic ulcer , 30 and ic ulcer .Here, the yield of our KPEVs was higher than the yield from animal samples and mammalian cells culture medium . In addithe Zingiberaceae family, share similar physical properties to our KPEVs [In this study, purified KPEVs have a low PDI value, exhibiting an acceptable size homogeneity for lipid-based nanoparticles . In addiur KPEVs , 36. Theur KPEVs , 10, 39.2 fluid extracts (SFEs) of KP contained the highest concentration of 5,7-dimethoxyflavone, showing more potent antiproliferative activity against HeLa and human gastric adenocarcinoma AGS cell lines [Our study was the first to present the analysis of the KPEV cargo, revealing a highest content of 5,7-dimethoxyflavone (DMF), the main methoxyflavone in the KP rhizome . Similarll lines . The amoll lines . Thirteell lines . Other mll lines . TherefoH. pylori infection [in vitro digestion assay shown that KPEVs were stable under gastric-like conditions as their size was only slightly enlarged and no effect to structural integrity. Under the same condition, the surface charge of the KPEVs was neutralized to a weak positive charge by the hydrogen ions in gastric acid (HCl). Our data is consistent with the finding by Mu et al. that edible plant-derived exosome-like nanoparticles can resist gastric pepsin solutions, exhibiting altered sizes and a remarkable reduction in negative charge in a stomach-mimicking solution [The KP bioactive compounds possessed a remarkable ability to treat gastrointestinal tract disorders, as evidenced by reducing the size of the gastric lesion, enhancing mucosal protection, exerting antiinflammatory effects, and inhibiting nfection , 18, 19.solution . TherefoThe critical challenge in applying PDEVs is their stability in different storage temperatures, durations, and freeze-thaw cycles. Most plant crude extracts, or EVs are generally kept at −20°C or −80°C, which would stably maintain their biological activities . KPEVs mEV stability has only been explored in mammalian cells. Munagala et al. has reported that bovine milk-derived exosomes are stably maintained at −80°C for up to 6 months and partially degraded at 4°C . Our datAlthough the lipid composition of PDEVs is different from animal and mammalian cells, we confirmed that labeled KPEVs were internalized into gastric cells. The mechanism of PDEV internalization into mammalian cells remains unclear. However, it has been noted that endogenous phosphatidic acid on the PDEVs surface is attributed to the internalization by the endocytosis pathway . PhosphaIn this study, we demonstrated that KPEVs were nanoparticles enclosed with 5,7-dimethoxyflavone, a major KP bioactive compound. These KPEVs can be internalized into gastric cells and are resistant to gastric acid. In addition, our study is the first to describe the storage stability in PDEVs by documenting the physical characteristics and lipid degradation of our KPEVs. The KPEVs are best maintained until 8 weeks at −20°C and −80°C with a single freeze-thaw cycle. Moreover, the cytotoxic effect of KPEVs is related to their concentrations. Therefore, KPEVs provide an alternative approach for enhancing gastric cancer therapy as a drug delivery vehicle.