ObjectiveTo observe the effects of Danggui Shaoyaosan on macrophage polarization and renal inflammation in db/db mice with diabetic kidney disease （DKD）， and to explore its renal protective effects and underlying mechanisms. MethodsEight db/m mice were assigned to the normal group， and forty db/db mice were randomly divided into a model group， low-， medium-， and high-dose Danggui Shaoyaosan groups （8.39， 16.77， 33.54 g·kg^-1）， and an irbesartan group （0.025 g·kg^-1）. All mice were administered treatment by gavage for 12 consecutive weeks. General conditions of the mice were observed during the intervention. At the end of the 12-week intervention， 24-h urine samples were collected using metabolic cages， after which the mice were anesthetized for sample collection. Blood was collected by enucleation and centrifuged to obtain serum for the determination of glycated serum protein （GSP）， serum creatinine （SCr）， blood urea nitrogen （BUN）， total cholesterol （TC）， and triglycerides （TG）. The urinary albumin-to-creatinine ratio （UACR） was measured. Renal pathological changes were observed using hematoxylin-eosin （HE） staining， periodic acid-Schiff （PAS） staining， and Masson staining. Enzyme-linked immunosorbent assay （ELISA） was used to detect serum tumor necrosis factor-α （TNF-α）， interleukin-10 （IL-10）， and monocyte chemoattractant protein-1 （MCP-1） levels. Immunofluorescence （IF） was performed to detect F4/80 expression in renal tissue， and immunohistochemistry （IHC） was used to assess CD206 expression. Real-time quantitative polymerase chain reaction （Real-time PCR） was employed to measure the mRNA expression of TNF-α， IL-10， inducible nitric oxide synthase （iNOS）， and arginase-1 （Arg-1）. Western blot analysis was used to detect the protein expression of iNOS， Arg-1， CD86， and CD206 in renal tissue. ResultsCompared with the normal group， the model group showed increased levels of GSP， UACR， SCr， BUN， TC， and TG， elevated levels of the inflammatory factor TNF-α and the chemokine MCP-1， and decreased IL-10 levels （P<0.01）. Pathological examination revealed glomerular hypertrophy， mesangial cell proliferation with marked mesangial expansion， inflammatory cell infiltration， vacuolar degeneration of renal tubular epithelial cells， prominent glycogen deposition， and increased collagen fiber deposition. In addition， relative F4/80 fluorescence intensity was enhanced， CD206 expression in the glomeruli and renal interstitium was reduced， and TNF-α and iNOS mRNA expression was increased. IL-10 and Arg-1 mRNA expression was decreased， iNOS and CD86 protein expression was increased， and Arg-1 and CD206 protein expression was decreased （P<0.05， P<0.01）. Compared with the model group， the Danggui Shaoyaosan groups and the irbesartan group showed decreased levels of GSP， UACR， SCr， BUN， TC， and TG， reduced serum TNF-α and MCP-1 levels， and increased IL-10 levels. Renal pathological damage was improved to varying degrees. Relative F4/80 fluorescence intensity was reduced， CD206 expression in the glomeruli and renal interstitium was increased， and TNF-α and iNOS mRNA expression was decreased. IL-10 and Arg-1 mRNA expression was increased， iNOS and CD86 protein expression was reduced， and Arg-1 and CD206 protein expression was increased （P<0.05， P<0.01）. ConclusionDanggui Shaoyaosan can improve renal function and alleviate renal pathological damage in db/db mice. Its mechanism may be related to inhibiting M1 pro-inflammatory macrophage polarization， promoting M2 anti-inflammatory macrophage polarization， reducing inflammatory responses， delaying the progression of renal fibrosis， improving renal pathological injury， and thereby exerting renal protective effects.

OBJECTIVE To investigate the improvement effects and mechanism of Achyranthes bidentata total saponins （ABS） extract on vascular endothelial dysfunction in spontaneously hypertensive rat （SHR） based on cytochrome P450 4A （CYP4A）/20-hydroxyeicosatetetraenoic acid （20-HETE）/G protein-coupled receptor 75 （GPR75） axis. METHODS Ten Wistar- Kyoto rats were taken as the normal control group. Forty SHR were first stratified by systolic blood pressure and then， within each stratum， randomly assigned using a random-number table to the model group （MOD group）， captopril positive control group （CAP group， 10 mg/kg）， ABS low- and high-dose extract groups （ABS-L group， ABS-H group， 60 and 120 mg/kg）， with 10 rats in each group. Animals in each group were given the corresponding drug or equal volume of pure water by gavage， once a day， for 28 consecutive days. After the last administration， systolic blood pressure of rats was measured. The levels of vasoactive substances， inflammatory factors and oxidative stress indicators in serum were measured. The pathological changes of rat thoracic aorta were observed. The level of reactive oxygen species （ROS） in aortic tissue was analyzed. The expressions of endothelial nitric oxide synthase （eNOS）， CYP4A， GPR75， nuclear factor-κB p65 （NF-κB p65）， phosphorylated NF-κB p65， p22phox， and reduced nicotinamide adenine dinucleotide phosphate oxidase 4（NOX4） in thoracic aorta tissue were detected. RESULTS After 28 d of treatment， compared with MOD group， the systolic blood pressure of rats in the ABS-L and ABS-H groups decreased significantly. The levels of 20-HETE， angiotensin Ⅱ， interleukin-1β， interleukin-6， tumor necrosis factor-α， intercellular cell adhesion molecule-1 and malondialdehyde in serum were significantly reduced （P＜0.05 or P＜0.01）， while the levels of nitric oxide， superoxide dismutase， glutathione peroxidase and catalase were significantly increased （P＜0.05 or P＜0.01）. Intimal damage of thoracic aorta was reduced， and endothelial cell morphology was improved. The expressions of ROS， CYP4A， GPR75， p22phox， NOX4 and the phosphorylation level of NF-κB p65 protein in thoracic aorta were down-regulated or reduced （P＜0.05 or P＜0.01）， while the expression of eNOS was up-regulated （P＜0.05 or P＜0.01）. CONCLUSIONS ABS extract may alleviate the inflammatory response and oxidative stress in SHR effectively by down-regulating the expression of CYP4A， reducing the production of 20-HETE， inhibiting the activation of GPR75， and subsequently suppressing the activation of downstream NF-κB and NOX4， thereby improving hypertension-related vascular endothelial dysfunction.

Objective: To observe the clinical efficacy and safety of Yiqi Tongluo Decoction in the intervention of early traditional Chinese medicine (TCM) syndromes after ischemic cerebrovascular disease (ICVD) intervention. Methods: From October 2020 to July 2023, a randomized, double-blind, placebo-controlled study was conducted to include 60 patients with qi deficiency, blood stasis, and phlegm obstruction syndrome after ICVD interventional therapy. They were assigned to the Yiqi Tongluo Decoction treatment group (30 cases) and the TCM placebo routine treatment control group (30 cases) according to the randomized block design. Both groups received routine standardized treatment of Western medicine, including dual antiplatelet, lipid regulation, and control of risk factors for cerebrovascular disease. The treatment group was treated with Yiqi Tongluo Decoction based on the control group. The course of treatment was 60 days and follow-up was carried out 2 and 6 months after the operation. The improvement of qi deficiency syndrome, blood stasis syndrome, phlegm syndrome score and TCM syndrome score, modified Rankin score (mRS), Barthel index (BI) score, Fatty acid-binding protein 4 (FABP4) level, incidence of transient ischemic attack (TIA) and ischemic stroke (IS) and incidence of adverse reactions, Head and neck CT angiography (CTA) or digital subtraction angiography (DSA) examination were collected. The clinical efficacy of the patients 2 months after the operation was taken as the main outcome index to preliminarily evaluate the early and long-term efficacy of Yiqi Tongluo Decoction after the ICVD intervention. The early and long-term clinical efficacy and safety of Western medicine standardized treatment combined with TCM Yiqi Tongluo Decoction on patients with qi deficiency, blood stasis and phlegm obstruction syndrome after ICVD intervention were evaluated. The safety of Yiqi Tongluo Decoction in the treatment of patients after ICVD intervention with white blood cell (WBC), C-reactive protein (CRP), fibrinogen (FIB), plasminogen time (PT), recurrence of cerebral ischaemia and restenosis in patients at 2 and 6 months after treatment were evaluated. Results: Compared to the control group, the TCM syndrome scores for qi deficiency, blood stasis and phlegm syndrome in the treatment group reduced significantly, the clinical efficacy improved significantly, the mRS score and FABP4 were reduced, and the BI score was increased. Adverse events such as cerebral ischaemia were fewer in the treatment group than in the control group, but the difference was not statistically significant; levels of CRP, WBC and PT were reduced, and levels of FIB were reduced at 6 months post-treatment, all P<0.01, and images were intuitively compared. The treatment group was superior to the control group. Conclusion Yiqi Tongluo Decoction combined with Western medicine standard treatment can improve the early clinical efficacy of ICVD patients with qi deficiency, blood stasis and phlegm obstruction syndrome after interventional surgery, improve neurological impairment and daily living ability, reduce the state of qi deficiency syndrome, blood stasis syndrome and phlegm syndrome after interventional surgery, and improve the clinical efficacy of TCM. At the same time, it can reduce the level of FABP4, the target of atherosclerosis and restenosis after interventional surgery, reduce the level of inflammation after interventional surgery in patients with ICVD, regulate coagulation function, and reduce the incidence of long-term recurrence of cerebral ischemia after interventional surgery, with good safety.

Objective To compare the success rate and stability of rat models of comorbid chronic pain and depression induced by different doses of complete Freund's adjuvant(CFA).Methods Sixty SD rats were divided randomly into a control group,low-dose CFA group(CFA-L),and high-dose CFA group(CFA-H)(n=20 rats per group).Rats in the CFA-L and CFA-H groups were injected with 50 and 100 μL CFA,respectively,and rats in the control group were injected with 0.9％sodium chloride solution.The general state,body weight,mechanical withdrawal threshold(MWT),and thermal withdrawal latency(TWL)were observed at 0,7,14,21,and 28 days after modeling.Depressive behavior was evaluated using the open field test(OFT),forced swim test(FST),and tail suspension test(TST).Glutamate(Glu)and γ-aminobutyric acid(GABA)levels in the anterior cingulate cortex were detected by enzyme-linked immunosorbent assay.Brain-derived neurotrophic factor(BDNF)expression in the anterior cingulate cortex was detected by immunohistochemistry,and pathological changes in the anterior cingulate cortex were observed by HE staining.Results(1)Regarding the general condition of the rats,the left ankle joint and toes were obviously red and swollen in the CFA-L and CFA-H groups on the 7th day after modeling,and the swelling was more severe in the CFA-H group.The redness and swelling of the left hind foot and ankle joint and toes gradually recovered in the CFA-L group on days 14,21,and 28 after modeling,but were still obvious in the CFA-H group,and the water and food intake decreased.(2)The body mass was significantly lower in rats in the CFA-H group compared with those in the blank and CFA-L groups on days 14,21,and 28 after modeling(P＜0.05,P＜0.05).(3)Regarding pain-related behavior,the MWT and TWL were significantly decreased in the CFA-L and CFA-H groups on the 7th and 14th days after modeling,compared with the control group(P＜0.05,P＜0.05).On day 21 after modeling,MWT was significantly lower in the CFA-H group than in the blank and CFA-L groups(P＜0.05,P＜0.05),and TWL was significantly lower in the CFA-L and CFA-H groups than in the blank group(P＜0.05,P＜0.05).On day 28 after modeling,MWT and TWL were significantly lower in the CFA-H group than in the blank and CFA-L groups(P＜0.05,P＜0.05).(4)In terms of depression-related behaviors,the total OFT movement distance was significantly lower in the CFA-H group than in the blank and CFA-L groups on day 7 after modeling(P＜0.05,P＜0.05).The total OFT distance and central dwell time were significantly lower in the CFA-H group than in the blank and CFA-L groups on days 14,21,and 28 after modeling(P＜0.05,P＜0.05),and the result in the FST and TST were significantly higher than in the blank and CFA-L groups(P＜0.05,P＜0.05).(5)Glu,GABA,and BDNF expression levels were significantly higher in the CFA-H group than in the blank and CFA-L groups(P＜0.05,P＜0.05),while GABA,Glu/GABA,and BDNF levels were significantly lower in the CFA-H group than in the blank and CFA-L groups(P＜0.05,P＜0.05,P＜0.05).(6)The CFA-L group showed less damage in the anterior cingulate cortex,more pyramidal cells,more arranged cells,clear nucleoli,and a small number of cells with karyokynesis and deep staining.Compared with the CFA-L group,rats in the CFA-H group showed a disordered cell arrangement in the injured area of the anterior cingulate cortex,a large number of pyknotic and hyperchromatic neurons,significantly fewer or absent pyramidal cells,and vacuoles,red blood cells,and neurofibrillary tangles in the interstitial space.Conclusions Injection of CFA 100 μL can be used to establish a rat model of chronic inflammatory pain and depression,showing hyperalgesia,depression-like behavioral changes,changes in levels of Glu,GABA,and BDNF in the anterior cingulate cortex,and pathological changes in the anterior cingulate cortex,consistent with the pathophysiological characteristics of chronic pain and depression.

AIM:Regulatory T cells(Tregs)are a specialized subset of CD4+T cells primarily involved in im-munosuppressive functions.AMP-activated protein kinase(AMPK)serves as a metabolic sensor that governs the differen-tiation,maturation,and immune functions of Tregs through metabolic reprogramming.However,the impact of AMPKα1(the catalytic subunit of AMPK)knockout specifically in Tregs on the host's immune microenvironment remains largely un-explored.METHODS:Histological changes in immune organs were assessed using HE staining.The types of immune cells and their relative population percentages in immune organs and blood were quantified through flow cytometry in both AMPKα1flox/flox(AMPKα1fl/fl)mice and Treg-specific AMPKα1 knockout mice(AMPKα1fl/flFoxp3cre mice).RESULTS:Compared to AMPKα1fl/fl mice,the percentage of eosinophils in the bone marrow of AMPKα1fl/flFoxp3cre mice was significant-ly reduced.Additionally,while the thymus of AMPKα1fl/flFoxp3cre mice exhibited normal structure,both its size and the ra-tio of thymus weight to body weight were significantly decreased.The knockout of AMPKα1 in Tregs led to a notable reduc-tion in the total percentage of immature double-negative(DN)cells.Consequently,the percentage of CD4+T cells derived from these DN cells also decreased,even though the percentages of DN1 and DN4 cells were higher in the thymus of AMPKα1fl/flFoxp3cre mice compared to AMPKα1fl/fl mice.Importantly,the proportion of Siglec-F+CD11b+eosinophils in the thymus was significantly lower in AMPKα1fl/flFoxp3cre mice.Knockout of AMPKα1 in Tregs resulted in a marked increase in the percentage of CD4+T cells in peripheral blood,alongside a decrease in the proportion of mature CD8+T cells.Similar-ly,the proportion of CD4+T cells in the spleen of AMPKα1fl/flFoxp3cre mice was elevated compared to AMPKα1fl/fl mice.In contrast,the proportion of neutrophils significantly decreased,while mononuclear cell proportions increased in the spleen of AMPKα1fl/flFoxp3cre mice.In lymph nodes,the medullary boundaries in AMPKα1fl/flFoxp3cre mice were blurred,and the lymphoid follicles were missing,a feature not observed in AMPKα1fl/fl mice.Furthermore,the knockout of AMPKα1 in Tregs reduced the CD3+T cell population,particularly the CD8+T cell population,in lymph nodes.Although the mature Treg cell population was significantly lower in AMPKα1fl/flFoxp3cre mice,the percentage of CD4+T cells was markedly in-creased.In contrast,there was no statistically significant difference in granulocyte populations between AMPKα1fl/flFoxp3cre and AMPKα1fl/fl mice.CONCLUSION:The populations of mature Tregs,CD8+T cells and eosinophils in various im-mune organs were significantly altered in mice with Treg-specific AMPKα1 knockout,suggesting a potential remodeling of the host immune microenvironment in response to inflammatory stimuli.

Objective To identify the mutation in pathogenic genes by analyzing the clinical features and genotype of a family with Al-port syndrome,in order to provide a theoretical basis in genetic counseling for guidance on the future pregnancies in this couple.Meth-ods Based on closely combined thorough re-examination for the report of previous whole-exome sequencing of the proband and the mu-tation site information,Sanger sequencing verification was conducted in the proband's family members.Additionally,a comprehensive assessment of the proband's clinical manifestations and family history was performed,and the previous whole-exome sequencing report was reanalyzed accordingly.Results In terms of the heterozygous mutation(NM_0000924:c.3289+1G＞A)in intron 35 of COL4A4 gene carried by the proband,this mutation was identified in the proband's father(Ⅲ7),grandmother(Ⅱ6),aunt(Ⅲ6),and two great-uncle(Ⅱ2 and Ⅱ5).All of them exhibited clinical manifestations carried the COL4A4:c.3289+1G＞A heterozygous mutation.However,no mutation was detected in the proband's mother(Ⅲ8),great-grandmother(Ⅰ2),great-aunt(Ⅱ3),and great-uncle(Ⅱ4),grandfather(Ⅱ7)who were clinically unaffected.Additionally,a heterozygous mutation(COL4A3:NM_000914:c.1956A＞G)was identified in ex-on 27 of the COL4A3 gene in the proband.Her mother(Ⅲ 8),the other grandmother(Ⅱ9)and aunt(Ⅲ9)all carried the mutation of COL4A3 but had no clinical manifestation.However,her father(Ⅲ7)did not carry this mutation.Conclusion The splicing site muta-tion COL4A4:NM_0000924:c.3289+1G＞A should be confirmed as the pathogenic cause of Alport syndrome in this family.The combi-nation of whole-exome high-throughput sequencing and Sanger sequencing can effectively diagnose Alport syndrome and provide genetic counseling for the couple's next pregnancy.

Objective To optimize the extraction process of chlorogenic acid and three other organic acids from Picris hieracioides L.with deep eutectic solvents using response surface methodology.Methods By comparing the extraction rate of organic acids in seven deep eutectic solvents,the best solvent combinations were identified,and the optimal extraction process of organic acids from Picris hieracioides L.was obtained by optimizing the liquid/feed ratio,extraction temperature,extraction time and other parameters using the response surface method on the basis of a one-way experiment.Results The deep eutectic solvent with 30%water content(choline chloride∶urea=1∶2)was effective in extracting organic acids from Picris hieracioides L.The optimal extraction process optimized by response surface method was:extraction temperature 30℃,extraction time 30 min,liquid-solid ratio 20∶1(mL·g-1),the extraction rate of organic acids under this condition was 1.092 9%.Conclusion The deep eutectic solvent optimized through response surface methodology is an efficient,green and eco-friendly extraction method.This study provides a theoretical foundation for the subsequent development and utilization of Picris hieracioides L..

Objective To screen natural small-molecule compounds with anti-Helicobacter pylori urease(HPU)activity based on traditional Chinese medicine active ingredients and to systematically investigate their inhibitory mechanisms against HPU,as well as their regulatory effects on cellular inflammation and oxidative stress following Helicobacter pylori(Hp)infection.Methods A multi-dimensional screening strategy was adopted.Firstly,virtual screening was performed on the traditional Chinese medicine monomer compound database based on the crystal structure of HPU,and the candidate molecules were selected in combination with bibliometric analysis.Subsequently,the modified Berthelot method was applied to verify urease inhibitory activity in vitro.Inhibition kinetics were analyzed with Lineweaver-Burk plots.The inhibitory sites were explored through sulfhydryl blocking agents and Ni2+competitive inhibitors,followed by molecular docking simulations with AutoDock Vina(version 1.2.3).A Hp-infected human gastric mucosal epithelial cells(GES-1)model was established.The compound's cytotoxicity was assessed with the CCK-8 assay and lactate dehydrogenase release assay.The mRNA expression levels of interleulain(IL)-6,IL-8,and IL-1β were quantified with quantitative real-time PCR(qRT-PCR).Intracellular reactive oxygen species(ROS)levels were measured with a DCFH-DA fluorescent probe.Results According to the screening results,the natural small-molecule compound rhodiosin(RHO)significantly inhibited HPU activity with a half-maximal inhibitory concentration(IC50)of(82.38±5.45)μmol/L.Enzyme kinetics analysis revealed that RHO acted as an anti-competitive inhibitor,showing an inhibition constant of(146.40±2.19)μmol/L;RHO-sulfhydryl/Ni2+-HPU interaction experiments confirmed that its target was located in the sulfhydryl group in the active center of HPU.Molecular docking simulations suggested that RHO is bound exactly to the Flap domain of the urease active pocket,with a binding energy of-8.678 kcal/mol.No significant cytotoxicity towards GES-1 cells was observed with RHO at 80 μmol/L in cellular experiments.Furthermore,RHO significantly down-regulates the mRNA overexpression of IL-6,IL-8,and IL-1β induced by Hp and reduces the production of ROS by 95%.Conclusion The monomer RHO of traditional Chinese medicine inhibits HPU through anti-competitive binding to the sulfhydryl site.It can effectively alleviate the inflammatory response and oxidative stress injury of GES-1 caused by Hp infection,providing a theoretical foundation for developing novel anti-Hp treatment strategies.

Objective:To investigate the relationship between serum annexin A1 (ANXA1), macrophage inflammatory protein 1-α (MIP-1α), soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) and pulmonary infection in elderly patients with coronary heart disease complicated with heart failure and their predictive value.Methods:A total of 197 elderly patients with coronary heart disease combined with heart failure admitted to the Second Hospital of Qinhuangdao from January 2021 to January 2023 were retrospectively selected, and divided into pulmonary infection group (36 cases) and non pulmonary infection group (161 cases) according to whether the patients had pulmonary infection during hospitalization. Serum ANXA1, MIP-1α and sTREM-1 levels were detected in two groups. Multivariate logistic regression model was used to analyze the influencing factors of lung infection in elderly patients with coronary heart disease combined with heart failure. The predictive value of serum ANXA1, MIP-1α and sTREM-1 levels on lung infection in elderly patients with coronary heart disease combined with heart failure was analyzed by receiver operating characteristic (ROC) curve.Results:The incidence of pulmonary infection in 197 elderly patients with coronary heart disease combined with heart failure was 18.27%(36/197). Compared with the non pulmonary infection group, the pulmonary infection group had higher levels of serum ANXA1, MIP-1 α, sTREM-1, C-reactive protein, procalcitonin, and higher proportion of New York Heart Association (NYHA) heart function grade Ⅳ and diabetes, and lower left ventricular ejection fraction (all P<0.05). Multivariate logistic regression analysis showed that NYHA cardiac function grade Ⅳ, diabetes mellitus and elevated levels of procalcitonin, ANXA1, MIP-1α and sTREM-1 were independent risk factors for pulmonary infection in elderly patients with coronary heart disease combined with heart failure (all P<0.05). ROC curve analysis showed that the area under the curve predicted by serum ANXA1, MIP-1α and sTREM-1 combined was 0.909, which was larger than that predicted by serum ANXA1, MIP-1α and sTREM-1 alone. Conclusions:Elevated levels of serum ANXA1, MIP-1α and sTREM-1 are independent risk factors for pulmonary infection in elderly patients with coronary heart disease combined with heart failure, and can be used as auxiliary predictors of pulmonary infection in elderly patients with coronary heart disease combined with heart failure.

Extracellular vesicles (EVs) are crucial for facilitating intercellular communication, promoting cell migration, and orchestrating the immune response. Recently, EVs can diagnose and treat tumors. EVs can be measured as biomarkers to provide information about the type of disease and therapeutic efficacy. Furthermore, EVs with lower immunogenicity and better biocompatibility are natural carriers of chemicals and gene drugs. Herein, we review the molecular composition, biogenesis, and separation methods of EVs. We also highlight the important role of EVs from different origins as biomarkers and drug delivery systems in tumor therapy. Finally, we provide deep insights into how EVs play a role in reversing the immunosuppressive microenvironment.