OBJECTIVE To focus on the classic NOD-like receptor protein 3 （NLRP3）/Caspase-1/gasdermin D （GSDMD） pyroptosis pathway and explore the mechanism by which Huatan qushi huoxue formula （HQHF） inhibits macrophage pyroptosis to ameliorate metabolic dysfunction-associated steatohepatitis （MASH）. METHODS RAW264.7 cells were divided into 5 groups： Control group （10% blank serum）， Model group [10% blank serum+5 μg/mL lipopolysaccharide （LPS）]， HQHF-L group （2.5% drug-containing serum+7.5% blank serum+5 μg/mL LPS）， HQHF-M group （5% drug-containing serum+5% blank serum+5 μg/mL LPS）， and HQHF-H group （10% drug-containing serum+5 μg/mL LPS）. After 24 h of routine culture post-administration， cells and supernatants were collected for assays. Cell morphology was observed via scanning electron microscopy and phase-contrast microscopy； localization and expression of gasdermin D-N （GSDMD-N） were observed by immunofluorescence. Interleukin-1β （IL-1β） and IL-18 contents in supernatants were detected by ELISA； mRNA and protein expressions of NLRP3， Caspase-1， and GSDMD were measured using real-time PCR and Western blot. RESULTS Compared with the Control group， the Model group showed typical pyroptotic morphology （cell membrane bulging and pore formation）， increased aggregation and fluorescence intensity of GSDMD-N on the cell membrane （ P ＜0.05）， significantly increased the contents of IL-1β and IL-18 in cell supernatants （ P ＜0.05）， and significantly up-regulated mRNA and protein expressions of NLRP3， Caspase-1， and GSDMD in cells （ P ＜0.05）. Compared with the Model group， the HQHF-L， HQHF-M and HQHF-H groups showed improved pyroptotic morphology， reduced membrane localization and significantly weakened fluorescence intensity of GSDMD-N （ P ＜0.05）， significantly decreased the contents of IL-1β and IL-18 in cell supernatants （ P ＜0.05）， and significantly down-regulated mRNA and protein expressions of NLRP3， Caspase-1， and GSDMD in cells （ P ＜0.05）. CONCLUSIONS HQHF inhibits LPS-induced macrophage pyroptosis， and its mechanism of improving MASH may be associated with the suppression of the activation of the classical NLRP3/Caspase-1/GSDMD pyroptosis pathway.

ObjectiveTo investigate the potential targets of 2，5-dihydroxybenzoic acid （2，5-DHBA） in the treatment of metabolic dysfunction-associated fatty liver disease （MAFLD） and the molecular mechanism by which it regulates the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） signaling pathway. MethodsA network pharmacology analysis was performed at first， and related databases were used to obtain the common action targets of 2，5-DHBA and MAFLD， followed by molecular docking and pathway enrichment analysis to predict the potential biological processes and signaling pathways regulated by these targets. Then mouse normal hepatocytes AML-12 were used for cell experiments， and CCK-8 assay was used to determine the three optimal intervention concentrations of 2，5-DHBA. AML-12 cells were divided into control group， model group （cells cultured in a medium containing 2 mmol/L free fatty acid ［FFA］ to establish a cell model of MAFLD）， and three 2，5-DHBA intervention groups （cells cultured with FFA and 2，5-DHBA at the three different concentrations of 1.25， 5， and 20 μmol/L， respectively）. Oil red O staining was used to observe intracellular lipid accumulation； the fluorescent probe DCFH-DA was used to measure the level of intracellular reactive oxygen species （ROS）； biochemical assays were used to measure the content of triglyceride （TG） and total cholesterol （TC）； quantitative real-time PCR and Western Blot were used to measure the expression levels of related genes and proteins. A one-way analysis of variance was used for comparison between multiple groups， and the Tukey’s test was used for further comparison between two groups. ResultsA total of 32 common targets were obtained for 2，5-DHBA and MAFLD. Within a concentration range of 1.25 — 20 μmol/L， 2，5-DHBA treatment， starting from the concentration of 2.5 μmol/L， increased the viability of AML-12 cells in a dose-dependent manner. The minimum effective concentration of 1.25 μmol/L， the intermediate gradient concentration of 5 μmol/L， and the maximum safe concentration within the effective range of 20 μmol/L were selected for low-， middle-， and high-dose intervention， respectively. Compared with the model group， 2，5-DHBA intervention at concentrations of 1.25， 5， and 20 μmol/L could significantly reduce lipid droplet accumulation in cells （all P<0.05）， and there were significant differences in TG and TC between the model group and the three intervention groups （all P<0.05）. Compared with the control group， the model group had a significant increase in ROS fluorescence intensity （P<0.05）， and compared with the model group， there was a dose-dependent reduction in intracellular ROS level after treatment with 5 μmol/L or 20 μmol/L 2，5-DHBA （P<0.05）. Compared with the model group， 2，5-DHBA treatment at concentrations of 5 and 20 μmol/L significantly downregulated the mRNA expression levels of the lipogenic genes SREBf1 and FASN and upregulated the mRNA expression levels of the key antioxidant gene Nrf2 and its downstream target gene HO-1 （all P<0.05）. Compared with the control group， the expression of p-PI3K/PI3K and p-Akt/Akt in the model group was significantly increased （all P<0.05）. Compared with the model group， the protein expression levels of p-PI3K/PI3K and p-Akt/Akt were significantly decreased after 5 μmol/L 2，5-DHBA treatment （P<0.05）. Compared with the control group， the expression levels of Nrf2 and HO-1 protein in the model group were significantly decreased （all P<0.05）. The protein levels of Nrf2 and HO-1 were significantly increased after treatment with 1.25 μmol/L 2，5-DHBA （P<0.05）. ConclusionNetwork pharmacology and cellular experiments confirm that 2，5-DHBA can alleviate lipid deposition and oxidative stress by regulating the PI3K/Akt signaling pathway， thereby exerting a therapeutic effect on MAFLD.

OBJECTIVE To investigate the ameliorative effect of Weiling decoction on metabolic associated fatty liver disease （MAFLD） based on the gut-liver axis theory.METHODS Male C57BL/6J mice were divided into a blank group （ n ＝9） and a modeling group （ n ＝47）. The modeling group was fed a high-fat diet for 12 weeks to establish the MAFLD model. Forty-five modeled mice were randomly divided into a model group， a positive control group [metformin， 0.2 g/（kg·d）]， and Weiling decoction low-， medium-， and high-dose groups [6.17， 12.34， 24.68 g/（kg·d）， calculated as crude drug]， with 9 mice in each group. Mice in each group received intragastric administration of the corresponding drug solution or normal saline once daily for 4 consecutive weeks. After the last administration， the body weight， liver weight， and liver index were compared among groups； the levels of liver function indicators （alanine transaminase， aspartate transaminase）， lipid indicators （total cholesterol， triglycerides， low-density lipoprotein cholesterol， high-density lipoprotein cholesterol）， inflammatory indicators [tumor necrosis factor-α， interleukin-6 （IL-6）， IL-1β]， oxidative stress indicators （malondialdehyde， superoxide dismutase， glutathione peroxidase）， and intestinal barrier indicators [lipopolysaccharide （LPS）， diamine oxidase， D-lactic acid] were measured； hepatic pathological changes were observed； the expressions of tight junction proteins [zonula occludens-1 （ZO-1）， occludin] in jejunal tissue and proteins related to lipid metabolism [sterol regulatory element-binding protein-1 （SREBP-1）， fatty acid synthase （FASN）， peroxisome proliferator-activated receptor α （PPARα）] and inflammatory pathways [Toll-like receptor 4 （TLR4）， myeloid differentiation primary response protein 88 （MyD88）， nuclear factor-κB p65 （NF-κB p65）] in liver tissue were detected； and changes in gut microbiota were analyzed.RESULTS Compared with the model group， hepatic steatosis and inflammatory injury were significantly alleviated in all Weiling decoction dose groups； body weight （at week 16）， liver weight， liver index， and the levels of liver function indicators， lipid indicators， inflammation indicators， oxidative stress indicators， and intestinal barrier indicators were all generally improved （ P ＜0.05）. The expressions of SREBP-1， FASN， TLR4， and MyD88 and the phosphorylation level of NF-κB p65 in liver tissue were generally decreased or downregulated （ P ＜0.05）， while the expressions of ZO-1 and occludin in jejunal tissue and PPARα in liver tissue were generally increased or upregulated （ P ＜0.05）. Compared with the model group， the Observed species， Chao1， and Ace indices of gut microbiota in the Weiling decoction high-dose group were significantly increased （ P ＜0.05）， and the relative abundances of Verrucomicrobiota， Akkermansia ， etc. were significantly increased， while those of Actinomycetota， Alistipes ， Faecalibaculum ， etc. were significantly decreased （ P ＜0.05）.CONCLUSIONS Weiling decoction can ameliorate MAFLD， and its effect may be associated with regulating gut microbiota， repairing the intestinal mechanical barrier， reducing LPS translocation， thereby inhibiting the TLR4/MyD88/NF-κB inflammatory pathway， alleviating oxidative stress， and regulating SREBP-1/FASN and PPARα-mediated lipid metabolism.

Metabolic dysfunction-associated fatty liver disease （MAFLD） has become the leading chronic liver disease worldwide. As a classic food-medicine homologous Chinese herb， hawthorn fruit has the functions of promoting digestion to resolve food stagnation and activating blood circulation to resolve blood stasis. Modern pharmacological studies have shown that hawthorn fruit and its active components exert diverse pharmacological activities in regulating lipid metabolism， improving insulin resistance， inhibiting inflammatory response， delaying hepatic fibrosis， and protecting hepatocytes. These effects precisely match the “multiple-hit” pathological features of MAFLD， and thus hawthorn fruit shows significant advantages in the prevention and treatment of MAFLD. Drug pair combination is a key strategy in traditional Chinese medicine to enhance efficacy and reduce toxicity. In clinical practice， hawthorn fruit is often combined with Salvia miltiorrhiza， Alisma orientalis， cassia seed， and Poria cocos， and such combination adheres to the principle of “mutual reinforcement and assistance” to enhance efficacy and expand their application in different syndromes of MAFLD. This article systematically reviews the core pharmacological mechanisms of hawthorn fruit in intervening against MAFLD and the research advances in its classic drug combinations， in order to provide a scientific reference for the in-depth development of hawthorn fruit and its combinations and the precise clinical intervention of MAFLD.

Objective:To evaluate the safety and efficacy of the probiotic Bifidobacterium longum subsp. longum BL21 (BL21) in preventing radiation-induced diarrhea (RID) in cervical cancer patients during radiotherapy (RT) and to investigate the intestinal microbiota in the patients. Methods:This study was a prospective, single-arm, phase Ⅱ clinical trial, involving cervical cancer patients treated with radical and adjuvant RT. From the first day of RT, participants took one pack of BL21 powder (containing 20 billion colony-forming unit(CFU) of Bifidobacterium longum subsp. longum BL21) orally every day until the end of RT. The occurrence of adverse events and RID during RT were assessed as per Common Terminology Criteria for Adverse Events ( CTCAE) v5.0. In this way, the safety and efficacy of BL21 in preventing RID were evaluated. Additionally, the intestinal microbiota in fecal samples collected from the patients before and after RT were analyzed using 16S rRNA sequencing. Results:A total of 35 cervical cancer patients were enrolled in this study, with 29 cases incorporated for the final analysis. No serious adverse event related to the administration of BL21 was observed. The patients exhibited slight RID, with the majority (22/29) developing no or grade 1 RID during RT. The microbiota in the fecal samples showed decreased alpha diversity after RT, as indicated by the Chao1 ( P = 0.002) and Shannon ( P = 0.005) indices. Furthermore, these samples exhibited a notably higher abundance of genus Clostridium (LDA score = 3.98). The fecal samples from patients with grade 1 RID or no RID post-RT exhibited higher alpha diversity than those from patients with grade 2 RID or above post-RT (Chao1: P = 0.07, Shannon: P = 0.28), as well as a high abundance of genera Gemmiger (LDA score = 4.48) and Dorea (LDA score = 3.83). Conclusions:The administration of BL21 to cervical cancer patients during RT is simple, convenient, safe, and effective in preventing RID, thus warranting further investigation.

Objective To investigate the prevalence and burden of retinoblastoma(RB)in China based on the Glob-al Burden of Disease Study 2021(GBD 2021),thus providing a theoretical foundation for the diagnosis and treatment of RB.Methods Relevant indicators such as the standardized incidence rate,standardized prevalence rate,standardized mortality rate,and standardized disability-adjusted life year(DALY)rate of RB in the total population,males,and females in China from 1990 to 2021 were analyzed to assess the prevalence and disease burden of this disease.The Joinpoint model was employed to analyze the annual percent change(APC)and the average annual percent change(AAPC)of the overall standardized incidence rate,overall standardized prevalence rate,overall standardized mortality rate,and overall standard-ized DALY rate of RB in China from 1990 to 2021.In addition,the changes in the overall standardized incidence rate,over-all standardized prevalence rate,overall standardized mortality rate,and overall standardized DALY rate were compared be-tween China and the globe from 1990 to 2021.Results Compared with 1990,the standardized incidence rate of RB in the total population,males,and females in China increased by 1.840 times,2.154 times,and 1.689 times,respectively,in 2021;the standardized prevalence rate of RB in the total population,males,and females in China increased by 1.876 times,2.133 times,and 1.701 times,respectively;the standardized mortality rate of RB in the total population,males,and fe-males in China decreased by 0.679 times,0.636 times,and 0.714 times,respectively;the standardized DALY rate of RB in the total population,males,and females in China decreased by 0.652 times,0.616 times,and 0.676 times,respectively.Joinpoint analysis showed that compared with 1990,the APPC of the overall standardized incidence rate and the overall standardized prevalence rate in China in 2021 increased by 3.21 and 3.23,respectively;the AAPC of the overall standard-ized mortality rate and the overall standardized DALY rate decreased by 3.61 and 3.46,respectively.Compared with 1990,the global overall standardized incidence rate and overall standardized prevalence rate increased by 0.237 times and 0.242 times,respectively.The global overall standardized mortality rate and overall standardized DALY rate decreased by 0.212 times and 0.195 times,respectively.Conclusion Compared with 1990,the standardized incidence rate and standardized prevalence rate of RB in the total population,males,and females in China increased in 2021,while the standardized mortal-ity rate and DALY rate decreased in 2021,indicating an increase in the prevalence of the disease but a decrease in the dis-ease burden.From 1990 to 2021,the difference between the overall standardized incidence rate and overall standardized prevalence rate in China and the global level first increased and then decreased;however,the overall standardized mortality rate and overall standardized DALY rate in China were always lower than the global level.

Objective To establish a stable cell line expressing luciferase using U87MG cells,and to establish a nude mouse glioblastoma in situ transplantation tumor model,in order to provide a suitable efficacy evaluation model for anti-glioblastoma drugs.Methods A stable and highly expressing luciferase U87MG cell line(U87MG-Luc)was prepared by infecting U87MG with lentivirus,and its stability was verified by repeated passages.U87MG-Luc single-cell suspension,which has been amplified and digested in vitro,was injected into the caudate nucleus of the right brain of nude mice using a brain stereotaxic device and a micro constant flow pump.Using small animal live imaging technology to observe the tumor formation and growth of U87MG-Luc inoculated,and confirm appropriate modeling conditions.Subsequently,the U87MG-Luc brain in situ tumor model was constructed to validate the efficacy of the first-line clinical treatment drug temozolomide,including indicators such as tumor growth status,animal weight,and survival time.Results Successfully established and screened U87MG-Luc stable cell line using lentiviral infection system.The luciferase expressed by stable strains was linearly related to cell seeding density,and the in vitro culture could stably express luciferase after continuous passage to the 21st generation.After inoculating U87MG-Luc cells at an appropriate concentration into the brain of nude mice,in situ brain tumor formation was achieved,with a high success rate(100％)of 2 × 105 cells/nude mouse.On the fifth day after inoculation,the brain tumor remained stable with no metastasis,and the tumor growth trend was good thereafter.In the U87MG-Luc brain in situ tumor model constructed in this study,temozolomide significantly inhibited tumor growth and prolonged the survival of tumor bearing nude mice(P＜0.001).Conclusion A stable and highly expressing luciferase U87MG-Luc cell line was successfully constructed,transduced,and screened based on the lentiviral expression system,and U87MG-Luc nude mouse brain in situ tumor model was constructed to provide a suitable pharmacological evaluation model for the discovery of anti-glioma drugs.

Colitis is a common inflammatory bowel disease whose pathogenesis is related to immune regula-tion and intestinal microbial homeostasis.Exosomes are membrane vesicles secreted by cells that contain proteins,lipids,and other substances.Stem cells and macrophages can participate in the regulation of related diseases by secreting exosomes.It has been found that exosomes of stem cell and macrophage origin are closely involved in the regulation of colitis,and exosomes derived from stem cells,especially mesenchymal stem cells,play an important role in anti-colitis.This article reviews the role and possible mechanisms of mesenchymal stem cell-based and macrophage-derived exosomes in the regulation of colitis,aiming to provide ideas for in-depth study of colitis and new strategies for its treatment.

Objective:To explore the therapeutic effect of Bupiqiangli compound on experimental myasthenia gravis with sub-clinical hypothyroidism in rats and the influence of hypothalamus-pituitary-thyroid-thymus(HPTT)axis.Methods:SPF Lewis rats were randomly divided into normal group,model group,Bupiqiangli compound low-dose,medium-dose and high-dose groups.Model rats were immunized with AchR-α subunit 97-116 peptide sequence to construct an experimental myasthenia gravis model,and then methimazole was used to prepare an experimental myasthenia gravis with subclinical hypothyroidism model.Bupiqiangli compound low-dose,medium-dose and high-dose groups were given 4.57 g/kg,7.12 g/kg and 9.49 g/kg of Bupiqiangli compound by gavage,nor-mal group and model group were given an equal volume of normal saline by gavage,once a day,for 4 weeks.After the last gavage,Lennon scores of rats in each group were recorded;HE staining was used to detect pathological changes of thymus and thyroid;ELISA was used to detect expression levels of acetylcholine receptor antibody(AchRab),thyroid stimulating hormone(TSH),thyrotropin releasing hormone(TRH),free thyroxine(FT4)and thyroxine(T4)in serum;mRNA level of TRH in hypothalamus and TSH in pituitary tissue were detected by qPCR;Western blot detected changes of protein expressions of Cleaved Caspase3,Bcl2 associated X protein(Bax)and B-cell lymphoma-2(Bcl2)in thymus.Results:Compared with normal group,model group showed obvious symp-toms of muscle weakness,Lennon score increased significantly(P<0.05),obvious pathological changes in thymus and thyroid tissues,while no significant changes in expressions of FT4 and T4 in serum(P>0.05),expressions of AchRab,TSH and TRH in serum were significantly increased(P<0.05),expressions of TRH in hypothalamus and TSH in pituitary were significantly increased(P<0.05),protein expressions of Cleaved Caspase3 and Bax in thymus were significantly decreased(P<0.05),while expression of Bcl2 protein in thymus increased significantly(P<0.05).Compared with model group,myasthenia symptoms of compound high-dose group were im-proved,Lennon score was significantly decreased(P<0.05),pathological changes of thymus and thyroid tissues were improved,ex-pressions of FT4 and T4 in serum had no significant changes(P>0.05),expressions of AchRab,TSH and TRH in serum were signifi-cantly decreased(P<0.05),expressions of TRH in hypothalamus and TSH in pituitary were significantly decreased(P<0.05),expres-sions of Cleaved Caspase3 and Bax in thymus were significantly increased(P<0.05),while expression of Bcl2 in thymus was signifi-cantly decreased(P<0.05).Conclusion:Bupiqiang compound can improve clinical symptoms of experimental myasthenia gravis with subclinical hypothyroidism in rats,and its mechanism may be related to the regulation of HPTT axis.

Objective To establish a stable cell line expressing luciferase using U87MG cells,and to establish a nude mouse glioblastoma in situ transplantation tumor model,in order to provide a suitable efficacy evaluation model for anti-glioblastoma drugs.Methods A stable and highly expressing luciferase U87MG cell line(U87MG-Luc)was prepared by infecting U87MG with lentivirus,and its stability was verified by repeated passages.U87MG-Luc single-cell suspension,which has been amplified and digested in vitro,was injected into the caudate nucleus of the right brain of nude mice using a brain stereotaxic device and a micro constant flow pump.Using small animal live imaging technology to observe the tumor formation and growth of U87MG-Luc inoculated,and confirm appropriate modeling conditions.Subsequently,the U87MG-Luc brain in situ tumor model was constructed to validate the efficacy of the first-line clinical treatment drug temozolomide,including indicators such as tumor growth status,animal weight,and survival time.Results Successfully established and screened U87MG-Luc stable cell line using lentiviral infection system.The luciferase expressed by stable strains was linearly related to cell seeding density,and the in vitro culture could stably express luciferase after continuous passage to the 21st generation.After inoculating U87MG-Luc cells at an appropriate concentration into the brain of nude mice,in situ brain tumor formation was achieved,with a high success rate(100％)of 2 × 105 cells/nude mouse.On the fifth day after inoculation,the brain tumor remained stable with no metastasis,and the tumor growth trend was good thereafter.In the U87MG-Luc brain in situ tumor model constructed in this study,temozolomide significantly inhibited tumor growth and prolonged the survival of tumor bearing nude mice(P＜0.001).Conclusion A stable and highly expressing luciferase U87MG-Luc cell line was successfully constructed,transduced,and screened based on the lentiviral expression system,and U87MG-Luc nude mouse brain in situ tumor model was constructed to provide a suitable pharmacological evaluation model for the discovery of anti-glioma drugs.