Objective To investigate the effects of Yishen Zhuyu Tongluo Decoction on chondrocyte pyroptosis and ferroptosis in rats with knee osteoarthritis(KOA).Methods Forty SD were divided randomly into a sham operation group(n=10)and a modeling group(n=30).A KOA model was established in the modeling group by anterior cruciate ligament transection of the right hind limb knee joint.The drawer test was used to confirm the successful establishment of the model.Post-surgery,the rats were subjected to 30 min of forced activity daily to induce KOA.Lameness and hopping movements were observed after 4 weeks,and hematoxylin and eosin staining confirmed cartilage surface damage,deformation,and inflammatory cell infiltration,indicating successful modeling.The model rats were then assigned randomly to a model group(n=8),celecoxib group(n=8),and Yishen Zhuyu Tongluo Decoction group(n=8).The sham operation and model groups received 10 mL/kg of saline by gavage,the celecoxib group received 12 mg/kg of celecoxib solution,and the Yishen Zhuyu Tongluo Decoction group received 5.4 g/kg of the herbal decoction,once daily for 8 weeks.After the interventions,the rats were anesthetized,blood was collected from the abdominal aorta and serum was separated,and the knee joints were isolated.Three samples were fixed in paraformaldehyde,while the remaining cartilage tissue was reserved.Pathological changes in joint cartilage were observed by hematoxylin and eosin and Safranin O-Fast Green staining.Bone microstructure was analyzed using micro-computed tomography.Serum interleukin(IL)-1β and IL-18 protein levels were detected by enzyme-linked immunosorbent assay.Relative mRNA expression levels of NLRP3,ASC,Caspase-1,GSDMD,IL-1β,IL-18,ACSL-4,FTH-1,GPX-4,and cyclooxygenase(COX)-2 were detected by reverse transcription quantitative real-time PCR,and protein expression of NLRP3,Caspase-1,and COX-2 were detected by immunohistochemistry.Results Compared with the sham operation group,model rats showed surface damage and deformation of cartilage tissue,disordered cell arrangement in all layers,significant loss of Safranin O-Fast Green staining,and sparse and irregular trabecular bone distribution.Serum levels of the inflammatory factors IL-1β and IL-18 were elevated(P＜0.01).mRNA expression levels of NLRP3,ASC,Caspase-1,GSDMD,IL-1β,IL-18,ACSL-4,and COX-2 were also significantly increased(all P＜0.01),while FTH-1 and GPX-4 mRNA expression levels were decreased(P＜0.01).Compared with the model group,rats in the celecoxib and Yishen Zhuyu Tongluo Decoction groups showed smoother and more intact cartilage surfaces,significantly increased cell counts,less loss of Safranin O-Fast Green staining,denser trabecular bone,and thicker cortical bone,with improved bone microstructure.Serum IL-1β and IL-18 levels were reduced(P＜0.01),NLRP3(P＜0.01,P＜0.05),ASC,Capase-1,GSDMD,IL-1β,IL-18,ACSL-4,and COX-2 mRNA levels were decreased(all P＜0.01),and relative expression levels of FTH-1 and GPX-4 mRNA were significantly increased(P＜0.01).Conclusions Yishen Zhuyu Tongluo Decoction can treat KOA by inhibiting chondroptosis and ferroptosis in chondrocytes,reducing serum IL-1β and IL-18 levels,and improving bone microstructure.

Objective To explore the molecular mechanism of autophagy mediated by the protein kinase B(AKT)/mammalian target protein of rapamycin(mTOR)pathway in the rehabilitation of muscle atrophy associated with rotator cuff tears(RCTs).Methods Forty male C57BL/6J mice were randomly assigned to the following four groups:sham group,RCTs group,RCTs+exercise group,and RCTs+exercise+rapamycin group,with 10 mice in each group.On the eighth week after grouping,healing of the bone-tendon interface and muscle cell atrophy were analyzed by histology.The mRNA expression levels of muscle-atrophy-related genes(Atrogin-1,Bnip 3,MuRF-1)in supraspinatus muscle tissue were measured by real-time quantitative reverse transcription polymerase chain reaction.The expression of LC3 and AKT/mTOR signal pathway proteins in the supraspinatus muscle tissue of the groups was detected by Western blot,and the degree of autophagy in each group was analyzed by transmission electron microscope.Results Compared with the sham operation group,in RCTs group's maturity score for the bone-tendon interface at the supraspinatus tendon anchorage and the cross-sectional area of the supraspinatus muscle fibers decreased significantly(P＜0.001),while muscle loss and the expression of Atrogin-1,Bnip 3,and MuRF-1 increased significantly(P＜0.001).Compared with the RCTs group,the RCTs+exercise group showed a significant increase in bone-tendon interface maturity score and cross-sectional area of the supraspinatus muscle fibers(P＜0.01)and a decrease in muscle loss and the expression of Atrogin-1,Bnip 3,and MuRF-1(P＜0.01).Compared with the sham group,the RCTs group's LC3Ⅰ/LC3Ⅱ and degree of autophagy in the supraspinatus muscle increased significantly(P＜0.001),while p-AKT/AKT and p-mTOR/mTOR expression decreased significantly(P＜0.01).Compared with RCTs group,the RCTs+exercise group's LC3Ⅰ/LC3Ⅱ and degree of autophagy decreased significantly(P＜0.01)and p-AKT/AKT and p-mTOR/mTOR expression increased significantly(P＜0.001).The addition of rapamycin significantly reversed the rehabilitation effect of exercise in the RCTs group.Conclusions This study confirmed the anti-atrophy effect of exercise rehabilitation in RCT diseases and showed that its mechanism is related to AKT/mTOR signal activation,which inhibits autophagy.

Objective To examine the effect of the demethylating agent 5-aza-2'-deoxycytidine(5-Aza-CdR)on pericyte-myofibroblast transition(PMT)in primary rat renal myofibroblasts.Methods Rat primary renal myofibroblasts were treated with 5-Aza-CdR 250 ng/mL for 72 h,and the degree of Epo promoter methylation was detected by pyrosequencing.Protein expression levels of α-smooth muscle actin(α-SMA),platelet-derived growth factor receptor-β(PDGFRβ)and DNA methyltransferase(Dnmt3a)were detected by immunofluorescence and Western blot,and erythropoietin(EPO)levels in the supernatant were detected.Results Compared with the control group,5-Aza-CdR treatment significantly decreased the expression of Dnmt3a and hypermethylation of the Epo promoter,and subsequently decreased the expression of α-SMA and the expression ratio of α-SMA to PDGFRβ in myofibroblasts.Meanwhile,5-Aza-CdR treatment increased the level of EPO in the cell supernatant.Conclusions 5-Aza-CdR can reverse PMT by inhibiting Epo promoter hypermethylation in primary renal myofibroblasts.

Objective To establish an efficient DNA extraction method for the batch identification of genotypes in model mice.Methods We extracted total DNA from transgenic mouse tails using four methods:alkaline simplified group,alkaline routine group,protease K cleavage group and DNA extraction kit group.The purity and concentration of DNA obtained by the four methods were measured,the effects of gel electrophoresis were evaluated,and the time and experimental costs of the four methods were compared.Results The protease K cleavage method produced the highest concentration of DNA,followed by the simplified alkaline boiling and routine alkaline boiling method.The reagent kit produced the highest DNA purity,followed by the simplified alkaline boiling method.The DNA templates obtained by the four method could be amplified by polymerase chain reaction and gel electrophoresis to obtain clear DNA target bands.In addition,the DNA template extracted by the simplified alkaline boiling method could be used for gene identification after storage at-20℃for 1 month,as well as requiring the least time and lowest costs.Conclusions The simplified alkaline boiling method is currently the simplest,fastest,and most economical DNA template-extraction method for batch identification of genotypes in model mice.

Pollen allergen-induced allergic rhinitis(AR),also known as seasonal allergic rhinitis(SAR),typically manifests during the period of pollen dissemination by anemophilous plants.The prevalence of SAR has more than doubled over the past three decades.The etiology of SAR is multifaceted,involving factors such as pollen allergens,environmental and climatic conditions,genetic predispositions,and the immunological status of the individual.Animal models provide a critical tool for elucidating the mechanisms underlying AR and advancing the development of effective preventive and therapeutic strategies.This review synthesizes the recent pertinent domestic and international literature on pollen-sensitized AR animal experiments.It systematically delineates the factors influencing the efficacy of these models,including the selection of animal strains,the production and associated challenges of sensitizing agents,specifically pollen antigens,the utilization and limitations of adjuvants,the procedural steps involved in model creation,and the method ologies for evaluating model effectiveness.The insights provided are intended to offer guidance and support for the development of appropriate animal models of pollen-induced AR,thereby facilitating both fundamental and applied research in this area.

Mitochondria act as the main energy supply station for cardiomyocytes and are thus crucial for maintaining normal cardiac function.Mitochondrial autophagy plays an important positive role in maintaining cardiomyocyte homeostasis and coping with stress.The progressive exacerbation of cardiovascular diseases presents a challenge to the homeostasis of mitochondrial autophagy through as-yet-unidentified pathological mechanisms,leading to mitochondrial damage,which may in turn trigger damage to cardiomyocytes.In addition,when mitochondrial autophagy fails to meet the physiological needs of the body,mitochondrial dysfunction may be triggered,which may in turn accelerate the progression of heart failure.In this review,we explore the specific roles of mitochondrial autophagy and mitochondrial dynamics in the heart,and discuss the mechanisms in the context of major cardiovascular diseases,focusing on the latest advances and important discoveries in this field.

In line with the rising global incidence of cancer,tumor metastasis has become the leading cause of mortality among cancer patients.Formation of the pre-metastatic niche(PMN)creates an optimal microenvironment for tumor cell colonization and metastasis,thus facilitating tumor dissemination.In this context,cancer-associated fibroblasts(CAFs)have garnered significant attention as a result of their multifaceted roles in tumor progression.CAFs enhance the formation of the PMN and promote the invasive behavior of tumor cells by remodeling the extracellular matrix,inducing epithelial-mesenchymal transition,stimulating angiogenesis and modulating the tumor immune microenvironment.These processes not only expedite metastasis but also enable tumor immune evasion.This paper reviews the fundamental mechanisms and functions of CAFs in tumor metastasis,examines their potential applications in cancer research and therapy,and offers new perspectives and directions for the development of anti-tumor metastasis strategies.

Objective To explore the effect and mechanism of lentinan(LNT)on hepatic tissue ferroptosis in mice exposed to sodium arsenite(SA).Methods C57BL/6N male mice were exposed to SA low-dose,SA high-dose,and LNT intervention combined with SA high-dose,then,hematoxylin and eosin(HE)staining was applied to assess pathological liver tissue damage;Enzyme-linked immunosorbent and Western blot were used to detect the content or expression of tumor necrosis factor α(TNFα),interleukin-6(IL-6),ferritinophagy or ferroptosis biomarkers.Results Compared with the control group,SA exposure induced the elevated levels of TNFα,IL-6,ferritinophagy biomarker ferritin heavy chain 1(FTH1)and microtubule-associated protein 1 light chain 3B(MAP1LC3B)in mice liver tissue,while levels the ferroptosis biomarker GPX4 decreased(P＜0.05).Compared with SA high-dose groups,LNT intervention showed the reduced pathological liver damage and the downregulated levels of TNFα,IL-6,FTH1,and MAP1LC3B,while the level of GPX4 upregulated(P＜0.05).Western blot experiment showed that LNT intervention antagonized the upregulated levels of FTH1,and autophagy biomarker LC3B/A,and antagonized the increased co-expressions of FTH1 with LC3B or Ub protein in SA high-dose group(P＜0.05).Conclusions LNT antagonizes SA-exposed hepatic pathological injury and ferroptosis in mice,possibly associated with inhibition of TNFα-ferritinophagy signaling.

Objective"Lie flat"has recently become a buzz word describing a specific psychological state in some individuals.However,its psychological meaning and mechanisms remain unclear.Lack of motivation under certain conditions may be one of the key psychological characteristics of this condition.Methods Sixteen male SD rats were randomly assigned to two food-restriction levels(90%and 80%of baseline weight,respectively),and subjected to a sucrose-pellet self-administration task.The establishment of the model is divided into three stages.The rats were trained to self-administer sucrose pellets in a high-reward learning stage and in a low-reward learning stage.We then assessed"lie flat"behavior in a preference-test stage,in which rats could choose between high-reward-and low-reward-paired nose-pokes in a discrete choice procedure.Results Combining self-feeding training with Western blot,the results showed that:(1)rats showed significantly decreased nose-poke behavior for low-reward,i.e."lie flat"behavior,but not for high-reward,when≥90%of the maximal reward was obtained in the high-reward task and the effort(X)in the low-reward task increased to the breaking point.(2)Dopamine D1R expression in the NAc was significantly higher in the"lie flat"group compared with the"not lie flat"rats,while D2R expression was similar in both groups.Conclusions Rats can show"lie flat"behavior,and up-regulation of D1R expression in the NAc may be a key part of the molecular basis responsible for this motivation deficiency in"lie flat"behavior.These results extend our understanding of"lie flat"behavior,and provide a new paradigm for the study of its mechanism.

A ketogenic diet(KD)refers to an eating pattern designed to achieve a low-calorie content,minimum carbohydrate intake,high-fat consumption,and standard protein levels.A ketogenic diet is used in clinical practice to treat conditions including heart disease,diabetes,obesity,autism,glioblastoma,and other cancers.Although a ketogenic diet has not been recommended for any neurological disorders except epilepsy,extensive recent research suggests that such a diet may have a neuroprotective effect and may thus represent a new dietary therapy for the treatment of Parkinson's disease(PD).In this review,we discuss in detail the mechanisms responsible for the neuroprotective effects of a ketogenic diet in Parkinson's disease,with the aim of providing references for future clinical and experimental studies.