Enhancement of porcine in vitro embryonic development through luteolin‑mediated activation of the Nrf2/Keap1 signaling pathway

Background Oxidative stress,caused by an imbalance in the production and elimination of intracellular reactive oxygen species(ROS),has been recognized for its detrimental effects on mammalian embryonic development.Luteolin(Lut)has been documented for its protective effects against oxidative stress in various studies.However,its specific role in embryonic development remains unexplored.This study aims to investigate the influence of Lut on porcine embryonic development and to elucidate the underlying mechanism.Results After undergoing parthenogenetic activation(PA)or in vitro fertilization,embryos supplemented with 0.5μmol/L Lut displayed a significant enhancement in cleavage and blastocyst formation rates,with an increase in total cell numbers and a decrease in the apoptosis rate compared to the control.Measurements on D2 and D6 revealed that embryos with Lut supplementation had lower ROS levels and higher glutathione levels compared to the control.Moreover,Lut supplementation significantly augmented mitochondrial content and membrane potential.Intriguingly,activation of the Nrf2/Keap1 signaling pathway was observed in embryos supplemented with Lut,leading to the upregulation of antioxidant-related gene transcription levels.To further validate the relationship between the Nrf2/Keap1 signaling pathway and effects of Lut in porcine embryonic development,we cultured PA embryos in a medium supplemented with brusatol,with or without the inclusion of Lut.The positive effects of Lut on developmental competence were negated by brusatol treatment.Conclusions Our findings indicate that Lut-mediated activation of the Nrf2/Keap1 signaling pathway contributes to the enhanced production of porcine embryos with high developmental competence,and offers insight into the mechanisms regulating early embryonic development.

Water Extract of Rice False Smut Balls Activates Nrf2/HO-1 and Apoptosis Pathways,Causing Liver Injury

Ustiloxins are vital cyclopeptide mycotoxins originally isolated from rice false smut balls that form in rice spikelets infected by the fungal pathogen Ustilaginoidea virens.The toxicity of the water extract of rice false smut balls(RBWE) remains to be investigated.Studies have shown that RBWE may be toxic to animals,but toxicological evidence is still lacking.In this study,we found that the IC50 values of RBWE to BNL CL.2 cells at 24 and 48 h were 40.02 and 30.11 μg/m L,respectively,with positive correlations with dose toxicity and time toxicity.After treatment with RBWE,the number of BNL CL.2 cells decreased significantly,and the morphology of BNL CL.2 cells showed atrophy and wall detachment.RBWE induced DNA presynthesis phase arrest of BNL CL.2 cells,increased the proportion of apoptotic cells and inhibited cell proliferation.RBWE up-regulated reactive oxygen species(ROS) levels and lowered mitochondrial membrane potentials.Additionally,Western blot and q RT-PCR results suggested that RBWE exerted the above effects by promoting the Nrf2/HO-1 and caspase-induced apoptosis pathways in vitro and in vivo.The contents of alanine aminotransferase,aspartate aminotransferase,alkaline phosphatase,and total bile acids in the serum of mice from Institute of Cancer were significantly up-regulated by RBWE.At the same time,RBWE can lead to increases in ROS and malondialdehyde contents,decreases in contents of oxidized glutathione,glutathione and reduced glutathione,as well as decrease in catalase and superoxide dismutase activities in mouse liver tissues,demonstrating that oxidative stress occurred in mice.Moreover,liver damage was further detected by haematoxylin-eosin staining and electron microscopy to verify the damage to the mice caused by RBWE.In general,RBWE may cause hepatotoxicity in vivo and in vitro via the apoptosis pathway,which provides a reference for hepatotoxicity and its mechanism of action.

Scutellarin alleviates complete freund’s adjuvant-induced rheumatoid arthritis in mice by regulating the Keap1/Nrf2/HO-1 pathway

Scutellarin(SCU)is a herbal flavonoid glucuronide with multiple pharmacological activities,including antioxidant,anti-inflammation,vascular relaxation,anti-platelet,and myocardial protection.However,the effect of SCU on complete Freund’s adjuvant(CFA)-induced rheumatoid arthritis(RA)had not been studied.In this study,we investigated the beneficial effects of SCU in the CFA-induced RA mice model and the anti-arthritic activity was evaluated by paw edema.Enzyme-linked immunosorbent assay(ELISA)was carried out to evaluate the plasma levels of immunoglobulin(Ig)G,IgE,tumor necrosis factor(TNF)-α,interleukin(IL)-1β,IL-6,receptor activator of nuclear factor-κB ligand(RANKL),and osteoprotegerin(OPG).Histological slides were prepared from the harvested paws of mice to determine the pathological changes in the joints.The proportions of T helper type 1(Th1)and T helper type 2(Th2)cells of CD4+T lymphocyte subsets were analyzed by flow cytometry.The expression of Kelch-like ECHassociated protein 1(Keap1),nuclear factor erythroid 2-related factor 2(Nrf2),and heme oxygenase-1(HO-1)was analyzed using real-time quantitative PCR(RT-qPCR)and western blotting assays.The present study demonstrated that SCU prevented CFA-induced RA,and inhibited the expression of inflammation factors,IgG,IgE,TNF-α,IL-1β,and IL-6.While SCU also reduced the RANKL level,it increased OPG expression in RA mice.The Th1/Th2 ratio was significantly lower in mice treated with SCU.Additionally,HO-1 expression was reduced while the expression of Keap1 and Nrf2 was elevated following SCU treatment.Results provide preliminary evidence to employ SCU in arthritis treatment which might be related to the regulation of Th1/Th2 balance and the Keap1/Nrf2/HO-1 pathway.

An exploration on the protective mechanism of Xuduan Zhongzi prescription against epididymis oxidative damage in oligoasthenospermia model rats based on Nrf2-NQO1/γ-GCS signaling pathway

Objective:To investigate the protective mechanism of Xuduan Zhongzi prescription against epididymal oxidative damage in oligoasthenospermia model rats.Methods:Forty SD rats were randomly divided into blank group,model group,Xuduan Zhongzi prescription group(10g/kg)and L-carnitine group(0.1g/kg).Except blank group,all induced oligoasmospermia.The blank group and model group were given normal saline intragastric administration,the Xuduan Zhongzi prescription group was given Xuduan Zhongzi prescription solution intragastric administration,and the L-carnitine group was given L-carnitine intragastric administration.HE staining was used to observe the epididymis structure after 8 weeks.The concentration and activity rate of epididymis sperm were measured by sperm quality.MRNA and protein expression levels of Nrf2,NQO1 andγ-GCs in epididymis were detected by RT-qPCR and immunohistochemistry.Results:①HE staining:in the blank group,the epididymis tubes were arranged tightly and regularly,the tissue structure was complete,the epithelial cells were arranged orderly,and the lumen sperm were numerous and evenly distributed.The epididymis of model group showed structural atrophy,loose arrangement,enlarged mesenchyme,increased cell debris and significantly reduced sperm cells.Compared with the model group,the lumen lesions of epididymis in Xuduan Zhongzi prescription group and L-carnitine group were significantly improved,and the amount of normal sperm in lumen was increased and the distribution was uniform.②Results of sperm quality comparison among each group:sperm density and sperm motility rate:compared with blank group,sperm density and sperm motility rate in other groups were significantly decreased(P<0.05),and sperm density and sperm motility rate in model group were significantly decreased(P<0.05);Compared with model group,the sperm density and motility rate in Xuduan Zhongzi prescription group and L-carnitine group were significantly increased(P<0.05).③RT-qPCR and immunohistochemistry:Compared with the blank group,the mRNA and protein levels of Nrf2,NQO1 andγ-GCs in epididymal rats in model group were significantly decreased(P<0.05),while the mRNA and protein levels of Nrf2,NQO1 andγ-GCs were significantly increased in L-carnitine group and Continua seed formula group(P<0.05).Conclusion:Xuduan Zhongzi prescription can reduce oxidative stress damage and improve sperm quality of oligoasthenospermia.The mechanism may related to promoting the activation of Nrf2-NQO1/γ-GCS pathway in epididymis of oligoasthenospermia rats,and up-regulate the expressions of Nrf2,NQO1 andγ-GCS proteins.

Mechanism of hesperidin improving myocardial ischemia/reperfusion injury in type 2 diabetic rats through SIRT1/Nrf2/HO-1 signaling pathway

Objective:To observe the protective effect of hesperidin on myocardial ischemia/reperfusion injury in type 2 diabetes mellitus and its effect on SIRT1/Nrf2/HO-1 signaling pathway.Methods:50 Sprague-Dawley(SD)rats were randomly assigned to the normal control group(NC),model group,ischemia-reperfusion group(IR),hesperidin group,SIRT1 inhibitor group and hesperidin plus SIRT1 inhibitor group.In addition to NC,the rats in the remaining groups were replicated by intraperitoneal of high-fat diet combined with injection of streptozotocin for type 2 diabetic rats.After then,the myocardial ischemia/reperfusion injury(MIRI)rat model was established by LAd for 30 minutes with 2 hours reperfusion.He staining was used to observe the pathological changes of myocardial tissue,and the levels of serum LDH,CK-MB and SOD,GSH and MDA in myocardial tissue were detected by kit methods,and the expression abundance of related proteins in 4-HNE and SIRT1/Nrf2/HO-1 signal pathway were detected by immunohistochemistry and Western blot;Results:Hesperidin could significantly inhibit cardiomyocyte necrosis and inflammatory cell infiltration,reduce LDH activity,CK-MB and MDA level,and increase SOD activity,GSH and 4-HNE level,the differences were statistically significant when compared with IR group(P<0.01).In addition,compared with the ischemia-reperfusion group,the expressions of SIRT1,Nrf2 and HO-1 proteins in hesperidin group were significantly up-regulated,the differences were statistically significant(P<0.01);Conclusion:Hesperidin inhibits oxidative stress by activating SIRT1/Nrf2/HO-1 signaling pathway,and play a protective effect of myocardial ischemia reperfusion injury in diabetic rats.

Effect of pestle intervention in type 2 diabetic peripheral neuropathy on Keap1/Nrf2/ARE pathway and the relationship with oxidative stress

Objective:To investigate the effect of pestle needle treatment on Nrf2 pathway and the relationship with oxidative stress in diabetic peripheral neuropathy.Methods:Patients with DPN who met the inclusion criteria were randomly divided into control and test groups with 30 patients in each group in a 1:1 allocation ratio.Both groups were given basic treatment,and the pestle group was treated with needle pestle therapy 5 times a week for a total of 4 weeks of intervention.Serum SOD and GSH PX levels were examined by colorimetry before and after intervention;Serum Keap1/Nrf2/ARE signaling pathway related factors expression levels were measured by ELISA;Keap1 and Nrf2 mRNA expression was determined by RT-PCR.Results:Compared with the control group,SOD and GSH-Px in the test group were significantly increased,Keap1 expression was decreased,Nrf2 expression was increased,Keap1 mRNA expression was significantly decreased,and Nrf2 mRNA expression was significantly increased.Conclusions:the pestle needle may enhance the body's antioxidant capacity by modulating the Keap1/Nrf2/ARE signaling pathway to enhance the production of its downstream antioxidant enzymes SOD and GSH Px,thereby protecting and repairing the damaged peripheral nerves in DPN patients.

Effects of nuciferine on Nrf2/HO-1 signaling pathway in adipose tissue of obesity model rats

Objective:This study aimed to explore the therapeutic effect of nuciferine on high-fat diet-induced obesity in rats and the influence of nuciferine on nuclear factor erythroid 2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)signaling pathway in the adipose tissue.Methods:A total of 40 male Sprague Dawley(SD)rats were evenly divided into the normal,model,positive control,and nuciferine groups,using the random number table method.Except for the normal group,rats in the other groups were fed with high-fat diet for 12 weeks to establish the obesity model.During the model establishment,rats in the positive control group received atorvastatin calcium 2 mg/kg,rats in the nuciferine group received nuciferine 20 mg/kg,and rats in the normal and model groups received normal saline 2 mL,daily through intragastric administration for 12 consecutive weeks.After model establishment and administration,the body weight,Lee’s index,and blood lipids of rats in each group were measured,and hematoxylin and eosin(HE)staining was performed on the liver and adipose tissues to evaluate the therapeutic effect of nuciferine on obesity rat model.Additionally,the levels of superoxide dismutase(SOD),malondialdehyde(MDA),and glutathione peroxidase(GSH-Px)in the serum of rats in each group were determined,and the gene expressions of Nrf2 and HO-1 in the adipose tissue of rats in each group were detected through quantitative polymerase chain reaction(qPCR)to investigate the mechanism of action of nuciferine in the treatment of obesity.Results:After 12 weeks of model establishment and administration,we observed that compared with the model group,nuciferine could significantly reduce the body weight,Lee’s index,and serum triglyceride(TG),total cholesterol(TC),and low-density lipoprotein cholesterol(LDL-C)levels and increase the serum high-density lipoprotein cholesterol(HDL-C)level in obesity rat model(P<0.05 or P<0.01).HE staining revealed that nuciferine could significantly alleviate liver steatosis in obesity rat model and improve the cell morphology in epididymal adipose tissue.Moreover,nuciferine could elevate serum SOD and GSH-Px activities in obesity rat model and lower the serum MDA level(P<0.05 or P<0.01).The qPCR indicated that nuciferine could upregulate the gene expression of Nrf2 and HO-1 in the adipose tissue of obesity rat model(P<0.05 or P<0.01).

Rosmarinic acid elicits neuroprotection in ischemic stroke via Nrf2 and heme oxygenase 1 signaling

Rosmarinic acid（RA） can elicit a neuroprotective effect against ischemic stroke, but the precise molecular mechanism remains poorly understood. In this study, an experimental ischemic stroke model was established in CD-1 mice（Beijing Vital River Laboratory Animal Technology, Beijing, China） by occluding the right middle cerebral artery for 1 hour and allowing reperfusion for 24 hours. After intraperitoneally injecting model mice with 10, 20, or 40 mg/kg RA, functional neurological deficits were evaluated using modified Longa scores. Subsequently, cerebral infarct volume was measured using TTC staining and ischemic brain tissue was examined for cell apoptosis with TUNEL staining. Superoxide dismutase activity and malondialdehyde levels were measured by spectrophometry. Expression of heme oxygenase-1（HO-1）, nuclear factor erythroid 2-related factor 2（Nrf2）, Bcl-2, Bax, Akt, and phospho-Ser473 Akt proteins in ischemic brain tissue was detected by western blot, while mRNA levels of Nrf2, HO-1, Bcl-2, and Bax were analyzed using real time quantitative PCR. In addition, HO-1 enzyme activity was measured spectrophotometrically. RA（20 and 40 mg/kg） greatly improved neurological function, reduced infarct volume, decreased cell apoptosis, upregulated Bcl-2 protein and mRNA expression, downregulated Bax protein and mRNA expression, increased HO-1 and Nrf2 protein and mRNA expression, increased superoxide dismutase activity, and decreased malondialdehyde levels in ischemic brain tissue of model mice. However, intraperitoneal injection of a HO-1 inhibitor（10 mg/kg zinc protoporphyrin IX） reversed the neuroprotective effects of RA on HO-1 enzyme activity and Bcl-2 and Bax protein expression. The PI3 K/Akt signaling pathway inhibitor LY294002（10 mM） inhibited Akt phosphorylation, as well as Nrf2 and HO-1 expression. Our findings suggest that RA has anti-oxidative and anti-apoptotic properties that protect against ischemic stroke by a mechanism involving upregulation of Nrf2 and HO-1 expression via the PI3 K/Akt signaling pathway.

Hesperetin induces glyoxalase 1 enhancement in SH-SY5Y cells cultured with high glucose via Nrf2/ARE activation

OBJECTIVE To investigate the neuroprotective effects of hesperetin on central neurons under chronic high glucose,and the relationship to glyoxalase 1(Glo-1),a cytoprotective enzyme.METHODS The human neuroblas⁃toma SH-SY5Y cells were divided into 5 groups:normal glucose,high glucose(HG),HG plus low,middle,or high concentra⁃tion of hesperetin(1,5,25μmol·L^-1).After treatment for 72 h,neuron damages,Glo-1 expressions and functions,as well as Nrf2/ARE pathway and its regulating mechanisms were examined.RESULTS Hesperetin increased cell viability and decreased lactate dehydrogenase release,which was accompanied by the elevated activity,protein,and mRNA levels of Glo-1 as well as the enhanced Glo-1 functions in SH-SY5Y cells cultured with HG.Moreover,hesperetin activated Nrf2/ARE pathway as evidenced by the raised Nrf2 and p-Nrf2 levels in nucleus and up-regulation of γ-glutamycysteine synthase(γ-GCS),a well-known target gene of Nrf2/ARE pathway.Nevertheless,pretreatment with a PKC inhibitor(Go 6983)or an Akt inhibitor(MK-22062HCl,reflecting GSK-3β activation)abolished the effect of hesperetin on protein expressions of Glo-1 and γ-GCS.CONCLUSION Hesperetin exerted the neuroprotection by promoting Glo-1 function in central neurons in long-term HG condition,which was mediated by activation of Nrf2/ARE pathway;moreover,the increased Nrf2 phosphorylation and nuclear translocation mediated by PKC activation and/or GSK-3β inhibition were involved in the activation of Nrf2/ARE pathway by hesperetin.

Liaoqiao aqueous extract inhibits B16 melanoma growth involving MAPKs/Nrf2/HO-1 mediated anti-oxidation and anti-inflammation

Aim Forsythia suspensa （Thunb.） Vahl, Lianqiao in Chinese, is one of the most fundamental herbs in traditional Chinese medicine （TCM） with heat-clearing and detoxicating properties. In this study, we aimed to study the antitumor activity of Lianqiao aqueous extract against melanoma using cancer cell line-based in vitro and mouse allografl tumor in vivo models. Furthermore, we also investigated the underlying molecular mechanisms, par- ticularly the involvement of anti-inflammation and anti-oxidation properties in its antitumor activity. Methods The proliferation of cancer cells was measured by MTT assay. The transplanted B16-F10 melanoma in C57BL/6 mice were established and used for the evaluation of in vivo antitumor effect of LQ. Tumor growth was monitored twice a week. Ki67 and CD31 were used to detect cancer cell proliferation and angiogenesis in tumor, respectively. The anti-oxidative property of LQ was determined by measuring the levels of ROS, MDA and GSH. The anti-inflamma- tory effect of LQ was evaluated by measuring TNF-α and IL-6 using ELISA kits. Other protein expression was deter- mined by Western Blot. Results LQ strongly inhibited the growth of B16-F10 cells in vitro and the tumor growth in vivo. The survival time of tumor-bearing mice was significantly prolonged by LQ. LQ inhibited cancer cell prolif- eration and angiogenesis in tumor as evidenced by decreased expressions of Ki67 and CD31. Levels of ROS, MDA TNF-α and IL-6 decreased, while GSH increased in LQ treatment group, indicating a strong anti-oxidative and an- ti-inflammatory activity of LQ. The expression of antioxidant proteins Nff-2 and HO-1, tumor suppressors P53 and p-PTEN, and the MAPK pathways in tumor tissues were upregulated by LQ treatment. Conclusions LQ exhibited strong antitumor activity against B16-F10 murine melanoma both in vitro and in vivo. The antitumor effect of LQ in- volved the decreased oxidative stress and inflammation in tumor, which is closely related to the heat-clearing and detoxicating properties of LQ.

Protective effects of peptide KSPLY derived from Hericium erinaceus on H_(2)O_(2)-induced oxidative damage in HepG2 cells

Reactive oxygen species(ROS)-induced oxidative damage is strongly associated with the pathogenesis of chronic diseases,and natural antioxidant peptides have good abilities of scavenging ROS.The antioxidant activity of peptide Lys-Ser-Pro-Leu-Tyr(KSPLY)derived from Hericium erinaceus remains unclear.In the present study,the antioxidant effect and mechanism of KSPLY on H_(2)O_(2)-induced oxidative damage in HepG2 cells were investigated.The results indicated that KSPLY exhibited the antioxidant capacity in H_(2)O_(2)-induced HepG2 cells by enhancing superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),and catalase(CAT)activities.In comparison with the H_(2)O_(2)-treated damage group,the apoptosis rate,ROS level,and malondialdehyde(MDA)content of HepG2 cells treated with KSPLY were significantly decreased.The H.erinaceus-derived peptide KSPLY pretreatment promoted the expression of detoxification and antioxidant enzymes via the Keap1/Nrf2 signal pathway,thereby inhibiting the generation of ROS and MDA.In conclusion,the H.erinaceus-derived peptide KSPLY effectively protected HepG2 cells against H_(2)O_(2)-induced oxidative damage,and it provided a theoretical basis for the further development of new natural antioxidants.

Engineered Bacillus subtilis alleviates intestinal oxidative injury through Nrf2-Keap1 pathway in enterotoxigenic Escherichia coli(ETEC) K88-infected piglet

Engineered probiotics can serve as therapeutics based on their ability of produce recombinant immune-stimulating properties.In this study,we built the recombinant Bacillus subtilis WB800 expressing antimicrobial peptide KR32(WB800-KR32)using genetic engineering methods and investigated its protective effects of nuclear factor-E2-related factor 2(Nrf2)-Kelch-like ECH-associated protein 1(Keap1)pathway activation in intestinal oxidative disturbance induced by enterotoxigenic Escherichia coli(ETEC)K88 in weaned piglets.Twenty-eight weaned piglets were randomly distributed into four treatment groups with seven replicates fed with a basal diet.The feed of the control group(CON)was infused with normal sterilized saline;meanwhile,the ETEC,ETEC+WB800,and ETEC+WB800-KR32 groups were orally administered normal sterilized saline,5×10^(10)CFU(CFU:colony forming units)WB800,and 5×10^(10)CFU WB800-KR32,respectively,on Days 1-14 and all infused with ETEC K881×10^(10)CFU on Days 15-17.The results showed that pretreatment with WB800-KR32 attenuated ETEC-induced intestinal disturbance,improved the mucosal activity of antioxidant enzyme(catalase(CAT),superoxide dismutase(SOD),and glutathione peroxidase(GPx))and decreased the content of malondialdehyde(MDA).More importantly,WB800-KR32 downregulated genes involved in antioxidant defense(GPx and SOD1).Interestingly,WB800-KR32 upregulated the protein expression of Nrf2 and downregulated the protein expression of Keap1 in the ileum.WB800-KR32 markedly changed the richness estimators(Ace and Chao)of gut microbiota and increased the abundance of Eubacterium_rectale_ATCC_33656 in the feces.The results suggested that WB800-KR32 may alleviate ETEC-induced intestinal oxidative injury through the Nrf2-Keap1 pathway,providing a new perspective for WB800-KR32 as potential therapeutics to regulate intestinal oxidative disturbance in ETEC K88 infection.

Garcinia xanthochymus extract protects PC12 cells from H2O2-induced apoptosis through modulation of PI3K/AKT and NRF2/HO-1 pathways

The aim of the present study was to investigate the protective effects and underlying mechanisms of Garcinia xanthochymus, a perennial medicinal plant native to Yunnan, China, against H2 O2-induced oxidative damage in rat pheochromacytoma PC12 cells. Preincubation of PC12 cells with fruit Et OAc fraction(fruit-EFr., 12.5–50 μmol·L^(-1)) of G. xanthochymus for 24 h prior to H_2O_2 exposure markedly improved cell viability and increased the activities of antioxidant enzymes(superoxide dismutase, catalase, and heme oxygenase-1 [HO-1]), prevented lactate dehydrogenase release and lipid peroxidation malondialdehyde production, attenuated the decrease of matrix metalloproteinases(MMP), and scavenged reactive oxygen species(ROS). Fruit-EFr. also reduced BAX and cytochrome C expression and improved BCL-2 expression, thereby decreasing the ratio of BAX to BCL-2. Fruit-EFr. activated the nuclear translocation of NRF2 to increase HO-1 and induced the phosphorylation of AKT. Its cytoprotective effect was abolished by LY294002, a specific inhibitor of PI3 K. Taken together, the above findings suggested that fruit-EFr.of G. xanthochymus could enhance cellular antioxidant defense capacity, at least in part, through upregulating HO-1 expression and activating the PI3 K/AKT pathway and that it could suppress H_2O_2-induced oxidative damage via PI3 K/AKT and NRF2/HO-1 signaling pathways.

Arsenic Trioxide Combining Leflunomide Activates Nrf2-ARE-HO-1 Signaling Pathway and Protects Heart Xenografts

Objective:To investigate the molecular mechanisms underlying the effects of arsenic trioxide(As_(2)0_(3))in combination with leflunomide on the hamster-to-rat heart xenotransplant.Methods:Transplantation of LVG hamster hearts to Lewis rats was performed by anastomosis of vessels in the neck using end-to-end anastomosis with a non-suture cuff technique.Four groups of recipient rats(n=6 in each)were treated with normal saline(control),As_(2)0_(3)[5 mg/(kg*day)intraperitoneally],leflunomide[5 mg/(kg*d)orally],or leflunomide[5 mg/(kg.d)+As_(2)0_(3)5 mg/(kg.d)]in combination.Donor hearts and/or rat spleens were harvested and analyzed 4 days after transplantation.Quantitative reverse-transcription polymerase chain reaction and Western blot analysis were performed to detect the expression of the nuclear factor erythroid-derived factor 2-related factor(Nrf2)and its target gene heme oxygenase-1(HO-1),Treg cell marker fork-head Box P3(FOXP3),apoptosis-associated proteins Bcl-2,Bax,and cleaved caspase-3.Immunohistochemical staining was used to detect the levels of inflammatory natural killer cell and macrophage infiltration,intercellular cell adhesion molecule-1(ICAM-1)and complement C3.Results:Expression of Nrf2-ARE-HO-1 signaling pathway was upregulated in heart xenografts in rats treated with As_(2)0_(3) plus leflunomide compared with control rats or rats treated with either drug alone(P<0.01),and this was accompanied by an increased Treg cells in the recipient rat spleen(P<0.01).In contrast,the expressions of Bax,cleaved caspase-3,ICAM-1,and complement C3,and infiltration of inflammatory cells in the xenografts were inhibited by As_(2)0_(3) plus leflunomide treatment(P<0.01).Conclusion:Combination treatment with As_(2)0_(3) and leflunomide protected hamster heart-xenografts in recipient rats.

鸢尾草和体外冲击波对去势大鼠骨折愈合的影响

[目的]探究鸢尾素联合体外冲击波(extracorporeal shock waves,ESW)治疗去势大鼠骨质疏松性骨折的作用与机理.[方法]80只SD大鼠随机分为5组,每组16只.分别为假手术组、模型组、模型+ESW组、模型+鸢尾素组、模型+ESW+鸢尾素组.除假手术组仅行手术切口外,其他各组先后行双侧卵巢切除和左侧股骨骨折内固定,术后分别给予相应处理,其中,ESW治疗每周1次,鸢尾素组骨折部位局部注射.治疗后8周处死动物,行骨密度、破骨细胞指数、micro-CT、Western blot和RT-qPCR检测.[结果]在骨密度和破骨细胞指数均由优至劣低依次为,假手术组>模型+ESW+鸢尾素>模型+ESW组,或模型+鸢尾素组>模型组(P<0.05).MicroCT检测,包括BS/BV、Tb.N、Tb.Th和Tb.Sp,由优至劣低依次为,假手术组>模型+ESW+鸢尾素>模型+ESW组,或模型+鸢尾素组>模型组(P<0.05).Western blot检测BMP2、VEGF、Nrf2F的OH-1的蛋白相对表达量由高至低依次为,假手术组>模型+ESW+鸢尾素>模型+鸢尾素组,或模型+ESW组>模型组(P<0.05).RT-qPCR检测Nrf2和OH-1mRNA相对表达量由高至低依次为,假手术组>模型+ESW+鸢尾素>模型+鸢尾素组,或模型+ESW组>模型组(P<0.05).[结论]鸢尾素联合ESW可激活Nrf2/OH-1通路并促进VEGF和BMP2蛋白的表达,促进骨质疏松大鼠的骨折愈合.