EXPLORING THE THERAPEUTIC POTENTIAL OF AM114: A BORONIC CHALCONE DERIVATIVE INDUCE APOPTOSIS AND SUPPRESS PROINFLAMMATORY CYTOKINES AND CHEMOKINES IN INTERLEUKIN-1? STIMULATED HUMAN THP-1 DERIVED MACROPHAGES

Objective: Chalcones and their derivatives display a wide range of pharmacological activities. This study examined the effects of AM114, a boronic-chalcone derivative, on human THP-1-derived macrophages with and without interleukin-1? (IL-1?) stimulation. Methods: AM114 and Aspirin-treated THP-1-derived macrophages underwent activation with or without interleukin-1?. The IC50 concentrations of AM114 and Aspirin were determined through an MTT test. Apoptosis was measured using various techniques, including staining with acridine orange/Ethidium bromide, Hoechst 33342, and rhodamine 123 assays. Caspase-3 activity was measured using the spectrofluorimetric technique, while DNA fragmentation was assessed via agarose gel electrophoresis. Pro-inflammatory cytokines such as interleukin-6 (IL-6) and chemokines like interleukin-8 (IL-8) were measured using enzyme-linked immunosorbent assays.Results: AM114 and Aspirin showed dose-dependent cytotoxic effects on THP-1 macrophages. Induction of apoptosis was detected in AM114-treated THP-1 macrophages activated with IL-1? compared to macrophages without IL-1?. The gradation of dye uptake, membrane blebbing, increased caspase-3 activity, and DNA fragmentation ensures the induction of apoptosis, which indicates the cell's morphological changes, biochemical processes, and mitochondrial activity. Treating AM114 in IL-1?-activated THP-1 macrophages significantly reduced pro-inflammatory cytokines (IL-6) and chemokines (IL-8), suggesting its anti-cytokine potential in inflammatory diseases.Conclusion: The study results emphasize that AM114 could act as an anti-inflammatory agent by triggering apoptosis and reducing the release of cytokines and chemokines in inflammatory conditions. As a result, it may be used as a therapeutic option for inflammatory diseases.

Effect of Modified Shenhong Tongluo Prescription on Cell Apoptosis in Rats with Myocardial Ischemia-reperfusion Injury by Bcl-2/Bax/Caspase-3 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of modified Shenhong Tongluo prescription on cell apoptosis in rats with myocardial ischemia-reperfusion injury （MIRI）. MethodSixty Sprague-Dawley （SD） rats were randomly divided into a blank group， a model group， low-， medium-， and high-dose groups of modified Shenhong Tongluo prescription， and a simvastatin group. Except for the blank group， a rat model of MIRI was prepared by ligating the left anterior descending coronary artery. Starting from the first day after successful modeling， the blank group （1.0 mL·kg-1 physiological saline）， model group （1.0 mL·kg-1 physiological saline）， low-， medium-， and high-dose groups of modified Shenhong Tongluo prescription （1.031， 2.063， and 4.126 g·kg-1 Shenhong Tongluo prescriptiona standard concentrate）， and simvastatin group （0.71 mg·kg-1 simvastatin） were orally administered once daily for 2 weeks. Hematoxylin-eosin （HE） staining was used to observe the pathological changes of cardiomyocytes. Enzyme-linked immunosorbent assay （ELISA） was used to detect the levels of serum creatine kinase isoenzyme （CK-MB）， interleukin-6 （IL-6）， and tumor necrosis factor-α （TNF-α）. TdT-mediated dUTP nick-end labeling（TUNEL） staining was used to detect the apoptosis rate of rat cardiomyocytes. Western blot was used to detect the expression levels of apoptosis-related proteins B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X protein （Bax）， and caspase-3. ResultCompared with the blank group， in the model group， HE staining showed disturbed arrangement of cardiomyocytes， incomplete fibers， focal necrosis of cardiomyocytes， and inflammatory cell infiltration； serum CK-MB， IL-6， and TNF-α levels were significantly increased （P<0.05）； apoptosis rate of cardiomyocytes was significantly increased （P<0.01）， with significantly increased expression levels of Bax and Caspase-3 proteins， and significantly decreased Bcl-2 expression （P<0.05）. Compared with the model group， the low-， medium-， and high-dose groups of modified Shenhong Tongluo prescription significantly reduced CK-MB， IL-6， and TNF-α levels （P<0.05）， significantly downregulated cardiomyocyte apoptosis rate （P<0.05）， significantly decreased Bax and Caspase-3 proteins， and significantly increased Bcl-2 expression levels （P<0.01）. In the modified Shenhong Tongluo prescription groups， the expression levels of Bax and Caspase-3 proteins significantly decreased with increasing dosage， while the expression level of Bcl-2 significantly increased with increasing dosage of modified Shenhong Tongluo prescription （P<0.05）. ConclusionShenhong Tongluo prescription can alleviate myocardial tissue pathological damage and reduce myocardial cell apoptosis， possibly by inhibiting Caspase-3 and Bax expression and promoting Bcl-2 expression.

Role of HK2 and VDAC1 in Diacetylmorphine-induced Cardiomyocyte Apoptosis / 昆明医科大学学报

Objective To investigate the role of HK2 and VDAC1 in diacetylmorphine-induced cardiomyocyte apoptosis.Methods A dose-escalation method was used to establish a rat model of diacetylmorphine addiction.Forty SD rats were randomly divided into three groups,the normal group(n=10)was injected with an equal amount of saline subcutaneously,the model group(n=15)was injected with 5 mg/kg of diacetylmorphine for the first time,and then the dose was increased by 2.5 mg/(kg·d)day by day for 20 days,and the group of model +10 D(n=15)continued to increase the dose based on the model group up to the 10th day.Lactate dehydrogenase(LDH)and glutamic oxaloacetic transaminase(GOT)were detected by ELISA;HE staining was used to observe the pathological changes of myocardial tissues in each group;TUNEL staining was used to detect apoptosis in myocardial tissues in each group;and immunohistochemistry,RT-q-analysis,and immunochemistry were used to detect apoptosis in myocardial tissues in each group.Immunohistochemistry,RT-qPCR and Western bl-ot were used to detect the mRNA and protein expression of HK2,VDAC1 and apoptosis-related factors.Results HE staining revealed that myocardial tissues exhibited different degrees of damage with the prolongation of diacetylmorphine intervention.Compared with the normal group,serum LDH,GOT content and myocardial apoptosis rate increased in the model group,mRNA and protein levels of HK2 and anti-apoptotic factor Bcl-2 decreased,mRNA and protein levels of VDAC1 and pro-apoptotic factors Bax and Caspase-3 increased,and the protein level of Clevead Caspase-3 increased;in the model +10 D group the above indexes,there was a statistically significant difference(P<0.05).Conclusion Diacetylmorphine can cause cardiomyocyte apoptosis,and VDAC1 may be involved in the process of cardiomyocyte apoptosis caused by diacetylmorphine.

Exercise-induced chronic fatigue impairs working memory in rats via basal ganglia output nuclei / 神经解剖学杂志

Objective:To investigate possible neuromodulatory mechanisms involved in the involvement of parvalbu-min(PV)expression in the basal ganglia output nuclei,entopeduncular nucleus(EPN)and substantia nigra pars etic-ulata(SNr),in exercise-induced chronic fatigue impairs working memory capacity.Methods:Male SD rats were divid-ed into control group and Fatigue group by random number method,and a three-stage incremental load treadmill training program was selected to establish a chronic exhaustion exercise-induced fatigue rat model.The working memory ability of rats was assessed by the Y-maze autonomous alternation experiment.Immunohistochemical staining was used to ob-serve the expression of parvalbumin(PV)positive neurons and cysteine aspartate-specific protease-3(caspase-3)in EPN and SNr of rats.Results:The accuracy of voluntary alternation in the fatigue group was obviously lower than that in control group(P＜0.05).The results of immunohistochemical staining showed that the density of PV positive neu-rons and the degree of positive fiber staining in EPN and SNr in the fatigue group were obviously lower than those in the control group(P＜0.05,P＜0.01).The number of caspase-3 positive cells per unit area of EPN and SNr in the fa-tigue group was obviously higher than that in the control group(P＜0.05,P＜0.01).Conclusion:The mechanism of impairing working memory in rats caused by exercise-induced chronic fatigue may be related to the apoptosis of PV posi-tive neurons in EPN and SNr.

The mechanism of target regulating of miR-421 to Menin/Caspase-3 pathway for depression / 实用医学杂志

Objective To explore the mechanism of miR-421 affecting the occurrence and development of depression.Methods A depressive rat model was established by single intraperitoneal injection of lipopolysaccharide(LPS),and depressive behavior was detected by glucose preference test and open-field test.miRNA microarray chips and RT-PCR were used to analyze the expression level of miR-421 in hippocampus of the depressed rats.TargetScan database and mi RDB database were used to predict the target genes of miR-421.Dual luciferase reporter gene assay was used to observe the binding of miR-421 to the target genes.The impact of over-expression and inhibition of miR-421 on target genes was observed,then the influence of over-expression and inhibition of target genes on downstream factors was observed,and the related mechanism of miR-421 on depression was explored.Results miRNA microarray chips and RT-PCR assay showed that miR-421 was highly expressed in the hippocampus of the depressed rats(P<0.001),Inhibition of miR-421 expression could significantly restore the body weight and exercise ability of the depressed rats(P<0.001).Binding targets of Menin and miR-421 were predicted by TargetScan database,and interaction between Menin and miR-421 was demonstrated by dual-luciferase reporter gene assay.Menin expression was down-regulated while miR-421 was overexpressed(P<0.001),whereas it was up-regulated as miR-421 was inhibited(P<0.001).qPCR indicated that expressions of Caspase-3 and NF-κB in the hippocampus of the depressed rats was significantly increased(P<0.001),and IL-1β expression in the hippo-campus was significantly increased(P<0.01).When the expression of Menin was inhibited,the expressions of Caspase-3,NF-κB and IL-1β were increased(P<0.001),while the expressions of Caspase-3,NF-κB and IL-1β were decreased when Menin was overexpressed(P<0.001).Conclusions Inhibition of miR-421 expression can increase Menin expression,decrease Caspase-3 content,and reduce neuroinflammatory response,thereby improving depressive symptoms.

Grape seed extract inhibits apoptosis of rat growth plate chondrocytes and promotes tibial bone growth / 中国组织工程研究

BACKGROUND:Grape seed extract can inhibit chondrocyte apoptosis and aging,and improve osteoarthritis.However,the effects of grape seed extract on the apoptosis of chondrocytes in the growth plate and tibial growth are still unclear. OBJECTIVE:To investigate the effect of grape seed extract on interleukin-1β-induced apoptosis in rat growth plate cells and on tibial bone growth. METHODS:(1)Cell experiment:Growth plate chondrocytes from Sprague-Dawley rats were isolated,cultured,and identified.The cells were then randomly divided into control group,model group,grape seed extract group,miR-138-5p NC group and miR-138-5p inhibitor group.In the model group,20 ng/mL interleukin-1β was used to induce apoptosis in rat growth plate chondrocytes.In the grape seed extract group,20 ng/mL interleukin 1β was added along with 10 μmol/L grape seed extract solution for 48 hours.Cells in the miR-138-5p NC and inhibitor groups were transfected with 5 nmol/L miR-138-5p NC and 5 nmol/L miR-138-5p inhibitor for 12 hours,respectively,followed by addition of 20 ng/mL interleukin-1β.qRT-PCR was used to detect miR-138-5p and caspase-3 expression.Luciferase reporter assay was used to analyze the relationship between miR-138-5p and caspase-3 targeting.Cell counting kit-8 was used to detect cell proliferation activity.Flow cytometry was used to detect cell apoptosis.Caspase-3 and Bcl-2 protein expressions were detected by western blot.(2)Animal experiment:The animals were divided into normal control group,grape seed extract group and miR-138-5p inhibitor group.The effects of grape seed extract on epiphyseal closure and tibial growth of the tibial plateau in rats were observed. RESULTS AND CONCLUSION:miR-138-5p had a targeting relationship with caspase-3.Compared with the control group,cell proliferation was significantly reduced,apoptosis was significantly increased(P＜0.01),miR-138-5p,Bcl-2 expression was reduced(P＜0.01),and caspase-3 expression was increased(P＜0.01)in the model group.Compared with the mod group,the grape seed extract group showed a significant increase in cell proliferation,a significant decrease in apoptosis(P＜0.01),an increase in miR-138-5p and Bcl-2 expression(P＜0.01)and a decrease in caspase-3 expression(P＜0.01).Compared with the grape seed extract group,the miR-138-5p inhibitor group showed a significant decrease in cell proliferation,a significant increase in apoptosis(P＜0.01),a decrease in miR-138-5p and Bcl-2 expression(P＜0.05 or P＜0.01),and an increase in caspase-3 expression(P＜0.05).Intragastric administration of grape seed extract could delay epiphyseal closure of the tibial plateau and promote tibial bone growth in rats,whereas miR-138-5p inhibitor intervention inhibited the effect of grape seed extract on tibial bone growth in rats.To conclude,grape seed extract can inhibit apoptosis of rat growth plate chondrocytes through regulating the miR-138-5p/caspase-3 pathway,improve epiphyseal closure of rat tibial plateau and promote tibial bone growth.

Effects of aerobic exercise on the expression of Notch1 and Caspase-3 in the hippocampus of Alzheimer's disease mice / 中国组织工程研究

BACKGROUND:β-amyloid protein and Tau protein have adverse effects on the cognitive function of Alzheimer's disease patients,and Notch1 and Caspase-3 can regulate the expression of β-amyloid protein and Tau protein.It is not clear whether Notch1 and Caspase-3 mediate the process of aerobic exercise to improve the cognitive ability of Alzheimer's disease patients.At present,there is a lack of studies on the effect of long-term aerobic exercise on the expression of Notch1 and Caspase-3 in the hippocampus of Alzheimer's disease mice. OBJECTIVE:To observe the expression of Notch1 and Caspase-3 in the hippocampus of Alzheimer's disease mice undergoing long-term aerobic exercise and to investigate the effects of Notch1 and Caspase-3 in Alzheimer's disease mice. METHODS:Wild type and APP/PS1 double-transgenic Alzheimer's disease mice aged 3 months were randomly divided into four groups:wild control group,wild exercise group,Alzheimer's disease control group and Alzheimer's disease exercise group,with 20 mice in each group.Mice in the control groups were not subjected to exercise,while those in the exercise groups received aerobic exercise intervention for 5 months.After the exercise intervention,Morris water maze was used to detect the spatial learning and memory ability of mice.Real-time PCR,immunofluorescence and western blot were used to detect the expressions of Aβ1-42,Tau,Notch1 and Caspase-3 in the hippocampal tissues of mice in each group. RESULTS AND CONCLUSION:The spatial learning and memory ability of Alzheimer's mice was significantly worse than that of wild-type mice(P＜0.05).The spatial learning and memory ability of mice in the exercise groups were significantly better than that in the corresponding control groups(P＜0.05).The expressions of Aβ1-42,Tau,Notch1 and Caspase-3 in the hippocampus were significantly higher in the Alzheimer's disease control group than the wild control group(P＜0.05)and were significantly lower in the Alzheimer's disease exercise group than the Alzheimer's disease control group(P＜0.05).To conclude,long-term aerobic exercise can improve the spatial learning and memory ability of Alzheimer's disease mice,which may be related to the decreased expression of Notch1,Caspase-3,Aβ1-42 and Tau protein in the hippocampus of Alzheimer's disease mice.

Effects of Ziziphi Spinosae Semen-Albiziae Flos on the IRE1α/ASK1/JNK Pathway of Endoplasmic Reticulum Stress in Hippocampus of Depression Model Rats / 中药新药与临床药理

Objective Based on the endoplasmic reticulum stress(ERS)inositol-requiring enzyme-1α(IRE1α)/apoptosis signal-regulated kinase 1(ASK1)/c-Jun N-terminal kinase(JNK)pathway to investigate the intervention effect of Ziziphi Spinosae Semen(ZSS)-Albiziae Flos(AF)on the depression model of rats,which were prepared by solitary rearing combined with chronic unpredictable mild stress(CUMS).Methods A total of 144 rats were randomly divided into normal group,model group,high-,medium-and low-dose groups of ZSS-AF,and Venlafaxine group.In addition to the normal group,the rats in other groups were isolated for 21 days combined with CUMS to prepare the depression model.The behavioral changes of rats were observed by open field test and Morris water maze test.The ultrastructural changes of hippocampal neurons were observed by transmission electron microscope.TUNEL staining was used to observe the apoptosis of nerve cells in hippocampus.The protein expression levels of IRE1α,phosphorylated(P)-IRE1α,ASK1,P-ASK1,JNK,P-JNK,B cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax)and cysteme aspartate specific protease-3(Caspase-3)in hippocampus were detected by Western Blot.The mRNA expression levels of IRE1α,ASK1,JNK,Bax,Bcl-2 and Caspase-3 in hippocampus were detected by Real-Time PCR.Results Compared with the normal group,the scores of horizontal movement and vertical movement in the open field test of rats in the model group were decreased(P＜0.01).In the water maze test,the escape latency was increased and the number of crossing the original platform was decreased(P＜0.01).The number of hippocampal apoptosis was increased(P＜0.01).The protein expression levels of P-IRE1 α/IRE1 α,P-ASK1/ASK1,P-JNK/JNK,Bax,Caspase-3 and mRNA expressions of IRE1α,ASK1,JNK,Bax,Caspase-3 in hippocampus were increased,while the protein and mRNA expression levels of Bcl-2 were decreased(P＜0.01).Compared with model group,the scores of horizontal movement and vertical movement in the open field test of rats in each dose ZSS-AF groups and Venlafaxine group were increased(P＜0.01).In the water maze test,the escape latency was decreased and the times of crossing the original platform was increased(P＜0.01).The number of hippocampal apoptosis was decreased(P＜0.01).The mRNA expression levels of P-IRE1α/IRE1α,P-ASK1/ASK1,P-JNK/JNK,Bax,Caspase-3 protein and IRE1α,ASK1,JNK,Bax,Caspase-3 in hippocampus were decreased,while the protein and mRNA expression levels of Bcl-2 were increased(P＜0.05,P＜0.01).The effect of medium-dose ZSS-AF group was better than that of high-and low-dose groups.Conclusion ZSS-AF may play an antidepressant role by regulating IRE1α/ASK1/JNK pathway of endoplasmic reticulum stress.

Hei Xiaoyaosan Regulates Fas/FasL/Caspase-8/Caspase-3 Signaling Pathway to Inhibit Neuronal Apoptosis in AD Rats / 中国实验方剂学杂志

ObjectiveTo explore the effect and mechanism of Hei Xiaoyaosan in regulating the tumor necrosis factor receptor superfamily member 6 （Fas）/Fas ligand （FasL）/cysteine protease-8 （Caspase-8）/cysteine protease-3 （Caspase-3） signaling pathway to intervene in neuronal apoptosis and prevent Alzheimer's disease （AD）. MethodNinety SPF-grade SD male rats of 4 months old were selected and randomly grouped as follows： 10 rats in the blank group， 10 rats in the sham group （bilateral hippocampus injected with 1 μL normal saline）， and 70 rats in the modeling group ［bilater hippocampus injected with 1 μL amyloid-beta protein 1-42 （Aβ1-42） solution for the modeling of AD］. Fifty successfully modeled rats were selected and randomly assigned into model， donepezil hydrochloride （0.45 mg·kg-1）， and high-， medium-， and low-dose （15.30， 7.65， 3.82 g·kg-1） Hei Xiaoyaosan groups. Rats were administrated with corresponding agents by gavage once a day for 42 days. Terminal-deoxynucleoitidyl transferase-mediated nick end labeling （TUNEL） was employed to observe the apoptosis of neurons in the cortex and hippocampus， and immunohistochemistry （IHC） was used to detect the expression of B-cell lymphoma-2 （Bcl-2） and Bcl-2-associated X protein （Bax） in the hippocampus. Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） was employed to determine the mRNA levels of Fas， FasL， and Fas-associated protein with death domain （Fadd）. Western blot was used to determine the protein levels of Fas， FasL， Fadd， Caspase-3， cleved Caspase-3， Caspase-8， and cleved Caspase-8. ResultCompared with the blank group and sham group， the model group showed increased apoptosis rate in the cortex and hippocampus （P<0.01）， elevated Bax level （P<0.01）， lowered Bcl-2 level （P<0.01）， up-regulated mRNA levels of Fas， FasL， and Fadd in the hippocampus （P<0.01）， and up-regulated protein levels of Fas， FasL， Fadd， cleaved Caspase-3， and cleaved Caspase-8 （P<0.01）. Compared with the model group， donepezil hydrochloride and Hei Xiaoyaosan at high and medium doses decreased the apoptosis rate in the cortex and hippocampus （P<0.05， P<0.01）， lowered the Bax level （P<0.01）， elevated the Bcl-2 level （P<0.01）， and down-regulated the mRNA levels of Fas， FasL， and Fadd and the protein levels of Fas， FasL， Fadd， cleaved Caspase-3， and cleaved Caspase-8 （P<0.05， P<0.01） in the hippocampus. Low-dose Hei Xiaoyaosan decreased the apoptosis rate in the cortex and hippocampus （P<0.05， P<0.01）， lowered the Bcl-2 level （P<0.01）， and down-regulated the mRNA levels of FasL and Fadd （P<0.05） and the protein levels of Fas， FasL， Fadd， cleaved Caspase-3， and cleaved Caspase-8 （P<0.05） in the hippocampus. ConclusionHei Xiaoyaosan can protect neurons in the cortex and hippocampus of AD rats by inhibiting the apoptosis mediated by the Fas/FasL/Caspase-8/Caspase-3 signaling pathway.

Salvianolic Acid F Regulates Bax/Caspase-3/GSDME Signaling Pathway to Inhibit Pyroptosis of HK-2 Cells / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of salvianolic acid F （Sal F） in repairing the high glucose-induced injury in human kidney-2 （HK-2） cells via the B-cell lymphoma-2 （Bcl-2）-associated X protein （Bax）/cysteinyl aspartate-specific proteinase 3 （Caspase-3）/gasdermin-E （GSDME） pathway. MethodThe cell counting kit-8 （CCK-8） was used to measure the relative viability of HK-2 cells exposed to high glucose and different concentrations （2.5， 5， 10， 20 μmol·L-1） of Sal F and the relative viability of HK-2 cells treated with Sal F for different time periods. The levels of lactate dehydrogenase （LDH） and interleukin-1β （IL-1β） in the supernatant of the cell culture were measured by the LDH assay kit and enzyme-linked immunosorbent assay （ELISA） kit， respectively. Flow cytometry combined with Annexin V-FITC/propidium iodide （PI） and Hoechst 33342/PI staining was employed to reveal the proportion of PI-positive HK-2 cells exposed to high glucose. Western blotting was employed to determine the protein levels of Bax， Bcl-2， cytochrome C， cysteinyl aspartate-specific proteinase （Caspase）-9， Caspase-3， and GSDME in the HK-2 cells exposed to high glucose and treated with Sal F. The 2，7-dichlorodihydrofluorescein diacetate fluorescence probe （DCFH-DA） and mitochondrial membrane potential assay kit （JC-1） were used to determine the production of reactive oxygen species （ROS） and the mitochondrial membrane potential in the HK-2 cells exposed to high glucose and treated with Sal F. ResultCompared with the blank group， the model group showed decreased cell viability （P<0.01）， elevated levels LDH and IL-1β， increased proportion of PI-positive cells （P<0.01）， up-regulated protein levels of Bax， cytochrome C， Caspase-9， Caspase-3， and GSDME （P<0.01）， down-regulated protein level of Bcl-2 （P<0.01）， decreased mitochondrial membrane potential， and excessive ROS accumulation. Compared with the model group， Sal F repaired the high glucose-induced injury in HK-2 cells （P<0.05）， lowered the levels of LDH and IL-1β （P<0.05， P<0.01）， and decreased the proportion of PI-positive cells （P<0.01）. In addition， Sal F down-regulated the protein levels of Bax， cytochrome C， Caspase-9， Caspase-3， and GSDME and up-regulated the protein level of Bcl-2 （P<0.05， P<0.01）， increased the mitochondrial membrane potential， and decreased the accumulation of ROS in HK-2 cells. ConclusionSal F can reduce the production of ROS， restore the balance of mitochondrial membrane potential， and inhibit pyroptosis via the Bax/Caspase-3/GSDME signaling pathway to repair the high glucose-induced injury in HK-2 cells.

Tripterine promotes pyroptosis of liver cancer cells by regulating ATF4/caspase-3/GSDME signaling pathway / 中国药理学通报

Aim To explore the new mechanism of triptolide (TRI) inhibiting the progression of hepatocellular carcinoma (HCC) . Methods Different concentrations (0, 0 . 5, 2, and 8 jjunol • L~) of TRI were administered to act on liver cancer cells, and then the cell phenotypes and possible mechanisms were explored using experimental methods such as CCK-8, cell cloning, Transwell, and protein immunoblotting; siRNA was used to interfere with the target gene GSDME and its role was determined. Finally, the mechanism of TRI inhibiting the growth of HCC cells in vivo was validated using a transplanted tumor model. Results TRI could inhibit the proliferation, cloning, and invasion of HCC cells, and promote cell apoptosis. Immunoblotting results showed that the expression of GSDME was significantly upregulated in HepG2 or He-pal-6 hepatocellular carcinoma after TRI treatment, while the expression of cleaved caspase-3 and PARP also significantly increased. Knocking out GSDME could partially reverse TRI-induced cell apoptosis. At the same time, cells knocked down by GSDME had stronger cloning and migration abilities, and the apoptosis rate was reduced compared to the TRI treatment group alone. In vivo experiments showed that TRI inhibited HCC tumor growth, and the TRI + siGSDME group had a faster tumor growth rate than the TRI treatment group alone did. In addition, after TRI stimulation, p-eIF2a and ATF4 in HepG2 and Hepal-6 cells significantly increased. The immunofluorescence results showed a dose-dependent increase in the number of ATF4 positive cells in HepG2 and Hepal-6 cells after TRI stimulation. Conclusion The inhibitory effect of TRI on the growth and invasion of liver cancer cells may be related to its regulation of the ATF4/caspase-3/GSDME signaling pathway and promotion of liver cancer cell apoptosis.

Polysaccharide of Alocasia cucullata Exerts Antitumor Effect by Regulating Bcl-2, Caspase-3 and ERK1/2 Expressions during Long-Time Administration / 中国结合医学杂志

OBJECTIVE@#To study the in vitro and in vivo antitumor effects of the polysaccharide of Alocasia cucullata (PAC) and the underlying mechanism.@*METHODS@#B16F10 and 4T1 cells were cultured with PAC of 40 µg/mL, and PAC was withdrawn after 40 days of administration. The cell viability was detected by cell counting kit-8. The expression of Bcl-2 and Caspase-3 proteins were detected by Western blot and the expressions of ERK1/2 mRNA were detected by quantitative real-time polymerase chain reaction (qRT-PCR). A mouse melanoma model was established to study the effect of PAC during long-time administration. Mice were divided into 3 treatment groups: control group treated with saline water, positive control group (LNT group) treated with lentinan at 100 mg/(kg·d), and PAC group treated with PAC at 120 mg/(kg·d). The pathological changes of tumor tissues were observed by hematoxylin-eosin staining. The apoptosis of tumor tissues was detected by TUNEL staining. Bcl-2 and Caspase-3 protein expressions were detected by immunohistochemistry, and the expressions of ERK1/2, JNK1 and p38 mRNA were detected by qRT-PCR.@*RESULTS@#In vitro, no strong inhibitory effects of PAC were found in various tumor cells after 48 or 72 h of administration. Interestingly however, after 40 days of cultivation under PAC, an inhibitory effect on B16F10 cells was found. Correspondingly, the long-time administration of PAC led to downregulation of Bcl-2 protein (P<0.05), up-regulation of Caspase-3 protein (P<0.05) and ERK1 mRNA (P<0.05) in B16F10 cells. The above results were verified by in vivo experiments. In addition, viability of B16F10 cells under long-time administration culture in vitro decreased after drug withdrawal, and similar results were also observed in 4T1 cells.@*CONCLUSIONS@#Long-time administration of PAC can significantly inhibit viability and promote apoptosis of tumor cells, and had obvious antitumor effect in tumor-bearing mice.

Synergistic sensitization of hUMSCs-derived supernatant combined with temozolomide in different glioma cell lines / 中国病理生理杂志

AIM:To explore the synergistic sensitization effect of human umbilical cord mesenchymal stem cell culture supernatant(hUMSC-CM)combined with temozolomide(TMZ)on various glioma cell lines,and to elucidate the underlying mechanisms.METHODS:The hUMSC-CM was harvested using two different serum deprivation tech-niques at 24 and 48 h,and was converted into freeze-dried powder,which was then given to rat malignant glioma cell line RG-2,human astrocytoma cell line U251 and human glioblastoma cell line LN-428 at 5 concentrations(0,1,3,6 and 9 g/L).The effectiveness and sensitivity of hUMSC-CM for inhibiting growth of glioma cells at 24,48 and 72 h were as-sessed using CCK-8 assay.Hematoxylin-eosin(HE)staining combined with CCK-8 assay was employed to evaluate the chemotherapy sensitivity of glioma cells after 48 h of treatment with TMZ at 6 concentrations(0,25,50,100,200 and 400 μmol/L).Two concentrations(3 and 9 g/L)of hUMSC-CM and 3 concentrations(50,100 and 200 μmol/L)of TMZ were chosen for concurrent treatment of glioma cells to assess the proliferation and pathological alterations.TUNEL staining was utilized to detect apoptosis.Flow cytometry was utilized to analyze cell cycle modifications.The expression alterations of apoptosis-inducing proteins,cleaved caspase-3,cleaved caspase-8 and cleaved PARP1,as well as autophagy-inducing proteins beclin-1 and LC3,were examined using Western blot to investigate the synergistic sensitization mechanism of hUMSC-CM combined with TMZ in vitro.RESULTS:The susceptibility of glioma cell lines to hUMSC-CM and TMZ varied,with RG-2 showing the highest sensitivity,followed by U251,and then LN-428.The inhibitory effect of hUMSC-CM(3 and 9 g/L)and TMZ(50,100 and 200 μmol/L)combined treatment on glioma cells was significantly greater than that that of single-agent treatments(P＜0.05),demonstrating a dose-and concentration-dependent enhancement.Notably,the combination of 9 g/L hUMSC-CM(C9)with 50 μmol/L TMZ(T50)effectively suppressed glioma cell growth.CCK-8 as-say indicated a significant reduction of cell viability in C9+T50 group compared with either C9 or T50 alone(P＜0.05).HE staining and TUNEL staining revealed pronounced morphological changes and significant apoptotic features in glioma cells treated with C9+T50.Flow cytometric analysis confirmed that C9+T50 induced cell cycle arrest in glioma cells.Fur-thermore,compared with control group,the levels of cleaved caspase-3,cleaved caspase-8,cleaved PARP1,beclin-1,and LC3-Ⅱ/LC3-Ⅰ were significantly elevated in the C9+T50-treated glioma cells(P＜0.01).CONCLUSION:(1)The concomitant administration of hUMSC-CM and TMZ exerts a broad inhibitory effect on glioma cells,with a synergistic sen-sitization observed across different cell lines.(2)The enhancement of glioma cell sensitivity to TMZ by hUMSC-CM may be attributed to the modulation of caspase-8/caspase-3/PARP1 signaling pathway and the induction of both apoptosis and autophagy in glioma cells.

Mechanism by which Notch1 interference regulates the apoptosis of pulmonary microvascular endothelial cells / 中国基层医药

Objective:To investigate the mechanism of how Notch1 interference regulates the apoptosis of pulmonary vascular endothelial cells.Methods:During January to December 2022, human pulmonary microvascular endothelial cells were transfected with Notch1 siRNA, and the cell viability in each group was evaluated using the Cell Counting Kit-8 assay. The level of reactive oxygen species was determined using flow cytometry, while cell apoptosis was assessed using the same technique. After treatment with Notch1 siRNA, the protein expression levels of Notch1, Bcl-2, and Caspase-3 in the human pulmonary microvascular endothelial cells were detected using western blot assay.Results:The expression level of Notch1 mRNA in human pulmonary microvascular endothelial cells was significantly lower in the blank control and si-Notch1 groups than that in the siNC group ( t = 11.25, 9.47, both P < 0.05). Additionally, the optical density value and Bcl-2 protein expression level in the lipopolysaccharide (LPS) + Notch1 siRNA group were significantly higher than those in the LPS and LPS + siRNA groups ( t = 11.26, 11.68, both P < 0.05). The level of reactive oxygen species and the apoptosis rate of cells were significantly lower in the LPS + Notch1 siRNA group compared with the LPS and LPS + siRNA groups ( t = 11.68, 11.87, both P < 0.05). Furthermore, the protein expression levels of Notch1 and Caspase-3 were also significantly lower in the LPS + Notch1 siRNA group compared with the LPS and LPS + siRNA groups ( t = 5.08, 6.60, 3.84, 5.83, all P < 0.05). Conclusion:Notch1 interference may interference in the apoptosis of human pulmonary microvascular endothelial cells through regulating the level of reactive oxygen species, downregulating the protein expression of Notch1 and Caspase-3, and upregulating the protein expression of Bcl-2. These actions may contribute to the treatment of chronic obstructive pulmonary disease.

Evaluation of Anti-tumor Properties of Herbal Formulations against Ehrlich Ascites Solid Tumor Mice Model

Saraca indica (Ashoka) is an important plant belonging to the family Fabaceae. It essentially contains glycosides, tannin, saponin, flavonoids, and sterol. It possesses a variety of activities such as analgesic, antipyretic, fungitoxic, anthelmintic, antidiabetic, larvicidal activity, antimicrobial activity, central nervous system depressant activity, antiulcer activity, anti-cancer, and anti-inflammatory activity. The anticancer principle from S. indica flowers indicated 50 percent cytotoxicity (in vitro) in Dalton’s lymphoma ascites and Sarcoma-180 tumor cells at a concentration of 38 mug and 54 mug, respectively, with no activity against normal lymphocytes but preferential activity for lymphocytes derived from leukemia patients. A new plant is also taken as they have no reported activity for in vivo in cancer. One such plant is Solanum xanthocarpum Schrad. and Wendl. (Family: Solanaceae). Various therapeutic properties are attributed to it, particularly in the cure of asthma, chronic cough, and catarrhal fever. Plant contains alkaloids, sterols, saponins, flavonoids, and their glycosides and also carbohydrates, fatty acids, and amino acids. The exact mechanism of action of Kantkari is still unknown due to not reported yet, so the present study was designed to investigate the anti-tumor potential of herbal drugs (Ashoka and Kantkari) against Ehrlich ascites solid tumor mice model. Solid tumors were induced by injecting Ehrlich ascites carcinoma (EAC) cells (15 × 106/mice) subcutaneously in right hind limb. Treatment with drugs (Ashoka and Kantkari) showed significant decrease in body weight, solid tumor volume, and tumor weight in EAC tumor-bearing mice at a dose of 400 mg/kg and 200 mg/kg, respectively. Ashoka increased the mean survival time of tumor-bearing mice and percentage increase in life span (was found to be 90.53% and Kantkari is 88.33%, respectively, which is lower than the standard drug Vincristine but Kantkari shows that the tumor inhibition is more significant than Ashoka. Results of with hematoxylin and eosin and immunohistochemistry explained the possible mechanism of action of drugs (Ashoka and Kantkari). Decreased expression of vascular endothelial growth factor indicated the anti-angiogenic property. Immunochemical analysis of solid tumor showed increased expression of caspase-3 suggested that drugs (Ashoka and Kantkari) induced apoptosis in tumor cells. Findings of the present study explored the possible anti-tumor properties of drugs (Ashoka and Kantkari) and can be room for further evaluations in clinical studies.

Immunoexpression of ALDH1A1, FGFR2, CD44 and Caspase-3 in Oral Squamous Cell Carcinoma and Leukoplakia: A pilot study

Current research highlighted the importance to recognize feasible biomarkers for early diagnoses and treatment in oral cancer. Our study analyzed the expression and spatial distribution of ALDH1A1, FGFR2, caspase-3, and CD44 in Oral Squamous Cell Carcinoma (OSCC) and leukoplakia with and without oral mucosal dysplasia. Paraffin-embedded samples of OSCC (n=5), leukoplakia with (n=5) and without (n=5) dysplasia obtained by incisional biopsies were processed using conventional histochemical techniques. Immunohistochemistry was performed using antibodies against ALDH1A1, FGFR2, caspase-3, and CD44. Images of the immunohistochemically stained tissue sections were analyzed according to the intensity of the immunostaining of each marker and classified in Scores. The Kruskal- Wallis test was performed (p≤0.05). Our results demonstrated a statically difference in the expression of all immunomarkers between OSCC and leukoplakia without dysplasia, being more significant in FGFR2 and ALDH1A1. Within the limitations of this study, our data showed that all biomarkers were overexpressed in OSCC and leukoplakia with oral mucosa dysplasia, suggesting that the presence of dysplasia is a significant clinic-pathologic predictor for malignant transformation.

La actual evidencia científica enfatiza la importancia de reconocer biomarcadores viables para el diagnóstico y tratamiento temprano del cáncer oral. Nuestro estudio piloto analizó la expresión y distribución espacial de ALDH1A1, FGFR2, caspasa-3 y CD44 en carcinoma oral de células escamosas (COCE) y en leucoplasia con o sin displasia de la mucosa oral. Las muestras incluidas en parafina de COCE (n=5), con (n=5) y sin (n=5) displasia fueron obtenidas mediante biopsias incisionales, las cuales se procesaron utilizando técnicas histoquímicas convencionales. El análisis inmunohistoquímico se realizó utilizando anticuerpos contra ALDH1A1, FGFR2, caspasa-3 y CD44. Las imágenes de las secciones de cada muestra fueron analizadas según la intensidad de inmunoexpresión de cada marcador y se clasificaron en diferentes escalas (scores). Se realizó la prueba de Kruskal-Wallis (valores de p<0,05). Nuestros resultados demostraron una diferencia estadística en la expresión de todos los inmunomarcadores entre COCE y las muestras con leucoplasia sin displasia, siendo más significativa en FGFR2 y ALDH1A1. Considerando las limitaciones de este estudio, los datos sugieren que la presencia de displasia en la mucosa oral es un importante predictor clínico-patológico de transformación maligna.

Anti-inflammatory and antioxidant potential of Artemisia dracunculus L. aqueousextract against acetic acid induced ulcerative colitis in male Wistar rats

Ulcerative colitis (UC) is one of the two types of inflammatory bowel disease (IBD) which is increasing worldover due to modern life style. Patients with UC are prone to develop colorectal cancer. While the disease severity decides the treatment option, researchers look towards herbal medicines with anti-inflammatory properties for minimal or nil side effects. Artemisia dracunculus L., commonly called Tarragon, is a medicinal herb used in traditional Asian medicine mainly in Iran, India, Pakistan and Azerbaijan due to its special compounds. In this study, we tried to elucidate the effects of aqueous extract of tarragon on acetic acid induced ulcerative colitis (UC) in rats. Male Wistar rats were grouped into four groups of eight each viz., control; experimental control (UC was induced via luminal instillation of 4% acetic acid); and UC induced + aqueous tarragon extract (100 mg/kg) or prednisolone (2 mg/kg) orally for ten consecutive days. Tissue specimens were collected after the experimental period for evaluation of caspase-3 and cyclooxygenase-2 (COX-2) expression by immunohistochemistry. Real-time PCR was used to monitor the levels of IL-1, IL-6 and TNF-? in colonic homogenates. Moreover, the levels of myeloperoxidase, nitric oxide and total antioxidant capacity were measured in colonic homogenates. The results showed that both treatment regimens could similarly reduce the severity of disease symptoms. Treatment with aqueous extract of tarragon caused a better improvement (P <0.05) in the levels of myeloperoxidase enzyme, and total antioxidant capacity of colonic homogenates compared to prednisolone. Nevertheless, the levels of the expression of caspase-3, and COX-2 and TNF-? were reduced in UC rats received prednisolone more than UC rats received aqueous extract of tarragon. The was no statistical difference in the levels of nitric oxide, IL-1 and IL-6 between UC rats received tarragon extract or prednisolone. Overall, these findings suggest that the aqueous extract of tarragon is a promising strategy to control ulcerative colitis. Aqueous extract can also be used as an anti-inflammatory and immune system stimulant in conditions where the immune system is damaged.

Intervention effect and mechanism of Zhongfeng yure decoction on ischemic stroke model rats / 中国药房

OBJECTIVE To study the intervention effect and mechanism of Zhongfeng yure decoction on ischemic stroke model rats. METHODS Totally 85 rats were randomly divided into sham operation group （normal saline， n＝15）， model control group （normal saline， n＝18）， Nimodipine tablet group （positive control， 10.8 mg/kg， n＝18）， high-dose group of Zhongfeng yure decoction （20.52 g/kg， n＝17） and low-dose group of Zhongfeng yure decoction （5.13 g/kg， n＝17）， respectively. After 7 days of preventive continuous administration （once a day）， except for the sham operation group， the rats’ middle cerebral artery occlusion （MCAO） model was established by the modified suture method in other groups. After modeling， the rats in each group continued to be administered for 3 days. During experiment， general condition of the rats was observed， and the neurological function score was performed. After the last administration， the organ index was calculated， the cerebral infarction area and pathological changes of brain tissue were observed. The levels of tumor necrosis factor-α （TNF-α） and interleukin 6 （IL-6） in brain tissue and serum， and the average optical density value of caspase-3 and phosphorylated protein kinase B（p-AKT） protein in brain tissue were detected. RESULTS Three days after modeling， compared with sham operation group， the neurological function score， in brain tissue index， spleen tissue index， proportion of cerebral infarction area， the levels of TNF-α and IL-6 in brain tissue and serum， and the average optical density value of caspase-3 protein in brain tissue were significantly increased in the model control group （P＜0.05 or P＜0.01）； karyopyknosis， diffuse edema and other lesions appeared in brain tissue. Compared with the model control group， the above indexes in each administration group were improved to varying degrees. Among them， there were significant regression in brain tissue index， spleen tissue index， proportion of cerebral infarction area， TNF-α level in brain tissue and serum， and the average optical density values of caspase-3 protein and p-AKT protein in brain tissue of rats in high-dose group of Zhongfeng yure decoction （P＜0.05 or P＜0.01）. CONCLUSIONS Zhongfeng yure decoction has a certain intervention and therapeutic effect on MCAO model rats. The mechanism may be to reduce the secretion of inflammatory factors TNF-α and IL-6， down-regulate the expression of caspase-3 protein in ischemic brain tissue， up-regulate the expression of p-AKT protein， so as to protect the neurons.

Maresin1 inhibits the NF-κB/caspase-3/GSDME signaling pathway to alleviate hepatic ischemia-reperfusion injury / 中华肝脏病杂志

Objective: To investigate the role of Maresin1 (MaR1) in hepatic ischemia-reperfusion injury (HIRI). Methods: The HIRI model was established and randomly divided into a sham operation group (Sham group), an ischemia-reperfusion group (IR group), and a MaR1 ischemia-reperfusion group (MaR1+IR group). MaR1 80ng was intravenously injected into each mouse's tail veins 0.5h before anesthesia. The left and middle hepatic lobe arteries and portal veins were opened and clamped. The blood supply was restored after 1h of ischemia. After 6h of reperfusion, the mice were sacrificed to collect blood and liver tissue samples. The Sham's group abdominal wall was only opened and closed. RAW267.4 macrophages were administered with MaR1 50ng/ml 0.5h before hypoxia, followed by hypoxia for 8h and reoxygenation for 2h, and were divided into the control group, the hypoxia-reoxygenation group (HR group), the MaR1 hypoxia-reoxygenation group (MaR1 + HR group), the Z-DEVD-FMK hypoxia-reoxygenation group (HR+Z group), the MaR1 + Z-DEVD-FMK hypoxia-reoxygenation group (MaR1 + HR + Z group), and the Con group without any treatment. Cells and the supernatant above them were collected. One-way analysis of variance was used for inter-group comparisons, and the LSD-t test was used for pairwise comparisons. Results: Compared with the Sham group, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), interleukin (IL)-1β, and IL-18 in the IR group were significantly higher (P < 0.05), with remarkable pathological changes, while the level in the MaR1 + IR group was lower than before (P < 0.05), and the pathological changes were alleviated. Compared with the Con group, the HR group had higher levels of IL-1β and IL-18 (P < 0.05), while the MaR1 + HR group had lower levels of IL-1β and IL-18 (P < 0.05). Western blot showed that the expressions of caspase-3, GSDME, and GSDME-N were significantly higher in the HR group and IR group than in the other groups; however, the expression was lower following MaR1 pretreatment. The Z-DEVD-FMK exploration mechanism was inhibited by the expression of caspase-3 in HIRI when using MaR1. Compared with the HR group, the IL-1β and IL-18 levels and the expressions of caspase-3, GSDME, and GSDME-N in the HR + Z group were decreased (P < 0.05), while the expression of nuclear factor κB was increased, but following MaR1 pretreatment, nuclear factor κB was decreased. There was no significant difference in the results between the MaR1 + H/R group and the MaR1 + H/R + Z group (P > 0.05). Conclusion: MaR1 alleviates HIRI by inhibiting NF-κB activation and caspase-3/GSDME-mediated inflammatory responses.

Effect of SMAC Gene on Sensitivity of Lung Adenocarcinoma Cells to Paclitaxel and Cell Viability Based on caspase-3/Bcl-2/Bax Signaling Pathway / 肿瘤防治研究

Objective To investigate the effect of the SMAC gene on paclitaxel sensitivity and cellular activity in lung adenocarcinoma cells based on the caspase-3/Bcl-2/Bax signaling pathway. Methods A paclitaxel-resistant cell line A549/Taxol was established for lung adenocarcinoma, and the cells were divided into four following groups: pcDNA-NC (transfected with pcDNA-NC blank vector), pcDNA-SMAC (transfected with pcDNA-SMAC vector), siRNA-NC (transfected with siRNA-NC empty virus vector), and siRNA-SMAC groups (transfected with siRNA-SMAC lentiviral vector). The SMAC mRNA expression in cells was detected by qRT-PCR; cell sensitivity was detected by MTT; cell proliferation ability was detected by cloning assay; cell invasion ability was detected by Transwell; apoptosis ability was detected by flow cytometry assay; and caspase-3, Bcl-2 and Bax protein expression in cells were detected by Western blot analysis. Results The SMAC mRNA expression was significantly lower in A549 cells compared with BEAS-2B cells (P < 0.05). The SMAC mRNA expression was significantly higher in the pcDNA-SMAC group than that in the pcDNA-NC group cells (P < 0.05). The SMAC mRNA expression was significantly lower in the cells of the siRNA-SMAC group (P < 0.05) than that in the siRNA-NC group. The SMAC mRNA expression was significantly lower in the cells of the siRNA-SMAC group (P < 0.05) than in the siRNA-NC group. Compared with the pcDNA-NC group, the cell IC50, cell clone number, cell invasion ability, and Bcl-2 protein and Bcl-2/Bax ratio were significantly lower in the pcDNA-SMAC group, the cell resistance index reversal was 2.51-fold, and the apoptosis ability and caspase-3, as well as Bax protein expression, were significantly higher (P < 0.05). Compared with the siRNA-NC group, cell IC50, cell clone number, cell invasion ability, and Bcl-2 protein and Bcl-2/Bax ratio were significantly higher in the siRNA-SMAC group, and apoptosis ability and caspase-3 and Bax protein expression were significantly lower (P < 0.05). Conclusion High expression of SMAC increases paclitaxel sensitivity, inhibits cell growth and invasion, promotes apoptosis in lung adenocarcinoma cells, and has a regulatory effect on the caspase-3/Bcl-2/Bax signaling pathway.