Pretreatment with low-frequency repetitive transcranial magnetic stimulation may influence neuronal Bcl-2 and Fas protein expression in the CA1 region of the hippocampus: A possible anti-epilepsy mechanism in a lithium-pilocarpine-induced epileptic rat mod

BAOKGROUND： Bcl-2 and Fas proteins are well known as anti-apoptotic and pro-apoptotic factors respectively. However, whether the anti-epileptic mechanism of low-frequency repetitive transcranial magnetic stimulation （rTMS） involves an anti-apoptotic effect via regulating Bcl-2 and Fas protein expression remains to be determined. OBJECTIVE： To verify the correlation between the anti-epileptic mechanism following pretreatment of low-frequency rTMS and anti-hippocampal apoptosis. DESIGN, TIME AND SETTING： A randomized controlled animal experiment was performed at Institute of Neurological Disorders, Affiliated Hospital of North Sichuan Medical College between September 2007 and March 2008. MATERIALS： Pilocarpine （053K13011） was provided by Sigma, USA; lithium was provided by Shanghai Biotechnology Co., Ltd., China; Dantec Maglite-r25 rTMS instrument was provided by Dundee, Denmark. METHODS： A total of 21 adult male Wistar rats were randomly divided into control （n = 6）, rTMS pretreatment （n = 9）, and sham-stimulation （n = 6） groups. The rTMS pretreatment group was pretreated with low-frequency rTMS （0.5 Hz, 75% threshold intensity, 20 times/bundle, and 5 bundles/day）, while the sham-stimulation group was sham-stimulated with a similar sound for 7 successive days to establish lithium-pilocarpine-induced epileptic state models. MAIN OUTCOME MEASURES： Epileptic stroke latency; neuronal morphology was observed using hematoxylin and eosin staining; mean positive-reactive cell number and mean absorbance of Bcl-2 and Fas protein in the hippocampal CA1 region was observed using immunohistochemistry. RESULTS： Epileptic latency in the rTMS pretreatment group was significantly enhanced （P 〈 0.01）, and a number of degenerated neurons were observed to be apoptotic. Bcl-2 protein expression increased at each time point, but Fas protein expression decreased （P 〈 0.01）. CONCLUSION： Low-frequency rTMS has an anti-epileptic effect, which may be via regulation of Bcl-2 and Fas protein expression in the hippocampal region.

Fas mRNA expression and calcium influx change in H_2O_2-induced apoptotic hepatocytes in vitro

AIM: To investigate the relationship between Fas gene expression and calcium influx change in peroxide-induced apoptotic hepatocytes and the possible molecular mechanism of Rxa in protecting hepatocytes.METHODS: Single-cell Fas mRNA expression in H2O2-exposed L02 hepatocytes with or without treatment of Rxa,an extract from an anti-peroxidant, Radix Salviae Miltiorrhizae,was determined by all-cell patch clamp and single-cell reverse transcriptase polymerase chain reaction (RT-PCR). Transient calcium influx change ([Ca2+]i) in the cells was evaluated with all-cell patch clamp micro-fluorescence single-cytosolic free Ca2+ concentration technique. Fas protein expression, early apoptotic index (annexin-V+) and cell membrane change inthe cells were evaluated by immunohistochemistry, flow cytometry (FCM) and scan electron microscopy respectively.RESULTS: In cells exposed to H2O2 for 2 h, the specific lane for Fas mRNA was vivid on electrophoresis, with increased Fas protein expression, [Ca2+]i (from 143.66±34.21 to 1115.28±227.16), annexin-V+ index (from 4.00±0.79 to 16.18±0.72) and membrane vesicle formation. However, in cells exposed to H2O2 but pre-treated with Rxa, there was no increase in Fas mRNA or protein expression and [Ca2+]i (103.56±28.92). Annexin-V+ index (8.92±1.44) was lower than the controls (P＜0.01), and the cell membrane was intact.CONCLUSION: H2O2 induces apoptosis of L02 cells by increasing cytosolic [Ca2+]i, and inducing Fas mRNA and protein expression. Rxa protects the L02 cells from apoptosis through anti-peroxidation, inhibition of calcium overloading and prevention of the activation of cytosolic Fas signal pathway.

Construction and identification of Fas-targeting siRNA-expressing plasmid

Objective: To study the therapeutic potential of Fas inhibition in different diseases, a Fas-targeting siRNA (small interfering)-expressing plasmid was constructed. Methods: The U6 promoter cassette and siFas (small interfering RNA that inhibit Fas expression) template sequence were obtained by PCR method. They were cloned into modified pcDNA3.1. The resultant plasmid pU6-siFas was transfected into P815 cells with lipofectin2000 and selected under G-418-containing culture medium. Fas inhibition in stably transfected cells was detected by immunocytochemistry. Results: The plasmid pU6-siFas efficiently reduced the expression of Fas and conferred G-418 resistance in P815 cells. Conclusion: The successful construction of the siRNA expressing plasmid will facilitate the application of RNA interference technique and lay the foundation for further study of Fas inhibition in the treatment of different diseases such as aplastic anemia and acute liver failure.

Effects of electroacupuncture on conjunctival cell apoptosis and the expressions of apoptosis-related proteins Caspase-3,Fas and Bcl-2 in rabbits with dry eye syndrome

Objective:By observing the effects of electroacupuncture(EA)on the apoptosis of conjunctival cells of rabbits with dry eye syndrome(DES)and the expressions of apoptosis-related proteins Caspase-3,Fas and Bcl-2,to discuss the mechanism of EA in the treatment of DES from the perspective of cell apoptosis.Methods:Male New Zealand rabbits were randomly divided into a normal group(NG),a model group(MG),an EA group(EAG)and a sham EA group(SEAG).DES rabbit model was developed by eye drop of 0.1%benzalkonium chloride.The rabbit tear secretion and tear film break-up time(BUT)were measured;terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL)assay was used to detect the apoptosis of conjunctival cells;the expressions of Caspase-3,Fas and Bcl-2 proteins in conjunctival cells were detected by immunohistochemistry.Results:Compared with the NG,the rabbit tear secretion decreased and the BUT was shortened in the MG(both P<0.01);compared with the MG and the SEAG,the rabbit tear secretion increased and the BUT was prolonged in the EAG(all P<0.05).Compared with the NG,the apoptosis of rabbit conjunctival cells increased(P<0.01),the expressions of Caspase-3 and Fas proteins increased(both P<0.05),and the expression of Bcl-2 protein decreased(P<0.01)in the MG;compared with the MG and the SEAG,the apoptosis of rabbit conjunctival cells decreased(both P<0.01),the expressions of Caspase-3 and Fas proteins decreased(all P<0.05),and the expression of Bcl-2 protein increased(both P<0.01)in the EAG.Conclusion:EA can inhibit the apoptosis of rabbit conjunctival cells,down-regulate the expressions of apoptosis-related proteins Caspase-3 and Fas,and up-regulate the expression of Bcl-2 protein,which may be one of the mechanisms of EA in treatment of DES.

Effects of Xinfeng capsule on the Fas/FasL-mediated apoptotic pathway in patients with rheumatoid arthritis

OBJECTIVE:To observe the effects of Xinfeng capsule on the apoptosis of peripheral blood CD4+ T lymphocytes and changes in the Fas/Fas L-mediated apoptotic pathway in patients with rheumatoid arthritis(RA).METHODS:A total of 28 RA patients were included in the study;they were randomly divided into the Xinfeng capsule(XFC) group(3 capsules,3 per day)and the leflunomide(LEF) group(1 pellet,once per night).The treatment course in each groups was 12 weeks.The normal control(NC) group consisted of10 healthy people.The apoptotic rate was examined using flow cytometry.Fas,Fas L,caspase 8,caspase 3,bcl-2,and bax m RNA were examined using q RT-PCR.Apoptotic proteins Fas,Fas L,caspase8,and caspase 3 were examined using western blotting.RESULTS:After treatment,patients in the two groups all showed some trend of improvement.Disease activity indexes,joint morning stiffness time,joint swelling/tenderness number,health assessment questionnaire(HAQ) score,RA quality of life(RAQOL) questionnaire,and self-rating anxiety scale(SAS),as well as all apoptotic related indicators were reduced in both groups after treatment with no significant difference between groups.But the improvement in terms of the self-rating depression scale(SDS) in the XFC group was better than in the LEF group.RA patients showed lower apoptotic rates in CD4+ T cells,lower bax,Fas,caspase 8,and caspase 3 m RNA,and less protein expression of Fas,caspase 8,and caspase3 than in the NC group.These indicators increased after treatment.However,the level of Bcl-2 m RNA was higher in the XFC group than in the NC group before treatment,and it subsequently decreased.The XFC group expressed lower Bcl-2 m RNA than the LEF group.Negative correlations were found between ESR and the apoptotic rate in CD4 + T cells,Fas,and caspase 3;CRP and Fas;and,swollen joint count and Bax,while positive correlations were found between ESR and Bcl-2.CONCLUSION:XFC can regulate the Fas/Fas L system and promote CD4+ T cell apoptosis and thus reduce the abnormal immune response,which can improve symptoms in RA patients.

Effect of Icariin on apoptosis and expression of Fas,Fas ligand,B cell lymphoma,and Bcl-2-associated X protein in CD4＋ T lymphocytes from patients with ankylosing spondylitis

OBJECTIVE:To investigate the effects of icariin on apoptosis and the expression of Fas, Fas ligand(Fas L), B cell lymphoma(Bcl-2), and Bcl-2-associated X protein(Bax) in CD4+ T lymphocytes from patients with ankylosing spondylitis.METHODS:Primary cultures of peripheral blood CD4+ T lymphocytes were established and treated with icariin at high, medium, and low doses(0.5,0.25, and 0.125 mg/mL).Sulfasalazine treated and helthy cells were used as controls.Apoptosis of treated cells was determined by flow cytometry.Reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assays were used to determine the effects of icariin on the expression of Fas, Fas L, Bcl-2, and Bax.The activity of caspase 8 and caspase 3 was determined by a colorimetric assay.RESULTS:The m RNA and protein expression of Fas,and activity of caspase 8 and caspase 3 in CD4+ T lymphocytes were increased by icariin(P < 0.05).Conversely, the m RNA and protein expression of Bcl-2 was decreased(P < 0.05).The expression of Fas L and Bax were not significantly different between groups.The proapoptotic effects of icariin were dose-dependent.CONCLUSION:Icariin induces the apoptosis of CD4 + T cells from patients with AS comparing to normal control.Therefore, the induction of apoptosis may be the likely mechanism of action of icariin's antirheumatics activities.

Study on the Effect of Moxibustion in Treating Rhreumatoid Arthritis Rats and Its Mechanism

Objective： To investigate the therapeutic efficacy of moxibustion in treating rheumatoid arthritis （RA） and its mechanism. Methods： A hundred male Wistar rats were randomized into 5 groups, 20 in each group. RA models were developed in all groups except for the normal group. When the models were successfully prepared, the rats in the normal group and the model group didn’t receive any treatment. Those in the moxibustion group received suspended moxibustion by moxa stick, those in the cigarette group received fumigation by cigarette; while those in the moxa oil group were treated by applying moxa essential oil to acupoints. After consecutive 15-day treatment, body weight, paw circumference, thymus and spleen indexes were compared among the five groups, and the expression of Fas/FasL protein in synovium of rat’s joint was detected by using immunohistochemical （IHC） method. Results： After treatment, the moxibustion group had body weight increased, joint swelling relieved, thymus index enhanced, spleen index dropped, and the expression of Fas/FasL protein raised. Regarding the above indexes, the moxibustion group and the cigarette group showed more significant improvements than the moxa oil group; the moxibustion group had more significant effect on the expression of Fas/FasL than the cigarette group. Conclusion： Moxibustion obviously improves the symptoms of RA rats, regulates the expression of Fas/FasL protein in synovium of rat’s joint, and induces apoptosis of synovial cells, which maybe one of the mechanisms of moxibustion in achieving the warming and unblocking effect.