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ObjectiveTo determine the mechanism of Yitangkang in correcting excessive apoptosis of skeletal muscle cells to improve insulin resistance （IR） by inhibiting the advanced glycation end product （AGE）/receptor for the advanced glycation end product （RAGE） signaling pathway. Method① In vitro experiments. Yitangkang-medicated serum was prepared. C2C12 cells were divided into a blank group， a model group， high-， medium-， and low-dose Yitangkang-medicated serum groups （40， 20， and 10 g·kg-1）， and a RAGE inhibitor group. The IR model was induced by palmitic acid in C2C12 cells except for those in the blank group. After the corresponding intervention methods were conducted，the cell viability and glucose consumption level of each group were determined. In addition，the apoptosis rate was determined using flow cytometry. The mRNA and protein expression levels of the important apoptotic proteins ［B-cell lymphoma 2 （Bcl-2）， Bcl-2-associated X protein （Bax）， p53， cysteinyl aspartate-specific protease-3 （Caspase-3）， and cysteinyl aspartate-specific protease-9 （Caspase-9）］ were determined using Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot. ② In vivo experiments. Ninety-six eligible Wistar rats were divided into a blank group， a model group， high-，medium-，and low-dose Yitangkang groups （40， 20， and 10 g·kg-1）， and a western medicine group （pioglitazone hydrochloride，1.35 mg·kg-1）. The IR model was induced using high-glucose and high-fat feed for diabetes combined with intraperitoneal injection of low-dose streptozotocin （STZ） in animals and verified by the hyperinsulinemic-euglycemic clamp （HEC） test. After the model was determined successfully， the rats in each group were given intragastric administration of drugs as required. Terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） assay was performed to determine the number of positive apoptotic cells in the skeletal muscle tissues of rats in each group，while Real-time polymerase chain reaction（Real-time PCR） and Western blot were performed to determine the mRNA and protein expression levels of the important apoptotic proteins Bcl-2， Bax， p53， Caspase-3， and Caspase-9. Result① In vitro experiments. compared with the blank group， the model groups showed increased apoptosis rate of C2C12 cells and decreased cell viability and glucose consumption （P<0.01）. Compared with the model group， the Yitangkang-medicated serum groups and the RAGE inhibitor group showed decreased apoptosis rate of C2C12 cells and increased cell viability and glucose consumption （P<0.01）. Compared with the blank group， the model group showed decreased expression levels of Bcl-2 mRNA and protein in C2C12 cells and increased mRNA and protein expression levels of Bax， p53， Caspase-3， and Caspase-9 （P<0.01）. Compared with the model group， the Yitangkang-medicated serum groups and the RAGE inhibitor group showed increased expression levels of Bcl-2 mRNA and protein in C2C12 cells （P<0.01） and decreased mRNA and protein expression levels of Bax， p53， Caspase-3， and Caspase-9 （P<0.05， P<0.01）. ② In vivo experiments. The number of positive apoptotic cells in the skeletal muscle tissues of rats in the model group significantly increased as compared with that in the blank group （P<0.01）. The number of positive apoptotic cells in the skeletal muscle tissues of rats in the Yitangkang groups and the western medicine group decreased as compared with that in the model group （P<0.01）. Compared with the blank group， the model group showed decreased expression levels of Bcl-2 mRNA and protein in skeletal muscle tissues of rats and increased mRNA and protein expression levels of Bax， p53， Caspase-3， and Caspase-9 （P<0.01）. Compared with the model group， the Yitangkang groups and the western medicine group showed increased expression levels of Bcl-2 mRNA and protein in skeletal muscle tissues of rats （P<0.01） and decreased mRNA and protein expression levels of Bax， p53， Caspase-3， and Caspase-9 （P<0.05， P<0.01）. The medium-dose Yitangkang showed a similar effect as RAGE inhibitor， and the effect was equivalent to that of pioglitazone hydrochloride. ConclusionYitangkang can inhibit skeletal muscle cell apoptosis by inhibiting the AGE/RAGE signaling pathway.
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OBJECTIVE： To investigate the mechanism of calycosin （CA） inhibiting the proliferation and migration of lung adenocarcinoma cells by regulating miR-21/PTEN signaling pathway. METHODS： Using lung adenocarcinoma SPC-A1 cells as objects， cell proliferation was detected by MTT method after treated with different doses of CA （5， 15， 25， 50， 75， 100 μg/mL） for 12， 24， 48， 72 h. Cell survival rate， 30％ cell growth inhibition concentration （IC30） and half inhibition concentration （IC50） were calculated. Transwell migration test was used to detect the migration of cells after treated with low-dose， medium-dose and high-dose of CA （50， 75， 100 μg/mL） for 24 h. The number of stained cells was recorded and inhibition rate of cell migration were calculated. Western blotting assay and real-time PCR were used to detect the expression of miR-21 as well as the proteins and their mRNAs expression of PTEN， VEGF， MMP-9 after treated with low-dose， medium-dose and high-dose of CA （50， 75， 100 μg/mL） for 24 h. After transfected with miR-21 mimics and miR-21 inhibitor， the effects of CA （75 μg/mL） on the expression of miR-21 and the protein expression of PETN， VEGF and MMP-9 were detected. RESULTS： After treated with 50， 75， 100 μg/mL CA for 12， 24， 48 h， 25， 50， 75， 100 μg/mL CA for 72 h， cell survival rate was decreased significantly （P＜0.05 or P＜0.01）. IC30 of CA were 82.24， 50.45， 46.34， 31.81 μg/mL ； IC50 of CA were 108.06， 73.35， 70.08， 49.89 μg/mL during 12-72 h. Compared with normal control group， the number of stained cells in CA groups， protein expression of VEGF in CA low-dose group， expression of miR-21 as well as proteins and their mRNAs expression of VEGF， MMP-9 in CA medium-dose and high-dose groups were decreased significantly； the medium-dose and high-dose groups were significantly less or lower than low-dose group； the high-dose group was significantly less or lower than medium-dose group （P＜0.05 or P＜0.01）. Cell migration rate of CA groups as well as protein and its mRNA expression of PTEN in CA medium-dose and high-dose groups were increased significantly； the medium-dose and high-dose groups were significantly higher than the low-dose group； the high-dose group was significantly higher than the medium-dose groups （P＜0.05 or P＜0.01）. After transfected with miR-21 mimics， expression of miR-21 as well as protein expression of VEGF and MMP-9 were increased significantly in miR-21 mimic group， compared with normal control group； protein expression of PTEN was decreased significantly （P＜0.01）. After intervened by CA， expression of miR-21 as well as protein expression of VEGF and MMP-9 in cells were decreased significantly， compared with miR-21 mimic group； protein expression of PTEN was increased significantly （P＜0.05 or P＜0.01）. After transfected with miR-21 inhibitor， expression of miR-21 as well as protein expression of VEGF and MMP-9 were decreased significantly in miR-21 inhibitor group， compared with normal control group； protein expression of PTEN was increased significantly （P＜0.05 or P＜0.01）. After intervened by CA， the expression of miR-21 and above protein had no significant change in cells， compared with miR-21 inhibitor group （P＞0.05）. CONCLUSIONS： CA can inhibit the proliferation and migration of lung adenocarcinoma SPC-A1 cells in a dose-dependent manner， which may be associated with the regulation of miR-21/PTEN signaling pathway.
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Objective To explore the effect of tert?butylhydroquinone(tBHQ)on ultraviolet B(UVB)?induced oxidative damages in human im?mortalized keratinocytes(HaCaT),and discuss its mechanism. Methods The cultured HaCaT cells were randomly divided into 4 groups:control group(G1),ultraviolet irradiation group(G2),25μmol/L tBHQ pretreatment before ultraviolet irradiation group(G3),and 50μmol/L tBHQ pre?treatment before ultraviolet irradiation group(G4). The content of reactive oxygen species was detected by DCFH?DA method,and the cell prolifera?tion was evaluated by MTT. Western blot was used to measure the protein expression of nuclear factor E2?related factor 2(Nrf2)in both nuclear fac?tions and whole?cell of HaCaT. The mRNA expressions of CAT and SRX were determined by real?time RT?PCR. Results The content of reactive oxygen species in HaCaT cells was increased,and the cell proliferation rate was decreased significantly after ultraviolet irradiation. The pretreatment of 25 and 50μmol/L tBHQ can inhibit the UVB?induced oxidative damage in a dose?dependent manner in HaCaT cells. Compared with G2 group, tBHQ pretreatment could dose?dependently increase the level of Nrf2 protein in nuclear factions and whole?cell of HaCaT,and also the mRNA ex?pressions of CAT and SRX. Conclusion UVB irradiation can induce oxidative stress damages of HaCaT cells. tBHQ may inhibit the UVB?induced oxidative damages through enhancing Nrf2 expressions and nuclear translocation,then activating the transcription of the downstream antioxidant en?zymes CAT and SRX.
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Objective to observe the influence of Candida albicans infection on t cell subsets in mice with spleen deficiency,so as to explore the immune mechanism. Methods totally 150 healthy SPF mice were randomly divided into four groups:control group(N1 group,n = 30);Candida albicans infection group(N2,n = 40),spleen deficiency model group(M1,n = 40),spleen deficiency model combined with Candida albicans in-fection group(M2,n = 40). N2 and M1 mice were infected by Candida albicans at a concentration of 2×108 CFU/mL. ten mice were randomly se-lected from each group at 7,14,and 21 days after infection respectively,and the index was detected. Flow cytometry was used to detect the percent-age of CD4+/CD8+t cells in intestinal mucosa of mice,and the expression level of IL-4 and IFN-γ mRNA was detected by Rt-PCR assay. the levels of IFN-γ and IL-4 were detected by Western blot assay. Results Compared with N1 group,the proportion of CD4+t cells in the lamina of the natu-ral layer of the small intestine was decreased(P < 0.01),the proportion of CD8+t cells was increased(P < 0.01),the ratio of the two was significant-ly decreased(P < 0.01),and the expression levels of IL-4,mRNA IFN-γ and protein in the small intestine tissues were increased in other groups (P < 0.01). In addition,the expression level of IFN-γ was significantly increased in M2 group,while the expression of IL-4 was significantly in-creased in N2 group. Conclusion the susceptibility of Candida albicans infection was increased in spleen deficiency mice,which may be closely related to the regulation of th1/th2 balance.
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Objective To observe the effects of Jianpi Yangxue Qufeng Formula (JPYXQF) on the AQP3 in mice with chronic eczema, and explore mechanism of action. Methods Fifty healthy male mice were randomly divided into 5 groups, namely normal group, model group, positive medicine group and JPYXQF high and low dose groups. Low-dose DNCB and Sennae Fominm were used to establish mice models of chronic eczema with spleen deficiency. JPYXQF groups were treated by JPYXQF for gavage, while the positive medicine group was treated by levocetirizine hydrochloride for gavage. The expression of AQP3 in mice skin tissue was detected by immunohistochemical method. At the same time, the pathological changes of skin were observed. Results The pathology of mice skin lesion showed that JPYXQF has certain recovery effects on the inflammation injury of skin lesion. Compared with the normal group, expression of AQP3 over expressed in model group. Compared with the model group, the expression of AQP3 in all treatment groups significantly decreased, and the staining intensity decreased. In the model group, the average optical density of AQP3 was significantly higher than that in the normal group (P<0.01). Compared with the model group, the treatment groups can reduce the expression of AQP3 in mice skin tissues (P<0.05). Conclusion JPYXQF can reduce the over expression of AQP3 in skin lesion, which is probably its mechanism for the treatment of chronic eczema.
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Objective To investigate the effects of Erlong Zuoci Pills on AQP4 expression in cochlear tissue of mice with elderly kidney deficiency deafness;To discuss the action mechanism. Methods Intraperitoneal injection of hydrocortisone method was used to duplicate mice models with kidney deficiency except the normal control group. After the models were established, mice were divided into model group and TCM group, 16 mice in each group. TCM group was gavaged by Erlong Zuoci Pills, model group and normal control group were gavaged by normal saline for 22 d. Cochlear stretched preparation technology was used to observe morphological changes in cochlear inner and outer hair cells, and supporting cells. Immunohistochemistry and Western bolt were used to detect protein expression of AQP4. Results Compared with normal control group, mice in model group missed inner and outer hair cells and supporting cells of the cochlea. Compared with model group, arrangement of cochlear inner and outer hair cells and supporting cells was neat and boundary was clear in TCM group. Compared with model group, protein expression of AQP4 in cochlear tissues in TCM group increased (P<0.01). There was no difference between TCM group and normal control group. Conclusion Erlong Zuoci Pills have significant therapeutic effect for elderly kidney deficiency deafness, and the treatment is related to the upregulation of protein expression of AQP4 in cochlear tissues.
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Objective To investigate the influence of the eye acupuncture therapy in the expressions of brain derived neurophic factor(BDNF)and tyrosine kinase receptor B(TrkB)in penumbra brain tissue of ischemic area in the rats with acute cerebral ischemia reperfusion inj ury,and to explore the related mechanism of protective effect on brain of eye acupunture.Methods 62 SD rats were randomly divided into blank control group (n=11),sham operation group (n=11)and model copy group (n=40).The rats in copy model group were used to establish models with suture method,and the successful model rats (n=32)were randomly divided into model group(n=16)and eye acupuncture group(n=16).The rats in eye acupuncture group were treated with eye acupuncture intervention;the acupoints were chosen according to human acupoint selection method, the liver, the upper-jiao, the down-jiao district,and renal were selected for acupuncture intervention. After reperfusion for 2 h,the acupuncture was performed once every 8 h,lasted for 10 times;30 min after the last intervention,the rats were sacrificed;the regional ischemia penumbra around the parts of the brain tissue was obtained and the expressions of BDNF and TrkB mRNA and protein in brain tissue of the rats were detected with RT-PCR and Western blotting method. Results Compared with blank control group,the expression levels of BDNF and TrkB mRNA and protein in brain tissue of the rats in sham operation group had no statistical significance(P>0.05);compared with sham operation group,the expression levels of BDNF and TrkB mRNA and protein in brain tissue of the rats in model group were significantly up-regulated(P<0.01);compared with model group,the expression levels of BDNF and TrkB mRNA and protein in brain tissue of the rats in eye acupuncture group were significantly up-regulated(P<0.05 or P<0.01).Conclusion The expressions of BDNF and TrkB are increased after cerebral ischemia and reperfusion injury.Eye acupuncture can protect the brain of MCAO/R rats,which may be related to the up-regulation of the expressions of BDNF and TrkB in the corresponding penumbra.
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Objective To investigate the effect of the eye-acupuncture therapy on serum TNF-α levels in rats with cerebral ischemia-reperfusion injury and the related mechanism. Methods Healthy SD rats were randomly divided into four groups: normal group, sham-operated control group, ischemia-reperfusion model group and eye-acupuncture group according to body weight. Sham-operated control group, ischemia-reperfusion model group and eye-acupuncture group were divided into the 3h group, the 24h group and the 72h group, a total of 10 groups (n=8). To establish the rat model of cerebral ischemia-reperfusion by suture method in ischemia-reperfusion model group and eye-acupuncture group. Eye-acupuncture was separately started immediately after reperfusion and at 30 min before sampling in the 3h eye-acupuncture group, besides, eye-acupuncture was separately taken every 12h in the 24h eye-acupuncture group and in the 72h eye-acupuncture group. The rats in normal group were not treated, those in sham-operated control group were inserted the fishing thread 1cm, and the others were identical with those in ischemia-reperfusion model group. At 3h, 24h, 72h after reperfusion, the neurophysical behaviours were accessed by ZeaLonga neurophysical impairment marks in ischemia-reperfusion model group and eye-acupuncture group. The method of ELISA was taken to detect the change of serum TNF-α levels in rats after the eye-acupuncture therapy. Results Compared with the ischemia-reperfusion model group, the neurologic impairment score of the eye-acupuncture group decreased obviously; The levels of serum TNF-αin ischemia-reperfusion model group at 3 h, 24 h, 72 h respectively were(76.803±18.325)pg/ml、(85.511±13.334)pg/ml、(86.831±9.232)pg/ml. The level of serum TNF-α in normal group was(24.304±6.511)pg/ml. The level of serum TNF-α in Sham-operated control group at 3 h, 24 h, 72 h respectively were(24.928±3.792)pg/ml,(27.533±5.362)pg/ml,(29.366±5.874)pg/ml. Ischemia-reperfusion model group compare with normal group and sham-operated control group, the difference were significant(P＜0.01). The levels of serum TNF-α in rats after the eye-acupuncture therapy at 3 h, 24 h, 72 h respectively were(40.185±3.335)pg/ml, (48.523±7.687)pg/ml, (51.611± 6.403)pg/ml. Compared with ischemia-reperfusion model group, the levels of serum TNF-α were strongly reduced(P＜0.01). Conclusion The eye-acupuncture therapy could play a role in improving cerebral ischemia-reperfusion injury evidently and the mechanism was related to its reducing serum TNF-α levels.