| Recent research shows that Chinese traditional medicine oral medicine used as one of the species, in diabetic treatment play an important role, and the active components of traditional Chinese medicine in the prevention and treatment of diabetes in the process of curative effect stability, low toxicity and fewer side effects, can use for a long time. We through the prophase work has been confirmed Reed rhizome ethanol extract on diabetes mice glucose-lowering lipid effect, also found its promote liver glycogen synthesis and in diabetic mice liver mitochondrial oxidative stress damage regulation and protection. As is known to all, glycogen synthetase and glycogen synthetase kinase-3β (GSK-3β) in these process plays a certain role, but the mechanism is still not clear.ObjectiveThis study intends to use molecular biology experimental technique, observation Reed rhizome ethanol extract of chain urea with bacteria hormone (STZ) induced diabetes mice liver glycogen synthetase and glycogen synthetase kinase-3β and the influence of the molecule biology index, this paper discusses Reed rhizome ethanol extract of specific hypoglycemic mechanism, clinical Chinese herbal medicine for the treatment of diabetes to provide theoretical basis and experimental basis.Methods(1) The Reed rhizome ethanol extract preparation:Reed rhizome (dry) cut into1-2cm broken section, soak in8times volume of95%ethanol12h, then respectively with six times volume and five times the volume of95%ethanol refluxing extraction, repeated3times (every time time is2h,1.5h,1h), filtering extract after merger, concentrated into concrete, seal, and save backup.(2) The STZ mice model induced diabetes preparation:mice feeding adaptability after1w, fasting24h, during free drinking water; Then intraperitoneal injection of citric acid buffer preparation of fresh STZ solution, each according to mice per gram weight0.12mg measurement (i.e.,0.12mg/g). Feeding conditions remain unchanged, feeding after60h fast can’t help water12h, tail vein take blood tests, fasting plasma glucose values (FBG), FBG was11.1mmol/L show that made die success.(3) Group and Treatment:laboratory mice were divided into normal control group, model group, Reed rhizome ethanol extract low dose group, Reed rhizome ethanol extract high dose group, drug control group. All mice daily lavage1time, continuous irrigation stomach15d, normal control group and model group lavage respectively and volume of distilled water a/d, drug withdrawal the next day, take off neck executed, and rapid cutting take liver, in liquid nitrogen, after transposed-80℃refrigerator save backup.(4) The RT-PCR method to detect glycogen synthetase and glycogen synthetase kinase-3α gene expression: Primer Premier3.0software design two gene and downstream Primer. Take25±2mg spare liver tissue, Trizol method extracting organization total RNA, nucleic acid protein content meter testing OD260/OD280value in the1.7-2.0in accord with experimental standard, by RT-PCR kit for the experiment.3%agarose gel electrophoresis detection products, image analysis system imaging, beta actin for reference, calculation of two genes in different groups of relative content.(5) Westen-blotting method to detect glycogen synthetase and glycogen synthetase kinase-3β protein expression:take100±2mg spare liver tissue, according to the small tissue protein extraction kit for extracting total protein, after PBS (precooling) cleaning2times, according to the100-200uL/g standard addition preliminary cold cracking fluid, percussion at room temperature to even, at0℃placed under the condition of30min, make its full cracking, full wavelength spectrophotometer to measure protein sample concentration. Take to measure protein boil2min, add sample40μg/hole,10%sds-page electrophoresis separation after turn to PVDF membrane, rabbit anti mice glycogen synthetase and glycogen synthetase kinase-3β polyclonal first antibody (1:500),4℃for the night, after washing join horseradish enzyme mark goat resistance to rabbit IgG (1:1000), wash the film. Image analysis system exposure imaging, β-actin for reference, calculation glycogen synthetase and glycogen synthetase kinase-3β protein relative content.The resultsP-actin is in reference. After Western blot detecting glycogen synthetase protein expression for the relative value of normal control group0.22±0.13, drug control group0.21±0.11, Reed rhizome ethanol extract high dose group0.19±0.10, Reed rhizome ethanol extract low dose group0.12±0.10,0.09±0.08model group. Statistical analysis, the normal control group, drug control group, Reed rhizome ethanol extract high dose group and model group (p<0.01), compared with statistically significant difference, Reed rhizome ethanol extract low dose group and model group (p<0.05), compared with statistics difference, Normal control group, drug control group, Reed rhizome ethanol extract high dose group comparison(p>0.05), no statistical significance. The normal control group, drug control group, Reed rhizome ethanol extract high dose group and Reed rhizome ethanol extract low dose group (p<0.05), compared with statistics difference. After RT-PCR amplification of glycogen synthetase mRNA normal control group0.44±0.15, drug control group0.42±0.13, Reed rhizome ethanol extract high dose group0.40±0.10, Reed rhizome ethanol extract low dose group0.22±0.12,0.17±0.16model group. Statistical analysis, the normal control group, drug control group, Reed rhizome ethanol extract high dose group and model group (p<0.01), compared with statistically significant difference, Reed rhizome ethanol extract low dose group and model group (p<0.05), compared with statistics difference, Normal control group, drug control group, Reed rhizome ethanol extract high dose group comparison(p>0.05), no statistical significance. The normal control group, drug control group, Reed rhizome ethanol extract high dose group and Reed rhizome ethanol extract low dose group (p<0.05), compared with statistics difference. Western blot after detecting glycogen synthetase kinase-3β protein expression of relative value for model group0.14±0.03, Reed rhizome ethanol extract low dose group0.13±0.07, Reed rhizome ethanol extract high dose group 0.10±0.04, drug control group0.09±0.02,0.07±0.03normal control group. Statistical analysis, the normal control group, drug control group, Reed rhizome ethanol extract high dose group and model group (p<0.01), compared with statistically significant difference, Reed rhizome ethanol extract low dose group and model group compared p>0.05, the difference being statistically significant; Normal control group, drug control group, Reed rhizome ethanol extract high dose group comparison(p>0.05), no statistical significance. The normal control group, drug control group, Reed rhizome ethanol extract high dose group and Reed rhizome ethanol extract low dose group (p<0.05), compared with statistics difference. Glycogen synthetase kinase-β RT-PCR amplification of glycogen synthetase kinase-3β mRNA, model group0.37±0.05, Reed rhizome ethanol extract low dose group0.28±0.09, Reed rhizome ethanol extract high dose group0.21±0.07, drug control group0.18±0.05,0.14±0.07normal control group. Statistical analysis, the normal control group, drug control group and model group (p<0.01), compared with statistically significant difference, Reed rhizome ethanol extract high dose group and model group (p<0.05), compared with statistics difference, Normal control group, drug control group, Reed rhizome ethanol extract high dose group comparison(p>0.05), no statistical significance; Reed rhizome ethanol extract low dose group and model group compared p>0.05, no statistical significance. The normal control group, drug control group and Reed rhizome ethanol extract low dose group (p<0.05), compared with statistics difference, Reed rhizome ethanol extract high dose group and Reed rhizome ethanol extract low dose group (p<0.05), compared with statistics difference.Conclusion1. Reed rhizome ethanol extract can increase the STZ induced diabetic mice liver glycogen synthase expression, at the same time reduce the GSK-3beta expression, objectively reduced glycogen synthetase phosphorylation inactivation, increased glycogen synthase relative content, the liver glycogen synthesis.3. Reed rhizome ethanol extract of STZ induced diabetes mice liver glycogen synthetase and glycogen synthetase kinase-3β expression influence has must concentration-response relationship. |
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