| Objectives(1)To investigate the effect of PERK-CHOP signal pathway on pancreatic tissue ERS in STZ-DM rats.( 2) To investigate the relationship between ERS which mediated by the PERK-CHOP signal pathway and acupuncture improve the apoptosis of pancreatic cells in STZ-DM rats.Methods(1)80 SPF grade(220±20)g male SD rats after 1 week adopted feed,10 rats were selected randomly as blank group, and feed 4 weeks by ordinary feed. 70 rats were treated with high fat high sugar diet and intraperitoneal injection of STZ once to establish the model of STZ-DM rats. 30 rats(RBG≥16.7mmol/L) were randomized into 3 groups, model group(n=10), acupuncture group(n=10) and metformin group(n=10). The acupuncture group received 4 courses of regular acupuncture treatment at Yishu Point and electroacupuncture treatment at Housanli Point and Neiting Point, each course consisted of seven consecutive days of treatment. The metformin group was given 100mg·kg-1 metformin HCL by gavage for 4 courses as acupuncture treatment group. The blank group and model group didn’t do any other treatment. All the groups were feed by ordinary feed while therapy.(2)Random blood glucose of each group was measured before and after therapy. Each rat was anesthetized by intraperitoneal injection of 10% chloral hydrate after 4th course of therapy. Then put the sacrificed rats on ice dish, removed the pancreatic bodies and tails of pancreas, and stored in liquid nitrogen, while one rat which was selected in each group randomly was stored in glutaraldehyde. We detected the gene expression and protein content of CHOP, PERK, Bcl-2 and Bax in pancreatic tissue of rats in each group by real-time fluorescent PCR and western blot, observed pancreatic ultrastructure through transmission electron microscope, and used SPSS17.0 forstatistical analysis of experimental data. The experimental data was expressed as X ±s. T test was used for comparing means between two groups, one-way variance(ANOVA) was used for comparing means among groups, and LSD test was used to compare two groups.Results(1)Random blood glucose: The RBG of blank group was significantly lower than the other 3 groups(7.180±0.329mmol/L vs 26.213±4.637mmol/L, P<0.01), showed models successfully established. At the 4th week after therapy, RBG of acupuncture treatment group was significantly decreased than before(21.833±5.369mmol/L vs 25.167±4.930mmol/L, P<0.05). Metformin group was also decreased significantly(22.938±5.292mmol/L vs 26.750±4.407mmol/L, P<0.05), and the difference between metformin group and acupuncture group was no statistical significance(P>0.05), showed that acupuncture treatment can reduce RBG. Blank group(7.180±0.329mmol/L vs 7.460±0.868mmol/L, P>0.05) and model group(26.303±3.593mmol/L vs 25.567±5.189mmol/L, P>0.05) were higher than before.(2)PERK-e IF2α-ATF4-CHOP signal pathway: At the 4th week after therapy, the expression of PERK m RNA in pancreatic tissue of rats in the model group was higher than the blank group(2.638±0.274 vs 1.000±0.066, P<0.01) which had statistical significance. The acupuncture group was significantly decreased compared with the model group(1.627±0.118 vs 2.638±0.274, P<0.01), but still higher than the blank group(1.627±0.118 vs 1.000±0.066, P<0.01) which had statistical significance, and was no statistical significance compared with the metformin group(1.627±0.118 vs 1.673±0.204, P>0.05). The expression of PERK protein in the model group was higher than the blank group(1.164±0.274 vs 0.465±0.089, P<0.01). The acupuncture group was significantly decreased compared with model group(0.783±0.117 vs 1.164±0.274, P<0.05), and was no statistical significance both compared with blank group(0.783±0.117 vs 0.465±0.089, P>0.05) and metformin group(0.783±0.117 vs 0.711±0.333, P>0.05). The expression of CHOP m RNA in pancreatic tissue of rats inthe model group was higher than the blank group( 3.067±0.171 vs 1.003±0.064, P<0.01) which had statistical significance. The acupuncture treatment group was decreased significantly compared with model group(1.472±0.153 vs 3.067±0.171, P<0.01), but was still higher than blank group(1.472±0.153 vs 1.003±0.064, P<0.01) which had statistical significance, and was no statistical significance compared with metformin group(1.472±0.153 vs 1.5988±0.172, P>0.05). The expression of CHOP protein in the model group was higher than the blank group(1.720±0.748 vs 0.916±0.453, P<0.05) which had statistical significance. The acupuncture treatment group was decrease compared with model group(1.071±0.700 vs 1.720±0.748, P>0.05) without a statistical significance, and was also no statistical significance compared with blank group(1.071±0.700 vs 0.916±0.453, P>0.05) and metformin group(1.071±0.700 vs 1.068±0.790, P>0.05). All above suggest the existence of ERS on pancreatic tissue in STZ-DM rats. And acupuncture can improve ERS by inhibiting PERK-CHOP signal pathway.(3)Pancreatic cells apoptosis: At the 4th week after therapy, the expression of Bax m RNA in pancreatic cells of rats in the model group was significantly higher than blank group(2.577±0.168 vs 1.005±0.120, P<0.01). The acupuncture group was significantly decreased compared with model group(1.518±0.203 vs 2.577±0.168, P<0.01), but also was higher than blank group(1.518±0.203 vs 1.005±0.120, P<0.01), and had no statistical significance compared with metformin group(1.518±0.203 vs 1.650±0.488, P>0.05). The expression of Bax protein in the model group was significantly higher than blank group(2.102±0.784 vs 0.795±0.313, P<0.01). The acupuncture group was also decreased compared with model group(1.095±0.049 vs 2.102±0.784, P<0.05), and was no statistical significance both compared with blank group(1.095±0.049 vs 0.795±0.313, P>0.05) and metformin group(1.095±0.049 vs 1.072±0.212, P>0.05). The expression of Bcl-2 m RNA in the model group had a significantly decrease compared with blank group(0.398±0.058 vs 1.005±0.094, P<0.01). The acupuncture group was increased significantly compared with model group(0.642±0.083 vs 0.398±0.058, P<0.01), but was still lower than blank group(0.642±0.083 vs 1.005±0.094, P<0.01), and was no statistical significance compared with metformin group(0.642±0.083 vs 0.620±0.049, P>0.05). The expression of Bcl-2 protein in the model group had a significantly decrease compared with blank group(0.412±0.250 vs 1.003±0.369, P>0.05). The acupuncture group had a significantly increase compared with model group(1.400±0.472 vs 0.412±0.250, P<0.01), and was no statistical significance both compared with blank group(1.400±0.472 vs 1.003±0.369, P>0.05)and metformin group(1.400±0.472 vs 1.119±0.073, P>0.05). All above showed that there were large number of cells apoptosis on pancreatic in STZ-DM rats. And acupuncture can inhibiting the apoptosis.(4)Morphological observation of pancreatic tissue cells under transmission electron microscope: Observed pancreatic ultrastructure through transmission electron microscope, there were abundant round shaped mitochondria and strip-shaped rough endoplasmic reticulum, and abundant zymogen granules aggregated around cytoplasm in the blank control group. In model control group, there were only a few of mitochondria with vacuolar degeneration, mitochondrial cristae distance expanded and disrupted, expansion and disruption of rough endoplasmic reticulum, margination and hyperchromatic phenomenon of nuclear chromatin, and decrease of zymogen granules. In acupuncture treatment group, the number of mitochondrion decreased with varying degrees of vacuolar degeneration, swell and mitochondrial cristae expansion in minority of mitochondria. And rough endoplasmic reticulum had expanded, deformed and arranged disorder slightly with disruption in a few of them. Zymogen granules were distributed around cytoplasm diffusely. In metformin group, there were vacuolar degeneration in a part of mitochondria, disruption of mitochondrial cristae, cell nucleus margination, expansion and disruption slightly in a few of rough endoplasmic reticulum, and decrease of zymogen granules.Conclusions(1)There is ERS on pancreatic tissue of STZ-DM rats, and may be mediated by thePERK-CHOP signal pathway.(2)Acupuncture can decrease the RBG of STZ-DM rats, and can protect the shape of the cells,and reduce the apoptosis of pancreatic cells.(3)Acupuncture can improve ERS by inhibiting PERK-CHOP signal pathway in STZ-DM rats’ pancreatic tissue,then reduce the apoptosis of pancreatic cells. |
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