ABSTRACT
ObjectiveTo investigate the effect of Zuoguiwan on pancreatic islet function in offspring of gestational diabetes mellitus （GDM） maternal rat model and explore the mechanisms of Zuoguiwan in improving pancreatic islet function based on postpartum pancreatic regeneration. MethodHealthy female SD rats with normal blood glucose levels were paired with male rats in a 2∶1 ratio and housed together. Pregnancy was confirmed based on vaginal plugs or vaginal smears. The pregnant rats were divided into the following groups： normal group， model group， insulin group （insulin Detemir， 20 U·kg-1）， low-dose Zuoguiwan group （1.89 g·kg-1）， and high-dose Zuoguiwan group （3.78 g·kg-1）. The GDM rat model was induced using streptozotocin in rats except for those in the normal group. The model was confirmed by blood glucose testing in the maternal rats. Except for the normal and model groups， the other groups received daily administration of corresponding treatments. At 21 days after birth， fasting blood glucose （FBG） and fasting serum insulin （FINS） levels were measured in 6 offspring from each group. The homeostasis model assessment of insulin resistance （HOMA-IR） was calculated， and an oral glucose tolerance test （OGTT） was performed on additional 12 offspring from each group. Blood samples were taken from the abdominal aorta of the offspring at postnatal day 22， and enzyme-linked immunosorbent assay （ELISA） was used to measure insulin， glucagon （GC）， pancreatic polypeptide （PPY）， and somatostatin （SS） levels in the serum. Hematoxylin-eosin （HE） staining was performed to observe pathological changes in the pancreatic tissue of the offspring. Immunofluorescence （IF） was used to observe the area and structure of the pancreatic islets. Western blot was used to detect the expression of key proteins involved in the development and functional expression of pancreatic β-cells， namely pancreatic and duodenal homeobox factor 1 （Pdx1）， Nkx6.1， and Glucose transporter 2 （Glut2）. ResultCompared with the normal group， the model group showed significant increases in FBG and FINS levels， and HOMA-IR （P<0.01）. Compared with the model group， the insulin group showed significant decreases in FBG levels and HOMA-IR （P<0.01）， the low-dose Zuoguiwan group showed a significant decrease in FBG levels （P<0.05）， and the high-dose Zuoguiwan group showed significant decreases in FBG and FINS levels， and HOMA-IR （P<0.01）. Compared with the normal group， the model group showed significant increases in OGTT 60-min blood glucose levels and AUC index （P<0.05， P<0.01）. Compared with the model group， the high-dose Zuoguiwan group showed significant decreases in OGTT60-min blood glucose levels and area under the curve（AUC） index （P<0.05， P<0.01）. HE staining of pancreatic tissue showed that compared with the normal group， the model group had a reduced number of islets and a loose arrangement of acinar cells. Compared with the model group， the groups with drug treatment showed increased number of islets and a compact arrangement of acinar cells. Compared with the normal group， the model group had significantly increased levels of insulin， GC， PPY， and SS in the serum （P<0.01）. Compared with the model group， the low-dose and high-dose Zuoguiwan groups and the insulin group showed significantly decreased serum levels of insulin， GC， PPY， and SS （P<0.05， P<0.01）. IF results showed that compared with the normal group， the model group had a significantly lower positive rate of insulin （P<0.05）. Compared with the model group， the low-dose and high-dose Zuoguiwan groups showed a significant increase in the positive rate of insulin （P<0.05）. There was no significant difference in the positive rate of GC among the groups. In terms of the proportion of insulin and GC in individual islets， compared with the normal group， the model group showed a significant decrease in the proportion of insulin （P<0.01） and a significant increase in the proportion of GC （P<0.01）. Compared with the model group， the low-dose and high-dose Zuoguiwan groups showed significantly increased proportion of insulin （P<0.01） and significantly decreased proportion of GC （P<0.01）. Compared with the normal group， the model group showed significantly decreased expression levels of Pdx1， Nkx6.1， and Glut2 proteins in the pancreatic tissue of GDM offspring （P<0.05）. Compared with the model group， the insulin group and the low-dose Zuoguiwan group showed significant increases in the expression levels of Pdx1 and Nkx6.1 proteins in the pancreatic tissue of GDM offspring （P<0.05）， and the low-dose and high-dose Zuoguiwan groups showed significant increases in the expression levels of Glut2 protein （P<0.05）. ConclusionZuoguiwan can promote pancreatic islet development in offspring of GDM maternal rat model， improve pancreatic islet morphology and function， and alleviate insulin resistance. Its mechanism of action may be related to the regulation of Pdx1， Nkx6.1， and Glut2 protein expression in the pancreatic tissue of offspring.
ABSTRACT
Objective:To observe the ferroptosis in hippocampal neurons of rats with multiple cerebral concussion (MCC) .Methods:Ninety clean grade male Wistar rats with a body mass of (250±10) g were randomly divided into control group (12 rats) and model group (78 rats) according to the random number table method. The rat of MCC model was prepared by hitting the frontotemporal lobe of rats with free fall method once a day for 3 consecutive days.Then the MCC model rats were randomly divided into 12 h, 24 h, 48 h, 72 h, 7 d and 14 d groups with 12 rats in each group. The balance beam experiment was used to detect the motor coordination function of the rats. The levels of interleukin-β(IL-1β), interleukin-6(IL-6), serum glutathione (GSH), neuron-specific enolase (NSE) in serum of rats were detected by ELISA.The content of iron ion in hippocampus was detected by colorimetry. The mRNA and protein levels of glutathione peroxidase 4(GPX4), ferritin heavy (FTH) and ferritin light(FTL) in the hippocampus were detected by RT-PCR and Western blot. Prussian blue staining was used to observe the iron deposition in brain tissue.The ultrastructural changes of hippocampal neurons and mitochondria were observed by transmission electron microscope. SPSS 24.0 statistical software was used for one-way ANOVA among groups, and LSD test was used for multiple pairwise comparison.Results:In the balance beam experiment, the passing time and motor coordination score of rats in each group were significantly different ( F=30.08, 60.34, both P<0.05). The passing time and motor coordination score of rats in the 48 h group ((87.00±4.74) s, (4.75±0.43)) were significantly higher than those in the control group ((35.13±6.99) s, (0.75±0.23)) (both P<0.05). There was significant difference in the total iron ion content, Fe 2+ content and Fe 3+ content in hippocampus of rats in each group ( F=25.20, 94.42, 40.25, all P<0.05), and the content of Fe 2+ in hippocampus of 48 h group was significantly higher than that of the control group ((10.17±0.05) ng/μL, (8.65±0.01) ng/μL)( P<0.05). In the results of RT-PCR and Western blot, the mRNA and protein levels of GPX4, FTH and FTL in hippocampus of each group were significantly different ( F=37.94, 82.09, 49.01, 71.63, 28.94, 15.78, all P<0.05). The mRNA level and protein level of GPX4 ((1.09±0.01), (0.23±0.01) )and FTL ((1.60±0.03), (0.64±0.02)) in 24 h group were significantly higher than those of the control group (GPX4: (1.00±0.02), (0.17±0.01)), FTL: ((1.00±0.04), (0.32±0.01))(all P<0.05). The mRNA level and protein level of FTH ((0.24±0.03), (0.07±0.01)) in 24 h group were significantly lower than those of the control group((1.00±0.01), (0.67±0.03))(both P<0.05). The results of electron microscope showed that the hippocampal neuronal cells of the model rats were reduced, the nucleolus was broken and the nuclear membrane was shrunk in varying degrees, the mitochondria were swollen and deformed and there were vacuoles, and the cristae in the mitochondria decreased or disappeared. Conclusion:The levels of inflammatory reaction and oxidative stress in the multiple cerebral concussion model rats increase, and the hippocampal neurons show the characteristics of ferroptosis, especially at 24 h and 48 h.