| Objective: To discuss the mechanism of berberine?BBR? for ameliorating gastrointestinal neurological disorder in early diabetic rats.Methods: Rats were assigned into three groups randomly, including the control group, the diabetic group and the BBR treated group. After fasting for 12 h, the diabetic rats and the BBR treated rats intraperitoneally injected with streptozotocin(STZ, 65mg·kg-1) to induce the diabetic model, drawing tail vein blood and detecting the glucose values after 72 h later. The diabetic rats model was regarded successful if the glucose value was over 16.70mmol·L-1. After 2 weeks of diabetes success, BBR(200mg·kg-1) was administered by gavage once a day for another 2 weeks. Using the HE staining, we observed the morphologic changes in gastric smooth muscle tissue; detected the changes of indicators related to cholinergic and nitrergic nerve, and studied the influence of BBR on that. Otherwise, using the electric field stimulation induced neurogenic reaction and organ bath, we investigated the effects of BBR on nerve functions in gastric smooth muscle, and explore the possible mechanism.Results:1 Effects of BBR by gavage on the body weigh, blood glucose and gastric fundus morphology in diabetic rats.1.1 Changes of the body weight and the blood glucose level in different group ratsCompared with the contemporaneous normal rats, the body weight were significantly decrease in both diabetic model group and BBR treated group rats?P<0.01?; compared with the diabetic model rats, body weight of rats in BBR treated group had no significant difference?P>0.05?. Compared with the contemporaneous normal rats, the blood glucose level had a significant increase in diabetic model group and BBR treated group rats?P<0.01?; compared with the diabetic model rats, the blood glucose level of BBR treated group rats were no significant difference?P>0.05?.1.2 The HE staining in gastric fundus sections of different group ratsEach group was stained clearly and visiblly, which both the nucleus and the basophilic granules were dyed into bluish violet, while the pink parts were cytoplasm or other organizations. Usually, neurons were shaped as circular or oval or irregular, concentrating in the myenteric nerve plexus, which had the clear cell nucleus and unambiguous nucleoli. Nissl bodies were that the basophilic granule scattered distribution in the cytoplasm. Take the HE staining results of control group as a reference, the cell nucleus distributed uniformly with clearly nucleoli in the gastric fundus smooth muscle of all groups; The neurons cell bodies of diabetic model group presented vascuolar degeneration; After the BBR oral administration, the number of vascuolar lesions was reduced. However, the number of neurons had no changes in gastric fundus smooth muscle of all groups.2 Effects of BBR by gavage on the related enzymes of gastrointestinal neurotransmitters NO and ACh in diabetic rats2.1 The drawing of standard curve about proteinDraw the protein standard curve with the standard concentration and the OD value of different concentration proteins as horizontal and ordinate. The equation of protein standard curve is y = 0.92 x + 0.042,R2 = 0.99.2.2 The changes of tNOS activity and nNOS in gastric fundus tissueCompared with the normal group, there were no significant difference on tNOS activity in gastric fundus tissue of diabetic group and BBR treated group rats?P>0.05?.Compared with the normal group, nNOS were decreased obviously in gastric fundus of diabetic rats?P<0.05?; but BBR had no effects on nNOS?P>0.05?.2.3 The changes of ChAT, AChE activity and ACh in gastric fundusCompared with the normal group, the ChAT activity was significantly increased in the gastric fundus of diabetic rats?P<0.05?; compared with the diabetic model group, the ChAT activity were significantly decreased in the gastric fundus of BBR treated rats?P<0.01?.Compared with the normal group, the AChE activity was significantly decreased in the gastric fundus of diabetic rats?P<0.05?; but BBR had no effects on AChE activity?P>0.05?.Compared with the normal group, the ACh was significantly increased in the gastric fundus of diabetic rats?P<0.01?; but BBR had no effects on ACh?P>0.05?.3. Influence of BBR on the neurogenic contraction and relaxation responses induced by EFS in gastric fundus circular muscle of rats3.1 The interaction effects of the inhibitory neurotransmitter NO and excitatory neurotransmitter ACh on the EFSWhen 100?mol·L-1 L-arg was administered in organ bath, the EFS-induced contractile responses were reduced significantly on the gastric fundus circular muscle of rats?P<0.01?, meanwhile the contraction amplitude became disorder. And when 10,100?mol·L-1 L-NAME were administered in organ bath, the EFS-induced contractile responses were increased markedly on the gastric fundus circular muscle of rats?P<0.01?.Given specimen 0.30?mol·L-1 carbachol for pre-contraction, neostingmine 0.01?mol·L-1 can significantly inhibited relaxation response of the rats gastric fundus circular muscle were induced by EFS?2HZ, 4HZ, 10 HZ decreased 13.8%, 11.2%, 14.2% respectively, P<0.01?.3.2 Effects of BBR, NOS inhibitor and AChE inhibitor on the contractile responses induced by EFS in gastric fundus circular muscle of ratsOn the gastric fundus circular muscle of rats, EFS-induced contractile responses were significantly increased by 1,10?mol·L-1 BBR, 10,100?mol·L-1 L-NAME and 0.003, 0.01?mol·L-1 neostigmine when administered in organ bath?P<0.05, P<0.01?. In present of 10?mol·L-1 L-NAME, 10?mol·L-1 BBR enhanced EFS-induced contraction amplitude further on the gastric fundus circular muscle of rats?P<0.05?.3.3 Influence of calcium channel blockers on the effects of BBREFS-induced contractile responses on the gastric fundus circular muscle were inhibited by both of 1?mol·L-1 amlodipine besylate and 10?mol·L-1 cilnidipine?decreased 43.9% and 44.7% respectively, P<0.01?, which they abolished the effects of BBR completely. In addition, 1?mol·L-1 amlodipine besylate significantly inhibited the effects of L-NAME and neostigmine on EFS-induced contractile response in gastric fundus circular muscle of rats?P<0.05, P<0.01?.4 Influence of BBR on the tNOS and AChE activity in vitro in the gastric fundus of rats0.01-1mmol·L-1 BBR and 1mmol·L-1 L-NAME did not affected the tNOS activity in the rats gastric fundus tissue?P>0.05?; but 10,100mmol·L-1 L-NAME decreased the tNOS vitality significantly?P<0.05?.0.01,0.10mmol·L-1 BBR and 0.01?mol·L-1 neostigmine did not affected the AChE activity in the rats gastric fundus tissue?P>0.05?; but 0.10,1?mol·L-1 neostigmine?P<0.05? or 1mmol·L-1 BBR?P<0.01? decreased the AChE vitality significantly.Conclusions:1 In the gastric fundus of STZ-induced early diabetic rats, the synthesis of excitatory neurotransmitter ACh is increased, and the elimination of that is reduced; and the synthesis of inhibitory neurotransmitter NO is decreased. The balance of the functions between NO and ACh is broken, and existing a negative correlation.2 The effects of BBR ameliorating neurotransmitters disorder, and promoting the release of ACh may primarily by influence the inflow of calcium in gastric fundus of early diabetic rats. There are less effects of BBR by gavage on the synthesis and elimination of ACh and NO in gastric fundus of diabetic rats. |
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