Effects And The Mechanism Of Electroacupuncture At Acupoint St-36 On ENS In Diabetic Rats

Background: Diabetes can result in loss of enteric neurons and subsequent gastrointestinal dysfunction, but the mechanism of enteric neuronal loss in diabetes is unclear. Glial cell line-derived neurotrophic factor (GDNF) and its downstream signal pathway PI3K/Akt may play an important role in enteric neuropathy in diabetes. Aim: To investigate the enteric neuropathy in diabetic rats and the role of glia cell line-derived neurotrophic factor (GDNF) and its signalling pathway PI3K/Akt in regulating enteric neurons survival.Methods: Male Sprague-Dawley (SD) rats were randomly divided into normal control group, diabetic groups (rats with diabetes for 4, 8 and 12 weeks respectively). The models of diabetic rats were established by peritoneally injection of streptozotocin and the control group were injected saline. Proximal and distal colon specimens were obtained from each rat. Phosphoinositol-3-kinase signaling pathway was analysed by Akt phosphorylation. Protein gene product 9.5 (PGP9.5) used as a pan-neuronal marker. The expressions of GDNF, phospho-Akt (p-Akt), neuronal nitric oxide synthase (nNOS) neurons, cholinergic [choline acetyltransferase (CHAT) stained] neurons and total neurons were measured by immunohistochemical streptavidinbiotin complex (SABC) methods, Western blotting and real-time polymerase chain reaction methods for each specimen.Results: (1) Expression of GDNF was significantly decreased in diabetes 8 and 12 weeks group compared with the control group in both proximal (P < 0.01) and distal (P < 0.01) colon. The change of GDNF expression was greater in the 12 weeks group than that in the 8 weeks group (P < 0.05). There were no significant differences between the 4 weeks group and the control group in expression of GDNF (P > 0.05). (2) The change trend of Akt phosphorylation was the same with GDNF. (3) The numbers of nNOS, CHAT neurons and total neurons in proximal and distal colon were decreased significantly during the course of diabetes (P < 0.05).Conclusions: Diabetes can significantly induce enteric neuropathy. This change may be mediated, in partly, via a reduction of GDNF and its main downstream signalling pathway PI3K/Akt, which is a survival signal for enteric neurons.Background: Electroacupuncture (EA) at acupoint ST-36 (Zusanli) has been used to alleviate gastrointestinal symptoms, improve gastrointestinal motility. Our previous study showed that diabetes can significantly induce enteric neuronal loss. But the effects of EA on enteric nervous system (ENS) have scarcely been investigated. Aim: To investigate the effects of EA at acupoint ST-36 on ENS.Methods: 64 SD male rats were randomly divided into eight groups: normal control group, diabetic group (DM), diabetic with chronic high frequency EA group (C-HEA), diabetic with chronic low frequency EA group (C-LEA) and diabetic with chronic sham EA group (C-SEA), diabetic with acute high frequency EA group (A-HEA), diabetic with acute low frequency EA group (A-LEA) and diabetic with acute sham EA group (A-SEA). Diabetes was induced by a single intraperitoneal injection of Streptozotocin (STZ) at 60 mg/kg and the controls were injected citrate buffer. The parameters of HEA included a frequency of 100HZ and an amplitude of 1mA, while the LEA were 10HZ and 1mA; the SEA was that needles were inserted into the acupoints but without current. The three long-term chronic EA groups were given high frequency or sham EA 30 min/d for 6 weeks after STZ injection and diabetes were diagnosed; the three short-term acute EA groups were given low frequency EA or sham EA 30 min/d since the 6th week after STZ injection and diabetes were diagnosed. The expressions of Protein gene product 9.5 (PGP9.5), neuronal NO synthase (nNOS) neurons, cholinergic (choline acetyltransferase [ChAT] stained) neurons were measured by immunofluorescence or immunohistochemistry, real-time PCR and Western blotting methods in proximal and distal colon tissues of each rat.Results: 1) The total neurons and the two types of enteric neurons (nNOS and CHAT neurons) in the mRNA and protein level were significantly decreased in six weeks diabetic group compared with the normal control group in both proximal (P<0.01) and distal colon (P<0.01). 2) Compared with all the other DM or DM with EA groups, the chronic HEA group could induce a more significant quantitative increase in the mRNA and protein level of the enteric neurons in both proximal (P<0.01) and distal colon (P<0.01).Conclusions: EA with high frequency at acupoint ST-36 can induce regeneration of lost enteric neurons in diabetic rats.Background: Our previous study showed that diabetes can significantly induce enteric neuronal loss and EA may induced regeneration of impaired enteric neurons. But the mechanisms of EA on enteric nervous system (ENS) have scarcely been investigated. Aim: To explore the possible mechanisms of EA at acupoint ST-36 on ENS.Methods: 64 SD male rats were randomly divided into eight groups: normal control group, diabetic group (DM), diabetic with chronic high frequency EA group (C-HEA), diabetic with chronic low frequency EA group (C-LEA), diabetic with chronic sham EA group (C-SEA), diabetic with acute high frequency EA group (A-HEA), diabetic with acute low frequency EA group (A-LEA) and diabetic with acute sham EA group (A-SEA). Diabetes was induced by a single intraperitoneal injection of Streptozotocin (STZ) at 60 mg/kg and the controls were injected citrate buffer. The parameters of HEA included a frequency of 100HZ and an amplitude of 1mA, while the LEA were 10HZ and 1mA; the SEA was that needles were inserted into the acupoints but without current. The three long-term chronic EA groups were given high frequency or sham EA 30 min/d for 6 weeks after STZ injection and diabetes were diagnosed; the three short-term acute EA groups were given low frequency EA or sham EA 30 min/d since the 6th week after STZ injection and diabetes were diagnosed. GDNF and Akt phosphorylation (p-Akt) were measured by real-time PCR and Western blot methods in proximal and distal colon tissues of each rat.Results: 1) GDNF and p-Akt in the mRNA and protein level were significantly decreased in six weeks diabetic group compared with the normal control group in both proximal (P < 0.01) and distal colon (P < 0.01). 2) Compared with all the other DM or DM with EA groups, the HEA group could induce a more significant quantitative increase in the mRNA and protein level of GDNF and p-Akt in both proximal (P < 0.01) and distal colon (P < 0.01).Conclusions: GDNF and PI3K/Akt signal pathway may play an important role in EA induced regeneration of impaired enteric neurons.