| Backgroud:Diabetic encephalopathy (DE), one of the serious complications of Diabetes Mellitus(DM), impaired cognition and memory of DM patients. Traditional treatment for DE is thecombination of thiazolidinedione (TZD), peroxisome proliferator-activated receptor(PPAR-γ) agonists to control blood glucose, with drugs to improve the function of braincells; however, there is still no specific medicine for DE therapy. Although the TZD drugsis effective to control blood glucose, the roles in prevention and treatment of neural injuryof DM patients is very limited, because TZD is difficult to pass the blood-brain barrier(BBB) due to its low liposolubility. Therefore, it has a very important clinical significanceto fill this gap to develop a therapeutic medicine for DE treatment, and this medicineshould have hypoglycemic effect as well as high liposolubility to pass the blood-brainbarrier.Aims：Alkyl-substituted benzimidazole group has low hydrophilic, high liposolubility and good biocompatibility. To this end, we synthesized benzimidazole derivative named ascompounds H2, H4and H6, by conjuncting the alkyl-substituted benzimidazole group toTZD group. The evaluation of these compounds on neural protection were carried outusing a neuron-injury model induced by high glucose stimulation in vitro, combined withimmunofluorescence staining, Western blot and flow cytometry methods. Among them,compound H2was identified to have the best neuroprotective effects. The preliminarypharmacokinetic results suggested that compound H2could pass the BBB of rats. Inaddition, the therapeutic effects of compound H2for diabetic encephalopathy wereinvestigated using a type2diabetes (T2DM) rat model. The results showed that H2significantly promoted the survival rate, decreased the level of blood glucose, andimproved learning and memory behaviors of T2DM rats. The underlying mechanisms ofH2neuroprotection were determined by biochemical markers related to inflammation andantioxidant system. We found that the levels of inflammatory cytokines IL-1β and TNF-decreased, while anti-inflammatory cytokine IL-10increased in the blood in H2-treatedT2DM rats. Furthermore, H2decreased the level of malondialdehyde (MDA), improvedthe antioxidant substances superoxide dismutase (SOD), catalase (CAT) and glutathioneperoxidase (GSH-Px) in the T2DM rats brain. This study provides preliminary basis toexplore a new candidate for the diabetes encephalopathy treatment.Methods:1. Synthesis of new benzimidazole derivative componds H2, H4and H61) Under acidic conditions, benzimidazoles were obtained by the condensationreaction using substituted anthranilic benzene and1-chloro-straight-chain carboxylic acid;2)5-methylenebis (4-hydroxyphenyl) thiophene-2,4-dione (H7) was harvestedthrough the reaction of4-hydroxybenzaldehyde with2,4-thiazolidine dione, then theintermediate5-(4-hydroxybenzyl) thiazolidine-2,4-dione (H8) was obtained by sodiumborohydride reduction;3) Synthesis of benzimidazole intermediates2-chloro-methyl-1H-benzimidazole (H1),2-(chloromethyl)-5-methyl-1H-benzimidazole (H3),2-(chloro-methyl yl)-1-methyl-1H-benzimidazole (H5); 4) Condensation reatcion of benzimidazole intermediates H1, H3and H5withthiazolidinedione intermediates H8to form the target compounds H2, H4, H6;2. The evaluation of the neuroprotective effects of new TZD derivative, compondsH2, H4and H6, in vitro1) Western blot analysisThe neuroprotective effects of compounds H2, H4and H6were investigated in thecultured cortical neurons. The neurons were stimulated with high glucose (30mM) for24h, followed by treatment with compounds H2, H4and H6at different concentrations(0.1,1.0,10μM) for another24h. Western blot analysis were used to detect the changes ofexpressions levels of aldose reductase (AR), hypoxia-inducible factor1-alpha (HIF-1),Bax and pro-caspase-3, which are related to the pathological progress of diabetes andneurotoxicity. Mannitol was used as the osmotic pressure control and prosome structurerosiglitazone (RGZ) was used as positive control.2) Immunofluorescence experimentsThe cultured mature neurons were stimulated with high glucose (30mM) for24h,followed by treatment with compound H2at different concentrations (0.1,1.0,10μM) foranother24h. The effects of H2on neuron morphology after treatment were checked byimmunofluorescence staining of neuronal cytoskeletal proteins microtubule-associatedprotein-2(MAP2). Mannitol and RGZ weres used as control.3) Flow cytometry experimentsThe effects of H2on neuron apoptosis were determined by flow cytometry afterneurons were stimulated with30mM glucose for24h followed by H2treatment at0.1,1.0,10μM for another24h. Mannitol and RGZ were both used as control.3. The pharmacokinetic study of compound H21) Reversed-phase liquid chromatography was used to detect the concentration of H2in samples, and the working system was used as following: mobile phase0.01mol/LNH4Ac: CH3OH (35:65, V/V), flow rate1mL/min, detection wavelength247nm. RGZwas used as the internal standard. Compound H2was adimistrated to normal rats byintraperitoneal injection at25mg/kg. Orbital blood was collected at0,0.083,0.25,0.5,1.0, 1.5,2.0,3.0and4.0h after H2administration, and the H2concentrations in blood weredetermined by HPLC assay, therefore, the pharmacokinetic parameters of H2in SD rats’plasma were obtained.2) Brain tissues were harvested after intraperitoneal injection of H2(25mg/kg) for0.25h and homogenized according to the preliminary experiments, HPLC assay was usedto detect H2concentrations in the brain tissue.4. The therapeutic effects of compound H2for diabetic encephalopathy treatment1) Establishment of T2DM and acute anxiety modelsTo establish type2diabetes (T2DM) and rat insulin resistance model, SD rats werefed continously with high sugar and fat diet for4weeks, then streptozotocin (STZ) wasadministrated to rats by intraperitoneal injection at30mg/kg, and the same diet wasmaintained for another8weeks to get T2DM model.To induced acute anxiety model, SD rats were constrainted in well-ventilated Perspexrestraining tubes for2h for two consecutive days.2) Morris water maze (MWM) testTo check the therapeutic effects of H2, T2DM rats were divided into DM controlgroup, H2-treated group (1mg/kg,3mg/kg,10mg/kg) and RGZ-treated group (3mg/kg)besides the normal control group, the animals were administrated with H2or RGZintragastricly for4weeks. Same volume saline was used as control. The effects of H2onspatial learning and memory were evaluated by the Morris water maze test, and the escapelatency and the number of crossing the platform were collected for analysis.3) Elevated plus maze (EPM) experimentThe elevated plus maze (EPM) was conducted to evaluated the potential side effectsof compound H2. SD rats were randomly divided into control group, H2-treated group (3mg/kg) and acute anxiety group. Individual animals were placed in the centre square,facing an open arm, and allowed to move freely for5min. Rats were videotaped using acamera fixed above the maze and analyzed with a video-tracking system. Open and closedarm entries (all four paws in an arm) and times were recorded. The number of entriesand time spent in open arms were used to measure anxiety degree, and the number of total entries in four arms was used for the detection of locomotor activity.4) Detection of the biochemical markersAfter the MWM test was finished, blood and brain tissue were harvested immediately.The concentrations of IL-1β, TNF-and IL-10in plasma, the activities of SOD, CAT,GSH-Px and MDA levels in brain homogenates were determined by kits purchased fromNanjing Jiancheng company.Results:1. Synthesis of componds H2, H4and H6Three target compounds of H2, H4, H6were synthesized, and their structures wereidentified by1H NMR and MS. In addition, single crystal of the intermediate H5andbyproduct H55were obtained during the synthesis process, and their structures werecharacterized by X-ray.2. The neuroprotective effects of new TZD derivative, componds H2, H4and H6, invitro1) Effects of compounds H2, H4and H6on neurons stimulated with high glucoseWestern blot results showed that the expression levels of AR, HIF-1and Bax incultured neurons increased markedly upon high glucose stimulation, while the level ofpro-caspase-3reduced, compared with normal controls. Treatment with compounds H2,H4and H6revesed the expression levels of AR, HIF-1, Bax and Pro-caspase-3to somedegree.30mM mannitol was set as a osmotic control to exclude the influence of osmoticpressure in culture system at the same time. The results showed that mannitol had noeffects on AR, HIF-1, Bax and Pro-caspase-3expression, similar to the results from thenormal control group. Therefore, the impact of osmotic pressure on the growth state of theneurons was excluded.2) Effects of compound H2on neuron morphologyThe morphological changes of the cytoskeleton in cultured neurons were observedafter30mM glucose stimulation for24h, which was characterized as discontinuous andbreak-like neurite by cytoskeletal protein MAP2staining. Treatment with compound H2for24h, the neurite morphology was rescued to normal. 3) Effects of compound H2on neuron apoptosisThere was no significant apoptosis in neurons upon high glucose stimulation for24h aswell as in other treated groups. It is possibile that early and acute apoptosis can not beinduced under high glucose condition in short time. Therefore, flow cytometry methodwas not sensitive to evaluate the neuroprotective effects of synthetic compounds in theearly phase.3. The pharmacokinetic study of compound H2A simple, safe and economical way was established in this study to detect theconcentrations of H2in the plasma and brain tissue using HPLC-UV method. Accurateand sensitive data were obtained for pharmacokinetic analysis. Administration of H2at25mg/kg(i.p), the parameters were obtained as following: the AUC (0-)14.581.45mg/L*h, Tmax0.17±0h, CLz/F1.73±0.17L/h/kg, t1/20.73±0.075h, Cmax26.39±1.02g/mL.The concentration of H2in brain after administration for0.0833h was1.57ng/g, and thisresult implicated that H2passed through the blood-brain barrier.4. The therapeutic effects of compound H2on the diabetic brain functions1) General observationType2diabetic rats showed a continued decline in weight, accompanied withpolydipsia, polyuria and polyphagia which was observed in diabetes patients, and thefasting blood glucose was more than7.8mmol/L. The weight of normal rats continued togrow and they survived healthily with smooth and glossy fur and in good mental status.The blood glucose decreased significantly in each treatment of H2and RGZ, comparedwith the T2DM group (p <0.01).2) Treatment of H2rescued deficits of learning and memory in T2DM ratsThe T2DM rats had impaired learning using the available visuospatial cues to locatethe submerged escape platform, as indicated by prolonged escape latency acrossconsecutive trials compared with the normal group (p <0.01), suggesting that T2DM ratsdevelops cognitive deterioration with increasing age. After medication of H2or RGZ for4weeks, the reduced escape latency across the trials of rats in H2groups (low, medium andhigh dose) and RGZ group were observed compared with the control T2DM group (p < 0.05, p <0.01, p <0.01and p <0.05). Furthermore, we confirmed the escape latency ofrats in high-dose H2group was significantly reduced compared with the RGZ-treated rats(p <0.05).The platform in the water maze was removed at day6, the numbers across theplatform in T2DM reduced markedly compared with the normal rats (p <0.01). Thenumbers across the platform increased significantly in H2-treated groups (medium andhigh dose), compared with RGZ-treated group (p <0.05, p <0.01).3) H2treatment prevented cataract development in T2DM ratsThere was visible lens opacification observed in T2DM rats from each group in thelate phase of the DM models. According to the lens grading system, the lens opacificationof the rats were graded. The worst grade of lens opacification happened in T2DM ratscompared with normal control and H2-treated rats (p <0.01). H2treatment (low, mediumand high dose) ameliorated significantly the severity of opacification compared withT2DM rats (p <0.05), and this effect was not observed in RGZ-treated group, implicatingthat H2treatment effectively prevent cataract development in diabetic rats.4) Elevated plus maze experimentThe acute anxiety rat model was established by restraint rats for two consecutive days,and the time spent in open arms increased compared with control rats (p <0.05). While,there was no significant difference of the number of entries and time spent in open armbetween H2-treated group and anxiety model group.5) Biochemical markersWe first checked the levels of inflammatory cytokines IL-1β and TNF-, andanti-inflammatory cytokine IL-10in rat plasma at the end of experiments. The levels ofIL-1β and TNF-in T2DM group were increased significantly compared with normalgroup (p <0.05, p <0.01), while levels of IL-10were decreased significantly (p <0.01).Compared with T2DM rats, levels of IL-1β decreased significantly in each of H2-treatedgroup (p <0.05). The levels of TNF-were significantly decreased in H2-treated group atboth middle and high dose compared with the T2DM group (p <0.05, p <0.01). Levels ofTNF-decreased markedly in high-dose H2group compared with RGZ-treated group (p < 0.05). The levels of IL-10in each H2-treated group were significantly increased comparedwith T2DM rats (p <0.05, p <0.05, p <0.01). Furthermore, the levels of IL-10were evenhigher in high-dose H2group than in RGZ-treated group (p <0.05).We next checked the activities of SOD, CAT and GSH-Px and the levels of MDA inbrain homogenate. The activities of SOD, CAT and GSH-Px decreased significantly inT2DM group compared with normal group (p <0.01). SOD acitvities increasedsignificantly in each H2-treated group compared with both T2DM group and RGZ-treatedgroup (p <0.05, p <0.01, p <0.05). The levels of CAT were significantly increased inmedium and high dose H2group compared with both T2DM and RGZ-treated group (p <0.05). Levels of GSH-Px activity in each H2-treated group were significantly highercompared with T2DM rats (p <0.05), and was significantly higher in high-dose H2groupcompared with RGZ group (p <0.05). Levels of MDA reduced significantly in eachH2-treated group compared with T2DM rats (p <0.01), and even lower significantly inmiddle and high dose H2group compared with the RGZ-treated group (p <0.05, p <0.01).Conclusion:1. Neuroprotective effects of compound H2in vitroWestern blot results showed that the expression level of AR and HIF-1, which arerelated to glucose metabolism, increased markedly in neurons upon the stimulation withhigh glucose for24h, as well as apoptosis-related protein Bax. While, compared withcontrol, the levels of pro-caspase-3were decreased, indicating that high glucose led toabnormal glucose metabolism of neurons followed by early neuronal injury. The syntheticcompounds H2, H4and H6decreased the expression of AR, HIF-1and Bax, therebyinhibiting the neuronal injury induced by high glucose. These results suggested thatsynthetic H2, H4and H6rescued abnormal glucose metabolism mediated by high glucose.Among these compounds, H2was the most effective one for neuroprotection.There were some morphological changes in neurons characterized as discontinuousand break-like neurite by cytoskeletal protein MAP2staining after treatment with highglucose. Treatment with compound H2for24h after high glucose injury, the neurite morphology was rescued to normal, implicating that synthetic H2may play aneuroprotective role.There was no significant apoptosis in neurons upon high glucose stimulation for24has well as in other different treatment groups. It is possible that neuronal injury is along-term process in diabetic patients, therefore, flow cytometry method was not sensitiveto evaluate the neuroprotective effects of these compounds in such a short-time treatment.2. The pharmacokinetics research of compound H2In the present study, H2content in the rat brain was1.57μg/g after administration for15min, and H2in plasma was26.4mg/mL at this corresponding time point, implicatingthat H2passed through the blood-brain barrier. It has been reported by other group that theconcentration of rosiglitazone, the positive control drug, in the cerebrospinal fluid wasonly0.0045%of that in blood. We failed to detect rosiglitazone in brain tissue other thanin blood using the same method in the present study, maybe due to rosiglitazoneconcentrations was below the detection line. Detailed reasons needs to be furtherinvestigated. Taken together, we identified the ability of the compond H2passing throughblood-brain barrier into the brain tissue, which was more effective than rosiglitazone.3. The mechanisms of compound H2in the treatment of diabetes brain dysfunctionEvidence showed that T2DM develops cognitive deterioration due to the centralnervous system (CNS) injury under high blood glucose. Morris water maze is a widelyused in the assessment of rodent learning and memory, objectively and accurately. Theescape latency and number of crossing the platform were used to evaluate the cognitivefunction of the animals. In this study, H2treatment improved the cognitive dysfunction ofT2DM rats, which was better than the positive control RGZ. The preliminary mechanismsof H2protection were further explored by detection of inflammatory andanti-inflammatory cytokines in blood, antioxidant system in brain. The results showed thatH2treatment inhibited IL-1β and TNF-expression, and promoted the anti-inflammatorycytokine IL-10expression, thereby reducing autoimmune and chronic inflammationreaction in diabetes. In addition, the activities of SOD, CAT and GSH-Px increased andMDA decreased significantly in H2-treated brains. Furthermore the improvement of H2at the medium and high dose was much better than RGZ-treated rats. All these datasuggested that H2could improve brain enzyme activity of antioxidant system to clear avariety of reactive oxygen species which is harmful to the health, as well as to reduceMDA, a fat oxidation toxic substance, to play a neuroprotective effect.It is reported by another group that the relief of anxiety is positive to the therapy ofdiabetic patients. Then we observed whether H2had potential therapeutic effects onanxiety. The results of EPM showed that there was no significant acute anxiolytic effect ofH2, and chronic anxiolytic effects need to be further elucidated.4. Prevention against cataract by H2Aged diabetic patients are often complicated by Ophthalmopathy. This phenomenonwas also confirmed in this study. We found visible lens opacification in T2DM rats ofeach group. H2treatment decreased the incidence rate and ameliorated significantlyseverity of opacification compared with T2DM rats. This data indicated that compoundH2might have potential therapeutic effects on cataract development.In summary, compound H2showed similar neuroprotective effects in vitro as RGZ.However, H2had greater efficacy of potential therapeutic effects on diabeticencephalopathy in T2DM rats as compared to RGZ. |
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