[Effect of "Xiusanzhen" on hippocampal muscarinic cholinergic receptor activity in Alzheimer disease rats].

OBJECTIVE: To explore the effect of "Xiusanzhen" [electroacupuncture (EA) of bilateral "Yingxiang" (LI 20) and "Yintang" (EX-HN 3)] on activities of hippocampal muscarinic cholinergic receptor (mAChR) and the involved neural path in Alzheimer Disease(AD)rats. METHODS: Forty Sprague Dawley rats were randomly divided into normal control, model, olfactory nerve severing (ONS)-EA of "Xiusanzhen" (ONS-EA) and EA of "Xiusanzhen" (EA) groups. AD model was established by intra-hippocampal injection (AP: 3.5 mm, L:2 mm, H: -2.8 mm) of Abeta(1-40) starch-like peptide (10 microg/2 microL) under the aid of a microsyringe installed in a brain stereotaxis instrument. For rats of the ONS-EA group, the olfactory nerve was severed by using a surgical knife after drilling a hole on the skull (5 mm anterior to the bregma, L, R: 2 mm). The mAChR density, and its maximum binding capacity (Bmax) and dissociation constant (Kd) of the hippocampus tissue were measured by using radio-ligand binding analysis and Lowry's microamount protein assay. RESULTS: In comparison with the normal control group, the hippocampal mAChR density and its Bmax in the model group were decreased remarkably (P < 0.05), while the Kd of M-receptor in the model group was increased remarkably (P < 0.05). In comparison with the model group, hippocampal mAChR density and its Bmax in the EA group were up-regulated obviously (P < 0.05), while the Kd of mAChR in the EA group was down-regulated significantly (P < 0.05). No significant differences were found between the model and ONS-EA groups in mAChR density and its Bmax and Kd (P > 0.05). CONCLUSION: "Xiusanzhen"-EA can effectively up-regulate hippocampal mAChR density and Bmax and down-regulate Kd of M-receptor of hippocampus in AD rats, which is dependent on the intact olfactory nerve pathway.

[Effect of "Xiusanzhen"on expression of hippocampal Bcl-2 and Bax proteins in Alzheimer disease rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) of "Xiusanzhen" [3 acupoints, i.e., bilateral "Yingxiang" (LI 20) and "Yintang" (EX-HN 3)] on expression of B-cell lymphoma/leukemia-2 (Bcl-2, an antiapoptosis protein) and Bax (a protein for promoting apoptosis) in the hippocampus in Alzheimer disease (AD) rats, so as to explore its clinical mechanisms underlying relieving AD. METHODS: A total of 40 Sprague Dawley rats were randomly divided into normal control, AD model, EA and olfactory nerve transaction(ONT) + EA groups, with 10 rats in each. AD model was established by injection of beta-amyloid (Abeta)(1-40) (2 microL containing 10 microg) into the hippocampus (Bregma: 3.5 mm, L: 2.0 mm, H: -2.8 mm below dura), and the ON was sectioned by using a fine needle. EA (80-100 Hz, 1-3 mA) was applied to bilateral LI 20 and EX-HN 3 for 10 min, once daily (except Saturdays and Sundays) for 6 weeks. The expression of Bcl-2 and Bax of hippocampus was measured by immunohisto-chemistry. RESULTS: Compared with the normal control group, the expression of Bcl-2 of hippocampus was significantly lower in the AD model group (P<0.05), and that of Bax in AD model group was much higher (P<0.01). In comparison with the model group, the expression of hippocampal Bcl-2 was up-regulated significantly (P<0.01), and that of hippocampal Bax protein downregulated considerably in the EA group (P<0.01). No significant differences were found between AD model and ONT + EA groups in the expression of hippocampal Bcl-2 and Bax proteins (P>0.05), suggesting a necessary premise condition for EA in regulating hippocampal Bcl-2 and Bax expression. CONCLUSION: "Xiusanzhen" can regulate the expression of hippocampal Bcl-2 and Bax proteins in AD rats, which may contribute to its clinical effect in relieving AD, and the therapeutic effect depends on the integrity of the olfactory nerve pathway.

[Effect of "Xiusanzhen" on learning-memory ability and hippocampal somatostatin and arginine vesopressin contents in vascular dementia rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) of "Xiusanzhen" [bilateral "Yingxiang" (LI 20) + "Yintang" (GV 29)] on learning-memory ability and hippocampal somatostatin (SS) and arginine vasopressin (AVP) contents in vascular dementia (VD) rats. METHODS: Forty SD rats were randomly divided into control, VD model, VD plus olfactory bulb lesion (OBL, destroyed by electro-coagulation) and EA groups, with 10 cases in each. VD model was established by 4-vessel occlusion. Morris maze tests were conducted for evaluating the rats' learning and memory ability. EA (1-3 mA, 80/100 Hz) was applied to bilateral LI 20 and GV 29 in VD + OBL and EA groups for 10 min, once daily (except Saturdays and Sundays) for 6 weeks. The contents of SS and AVP in hippocampus tissue were measured by radioimmunoassay. RESULTS: In comparison with control group, the average escape latency (AEL) prolonged significantly and the target-platform crossing times (TPCT) decreased remarkably in VD model group (P < 0.01). In comparison with VD model group, the AEL was shortened and TPCT increased remarkably in EA group (P < 0.01). No significant differences were found between VD model and VD + OBL groups in the AEL and the TPCT (P > 0.05). Hippocampal SS and AVP contents in VD model group were significantly lower than those in control group (P < 0.05), while those in EA group were markedly higher than those in VD model group (P < 0.05). No significant differences were found between VD model and VD + OBL groups in SS and AVP contents (P > 0.05). CONCLUSION: EA of "Xiusanzhen" can improve VD rats' learning-memory ability, which may be related to its effects in raising hippocampal SS and AVP contents and to the intact olfactory pathway.

[Effect of "Xiusanzhen" on learning-memory ability and hippocampal ChAT and AChE activity in Alzheimer disease rats].

OBJECTIVE: To explore the effect of "Xiusanzhen" [bilateral "Yingxiang" (LI 20) + "Yintang" (EX-HN 3)] on learning-memory ability and choline acetyl transferase (ChAT) and acetyl cholinesterase (AChE) activity of hippocampus tissue in Alzheimer Disease (AD) rats. METHODS: A total of 40 Sprague Dawley rats were randomly divided into control, AD model, AD + olfactory-bulb lesion (OBL, induced by electrocoagulation method) + EA and "Xiusanzhen" (AD + EA) groups. AD model was established by 4-vessel occlusion. Morris water maze tests were conducted for evaluating the rat's learning-memory ability. The activity of ChAT and AChE of hippocampus tissue was detected by colorimetric method. RESULTS: In comparison with control group, the average escape latency (AEL) prolonged significantly and the target-platform crossing times decreased remarkably in AD model group (P<0.01). In comparison with AD model group, the AEL shortened and the target-platform crossing times increased remarkably in AD + EA group (P<0.01). No significant differences were found between AD model and AD + OBL + EA groups in AEL and the target-platform crossing times (P>0.05). The ChAT and AChE activity of hippocampus in AD model group was significantly lower than that of control group (P<0.05), while that of ChAT and AChE in AD + EA group was significantly higher than that in AD model group (P<0.05). No significant difference was found between AD model and AD + OBL + EA groups in ChAT and AChE activity of hippocampus (P>0.05). CONCLUSION: EA of "Xiusanzhen" can markedly raise the learning-memory ability in AD rats, which may be related to its effect in increasing the ChAT and AChE activity of hippocampus in AD rats. Its therapeutical effect also depends on the intact of the olfactory pathway.

[Clinical investigation on electroacupuncture treatment of vascular dementia with "Xiusanzhen"].

OBJECTIVE: To observe the effect of electroacupuncture (EA) in the treatment of vascular dementia (VD) so as to explore an effective therapy for it. METHODS: Sixty cases of VD were randomly divided into acupuncture group and medication (Duxil, 40 mg/time, 2 times/d for 10 weeks) group, with 30 cases in each group. "Xiusanzhen" means that two acupuncture needles were penetrated subcutaneously from bilateral Yingxiang (LI 20) to Shangyingxiang (EX-HN 8) respectively, and the 3rd needle was penetrated from the midpoint between two Yangbai (GB 14) through Yintang (EX-HN 3) to the nasal root, followed by applying EA stimulation to the acupoints. Hasegawa's Dementia Scale (HDS), Mini-mental state examination (MMSE), and Functional Activities Questionnaire (FAQ) were used for assessment of the VD patient's state before and after the treatment. RESULTS: After the treatment, the total effective rates of acupuncture and medication groups were 80.00% and 73.33% respectively. The effective rate of acupuncture group was significantly superior to that of medication group (P<0.05). Before treatment, scores of HDS, MMSE and FAQ in both groups had no significant differences (P>0.05); after the treatment, the scores of HDS and MMSE increased significantly, and that of FAQ decreased remarkably in both groups (P<0.01), and the difference of scores (or absolute value of FAQ) between post-treatment and pre-treatment in EA group was obviously higher than that in medication group (P<0.05). CONCLUSION: Acupuncture of "Xiusanzhen" is effective in the treatment of VD patients.

Application of in-gel protease assay in a biological sample: characterization and identification of urokinase-type plasminogen activator (uPA) in secreted proteins from a prostate cancer cell line PC-3.

A considerable problem in proteomics is to separate and identify functional proteins that participate in specific biological processes. To expedite the analysis of active proteases, we have developed a substrate-specific, sensitive in-gel trypsin activity assay after two-dimensional (2-D) separation in a sodium dodecyl sulphate (SDS)-polyacrylamide gel [22]. Using this method, we detected and characterized Arg-specific protease activity in the secreted protein sample of a prostate cancer cell line, PC-3, in 1-D and 2-D gels. Mass spectrometry (MS) identified the protease as urokinase-type plasminogen activator (uPA). Western blotting using anti-uPA antibody and protease inhibition tests confirmed the identification. Since no antibody was involved in the procedure, the result clearly demonstrates the feasibility of this method for identifying novel proteases in biological samples.