[RhoA/ROCK pathway involved in effects of Sanjiao acupuncture on learning and memory and synaptic plasticity in Alzheimer's disease mice].

OBJECTIVE: To observe the effect of Sanjiao acupuncture(triple energizer acupuncture)on the small G protein guanosine triphosphate enzyme subfamily protein RhoA/Rho kinase (ROCK) pathway in Alzheimer's disease mice, and explore its effect on learning and memory function and neurosynaptic plasticity. METHODS: Forty SAMP8 senile dementia mice were randomly divided into model, Sanjiao acupuncture (acupuncture), non acupoint acupuncture (non-acupoint) and fasudil groups, with 10 mice in each group, another 10 SAMR normal aging mice were selected as normal aging group. Mice in the acupuncture group were treated with acupuncture intervention on "Danzhong"(CV18), "Zhongwan"(CV13), "Qihai"(BL24) and bilateral "Xuehai"(SP10) and "Zusanli" (ST36). Mice in the non-acupoint group were treated with acupuncture at each of the left and right non-acupoints under the ribs and mice in the fasudil group were intraperitoneally injected with fasudil (25 mg/kg). The mice in each group were given medicine or acupuncture on the second day after grouping for 28 continuously days, once a day. Morris water maze test was used to test the learning and memory ability of mice. HE staining was used to observe the pathological changes of neurons in hippocampus. The number of hippocampal neuron dendritic spine was detected by FD fast Golgi staining kit. The contents of ß-amyloid 42 (Aß42) and phosphorylated tau protein (p-tau) in hippocampus were detected by ELISA. Western blot was used to detect the protein relative expression levels of RhoA, ROCK, F-actin and p-cofilin in hippocampus. RESULTS: Compared with those in the normal aging group, the hippocampal neurons of the model group were disorderly arranged, the number of neuron was reduced, the escape latency, hippocampal Aß42 and p-tau contents, RhoA and ROCK protein expressions increased (P<0.05), the number of crossing the original platform, the number of neuronal dendritic spines, expressions of F-actin and p-cofilin decreased (P<0.05). After the interventions, there was no statistically significant difference in the above indicators in the non-acupoint group relevant to the model group (P>0.05). The acupuncture group and fasudil group improved the hippocampal pathological damage. The escape latency, hippocampal Aß42 and p-tau contents, the expressions of RhoA and ROCK protein decreased (P<0.05), and the number of crossing the original platform, the number of hippocampal neuron dendritic spines, expressions of F-actin and p-cofilin increased (P<0.05) in both of the acupuncture and fasudil groups in contrast to the model and non-acupoint groups. Compared with the acupuncture group, there was no significant difference in the above indicators in the fasudil group (P>0.05). CONCLUSION: Sanjiao acupuncture may inhibit the activation of the RhoA/ROCK pathway, so as to improve the learning and memory function of AD mice, increase the number of hippocampal neuron dendritic spines, and promote synaptic plasticity.

[Acupuncture Regulated the Expressions of Hsp84 and Hsp86 and Postponed Brain Aging in SAMP8].

Objective To observe the correlations between heat shock protein 84 ( Hsp84)/ Hsp86 and brain aging in senescence accelerated mouse prone 8 (SAMP8) and the regulation effects of acupuncture. Methods Ten senescence accelerated mouse resistant 1 (SAMR1) were recruited as a normal control group. Another 30 SAMP8 mice were divided into the blank control group, the acupuncture group, and the non-acupoint group by random digit table, 10 in each group. Mice in the acupuncture group received treatment with "Sanjiao" acupuncture method. Mice in the non-acupoint group were needled at two fixed non-acupoints located at bilateral hypochondrium of the body. Catching stimulus at equal volume was given to mice in the rest two groups. All intervention was performed once per day for 15 successive days. Neuromuscular coordination of mice was evaluated. Levels of oxidative stress and protein carbonyl were determined. mRNA and protein expression levels of Hsp84 and Hsp86 in the hippocampus of mice and Neuro-2a cells were detected using Real-time PCR and Western blot. Results Compared with the normal control group, the success rate of tight rope experiment was lowered (P <0. 01) , levels of super- oxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the hippocampus were reduced (P < 0. 01) , the levels of superoxide anion and protein carbonyl increased (P <0. 01, P <0. 05), mRNA and protein expression levels of Hsp84 and Hsp86 in the hippocampus decreased (P <0. 01) in the blank con- trol group. Compared with the blank control group and the non-acupoint group, the success rate of tight rope experiment was elevated (P <0. 05) , levels of SOD and GSH-Px were increased (P <0. 05, P < 0. 01) , the levels of superoxide anion and protein carbonyl decreased (P <0. 01 , P <0. 05), and expres- sion levels of Hsp84 and Hsp86 in the hippocampus increased (P <0. 01) in the acupuncture group. mR- NA expression levels of Hsp84 and Hsp86 were decreased in Neuro-2a cells after treated with AP21-35 (P <0. 01). Conclusions Increased oxidative damage of protein and decreased expression levels of Hsp84 and Hsp86 might be partial reasons for resulting in accumulation of denatured protein and brain aging in SAMP8. Acupuncture could delay brain aging by regulating the expressions of Hsp84 and Hsp86.

[Effect of acupuncture on transmembrane signal pathway in AD mice: an analysis based on lipid-raft proteomics].

OBJECTIVE: To reveal the transmembrane signal pathway participating in regulating neuron functions of treating Alzheimer's disease (AD) by acupuncture. METHODS: SAMP8 mice was used for AD animal model. The effect of acupuncture method for qi benefiting, blood regulating, health supporting, and root strengthening on the amount and varieties of transmembrane signal proteins from hippocampal lipid rafts in SAMP8 mice was detected using HPLC MS/MS proteomics method. RESULTS: Compared with the control group, acupuncture increased 39 transmembrane signal proteins from hippocampal lipid rafts in SAMP8 mice, of them, 14 belonged to ionophorous protein, 8 to G protein, 8 to transmembrane signal receptor, and 9 to kinase protein. Totally 3 main cell signal pathways were involved, including G-protein-coupled receptors signal, enzyme linked receptor signal, and ion-channel mediated signal. Compared with the sham-acupuncture group, acupuncture resulted in significant increase of kinase signal protein amount. From the aspect of functions, they were dominant in regulating synapse functions relevant to cytoskeleton and secreting neurotransmitters. CONCLUSION: The cell biological mechanism for treating AD by acupuncture might be achieved by improving synapse functions and promoting the secretion of neurotransmitters through transmembrane signal transduction, thus improving cognitive function of AD patients.

Effects of Ganfukang on expression of connective tissue growth factor and focal adhesion kinase/protein kinase B signal pathway in hepatic fibrosis rats.

OBJECTIVE: To investigate the effect of Ganfukang (GFK) on connective tissue growth factor (CTGF) and focal adhesion kinase (FAK)/protein kinase B (PKB or Akt) signal pathway in a hepatic fibrosis rat model and to explore the underlying therapeutic molecular mechanisms of GFK. METHODS: Fifty SD rats were randomly divided into five groups as follows: the control group, the model group (repeated subcutaneous injection of CCl4), and the three GFK treatment groups (31.25, 312.5, and 3125 mg/kg, intragastric administration). Reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemistry were used to examine the expression of CTGF, integrin α5, integrin ß1, FAK/Akt signal pathway, cyclinD1, and collagen in the different-treated rats. RESULTS: GFK attenuated the up-regulation of CTGF, integrin α5, and integrin ß1 in hepatic fibrosis rats and suppressed both the phosphorylation of FAK and the phosphorylation of Akt simultaneously (P<0.01). At the same time, the expression of cyclinD1, collagen I, and collagen III was decreased by GFK significantly (P<0.01). CONCLUSIONS: CTGF and FAK/Akt signal pathway were activated in the CCl4-induced hepatic fibrosis rats, which contribute to increased expression of cyclinD1 and collagen genes. The mechanisms of the anti-fibrosis activity of GFK may be due to its effects against CTGF and FAk/Akt signal pathway.

[Combination of acupoints based on the theory of qi tonification, blood regulation and strengthening the primary source].

The acupuncture technique of tonifying qi, regulating blood and strengthening the primary source of human life is established on the base of the new pathology of dementia due to dysfunction of sanjiao by Prof. HAN Jing-xian. It is held that Tanzhong (CV 17), Zhongwan (CV 12), Qihai (CV 6), Xuehai (SP 10), Zusanli(ST 36) and Waiguan (TE 5) were the basic points for treatment of dementia. There are 3 characteristics of the point combination. Firstly, emphasis should be put on regulation of the stomach and the spleen so as to promote transportation, transformation and distribution; secondly, monarch, minister, assistant and guide points should be selected on the base of meridians, and more attention should be paid to the entity and connections among organs; and thirdly, treatment should be given according to physiological functions of the viscera.

Herb medicine Gan-fu-kang attenuates liver injury in a rat fibrotic model.

AIM OF THE STUDY: To verify therapeutic effects of Gan-fu-kang (GFK), a traditional Chinese medicine compound, in a rat model and to investigate the underlying mechanisms. MATERIALS AND METHODS: Liver fibrosis was established by 12 weeks of carbon tetrachloride (CCl(4)) treatment (0.5mg/kg, twice per week) followed by 8 weeks of "recovery" in rats. Rats randomly received GFK (31.25, 312.5 and 3125 mg/kg/day, p.o.) or vehicle from weeks 9 to 20, and were sacrificed at the end of week 20 for histological, biochemical, and molecular biological examinations. In a separate set of experiments, rats received 12 weeks of CCl(4) treatment, concomitant with GFK (312.5mg/kg/day, p.o.) during the same period in some subjects, but were then sacrificed immediately. An additional group of rats receiving no CCl(4) treatment served as normal controls. RESULTS AND CONCLUSIONS: (1) CCl(4) treatment resulted in severe liver damage and fibrosis. (2) In the main block of the 20-week study, GFK attenuated liver damage and fibrosis. (3) In the 12-week study, GFK produced prevention effect against hepatic injury. (4) GFK suppressed the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1), type I collagen, platelet-derived growth factor-BB (PDGF-BB)/PDGF receptor-beta chains (PDGFRbeta) and mitogen-activated protein kinases (MAPKs)/active protein-1 (AP-1) signal pathways. Taken together, these results indicated that GFK could attenuate liver injuries in both settings. Our findings also suggest that the AP-1 pathway is the likely molecular substrate for the observed GFK effects.

Effects of traditional chinese medicine on endotoxin and its receptors in rats with non-alcoholic steatohepatitis.

AIMS: The aim of this research is to study the effects of traditional Chinese medicine on endotoxin and its receptors in rats with nonalcoholic steatohepatitis (NASH). METHODS: Fifty-six SD rats were divided into seven groups. All the animals were fed high fatty diet for 12 weeks. Rats with non-alcoholic steatohepatitis (NASH) were treated with traditional Chinese medicine according to low-dose, middle-dose, high-dose and Lipitor from fifth week. All rats were killed at the end of 12th week. The liver pathology changes were observed under light microscope. The levels of serum lipoid, alanine aminotransferase (ALT), endotoxin (ET), tumor necrosis factor-alpha (TNF-alpha) and interleukine-1beta (IL-1beta) were determined. The expressions of CD14 and nuclear transcriptional factor kappaB (NF-kappaB) were observed by immunohistochemistry. The expressions of lipopolysaccharide binding protein (LBP), toll-like receptor-4 (TLR-4), myeloid differentiation-2 (MD-2) and induced nitric oxide synthase (iNOS) mRNA were detected by the reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The levels of serum endotoxin in the middle dose group (0.0225 +/- 0.0112 EU/l) were lower than those in high fatty diet model group (0.2249 +/- 0.0982 EU/l) at 12th week, the difference was significant (P < 0.01). In the middle dose group, mean values of serum TNF-alpha and IL-1beta levels decreased dramatically (1.604 +/- 0.302 ng/ml and 0.052 +/- 0.024 ng/ml) compared with those in the high fatty diet model group (4.029 +/- 1.180 ng/ml and 14.944 +/- 0.491 ng/ml; P < 0.01 and P < 0.01). The expressions of CD14 and NF-kappaB in the middle dose group decreased compared with those in the high fatty diet model group. The expressions of LBP mRNA (0.284 +/- 0.105) and TLR-4 mRNA (0.290 +/- 0.123) in the middle dose group down regulated compared with those in the high fatty diet model group (1.060 +/- 0.158 and 1.261 +/- 0.368; P < 0.01 and P < 0.01). In the middle dose group MD-2 and iNOS gene expressions were 0.132 +/- 0.058 and 0.164 +/- 0.061, respectively, which were significantly lower compared with the high fatty diet model group (0.795 +/- 0.294 and 1.029 +/- 0.388; P < 0.01 and P < 0.01). CONCLUSIONS: The mechanism of non-alcoholic steatohepatitis (NASH) maybe related to increasing the levels of serum endotoxin, upregulating endotoxin receptors of hepatic tissue and enhancing liver inflammatory injury. Traditional Chinese medicine is a good treatment for non-alcoholic steatohepatitis (NASH). It can produce a marked effect via relieving LPS-induced liver injury.

Effect of Chinese traditional compound, Gan-fu-kang, on CCl(4)-induced liver fibrosis in rats and its probable molecular mechanisms.

AIM: To explore the antifibrotic effect of traditional Chinese medicine compound Gan-fu-kang (GFK) on CCl(4)-induced liver fibrosis in rats and its probable mechanisms. METHODS: The effects of GFK on CCl(4)-induced liver fibrosis were tested in rats. The liver histopathology was examined by light microscope, polaring microscope and electron microscope. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were assayed and the content of albumin (ALB) and hydroxyproline in the liver was measured. The expression of transforming growth factor-beta(1) (TGF-beta(1)) and laminin (LN) was determined by immunohistochemistry. Semi-quantitive computation of collagen types I and III and laminin was done. The expression of MMP-2 and TIMP-1 was assayed by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Upon pathological examination, GFK treatment had significantly reversed liver fibrosis. Hepatic extracellular matrix (ECM) deposition was significantly reduced, as evidenced by the reduction of the content of hydroxyproline, collagen types I and III, and laminin. Hepatic function was improved by GFK treatment, as evidenced by the increase of plasma ALB and A/G, and by the decrease of serum ALT and AST. TGF-beta(1) in liver was significantly reduced. A significant expression of MMP-2 and TIMP-1 mRNA in liver were downregulated after GFK treatment. CONCLUSION: The traditional Chinese medicine compound recipe GFK has an antifibrotic effect on CCl(4)-induced liver fibrosis in rats, which improves hepatic function and lessens the deposition of collagen in the liver. The probable antifibrotic mechanisms were: inhibiting the expression of TGF-beta(1) and decreasing expressions of MMP-2 and TIMP-1.

[Inhibitory effects of salviae miltiorrhizae and beta-aescinom natrium on the postburn acute lung injury in rats].

OBJECTIVE: To explore the inhibitory effect and its mechanism of salviae miltiorrhizae and beta-aescinom natrium on the postburn acute lung injury in rats. METHODS: Forty-five rats were randomly divided into sham control (C, n = 9), sodium chloride group (S, n = 9), salviae miltiorrhizae group (M, n = 9), beta-aescinom natrium group (A, n = 9), and combination group (MA, n = 9). The rats in M, A and MA groups were subjected to 30% TBSA III degree scald on the back, and all the rats were sacrificed at 24 PBH. The blood and pulmonary tissue samples were harvested from the rats at 24 PBH for the determination of leukocyte adhesiveness/aggregation (LAA) in peripheral blood, myeloperoxidase (MPO), malondialdehyde (MDA) and superoxide dismutase (SOD) contents, and the ratio of wet to dry weights (W/D) of lung tissue. RESULTS: Compared with those in S group, the LAA in blood and the pulmonary tissue contents of MPO, MDA and W/D rate in M and A groups, and especially in MA group, were decreased significantly, but the SOD content in pulmonary tissue increased obviously in M and A groups, especially in MA group. Furthermore, blood LAA was positively correlated with pulmonary tissue MDA content. CONCLUSION: Postburn intra-pulmonary agglutination and aggregation of PMNs and pulmonary injury by oxygen free radicals (OFRs) and their products could be inhibited by either Salviae Miltiorrhizae or beta-aescinom natrium. In addition, these agents could also increase the tissue content of antioxidant capacity and decrease pulmonary microvascular permeability and lung water content. The results indicated that all the agents used might be effective in prevention and treatment of postburn pulmonary injury, especially when used together.