Hydroxysafflor yellow A attenuates lymphostatic encephalopathy-induced brain injury in rats.

Hydroxysafflor yellow A (HSYA) is a main chemical component of the flower of Carthamus tinctorius. The present study investigated whether HSYA could attenuate brain injury induced by lymphostatic encephalopathy (LE). This was induced in adult male Wistar rats by cervical lymphatic blockade (CLB). Heart rate variability (HRV) was used as an indirect measurement of the regulatory function of the autonomic nervous system by recording the ECG signals from rats. It was shown that treatment with HSYA (5 mg/kg, i.p.) significantly alleviated the neurological deficits observed in rats with LE. Histological staining revealed that HSYA treatment attenuated LE-induced cell apoptosis in the rostral ventrolateral medullus (RVLM). Animals in the LE groups exhibited impaired regulatory roles of the autonomic nervous system in cardiovascular function, which was suppressed by pretreatment with HSYA. Additionally, HSYA administration significantly prevented the decrease of endothelial nitric oxide synthase (eNOS) mRNA and protein expression in the RVLM of rats with LE. These findings suggest that HSYA might provide neuroprotection against LE-induced brain injury and the associated functional alterations, which is likely regulated by the nitric oxide pathway.

HIF-1-α and survivin involved in the anti-apoptotic effect of 2ME2 after global ischemia in rats.

Survivin is an anti-apoptotic gene that decreases the apoptosis by depressing the expression of caspase-3. Hypoxia-inducible factor-1-alpha (HIF-1-alpha) is a transcription factor specifically activated by hypoxia. 2-methoxyestradiol (2ME2) is an estradiol derivative and a known HIF-1-alpha inhibitor. 2ME2 decreased apoptosis by inhibiting HIF-1-alpha. The aim of the present study was to investigate if survivin is involved in the anti-apoptotic effect of 2ME2. Male adult rats were used to make the global ischemia (GI) model. Ten minutes after GI, 2ME2 was injected intraperitoneally (16 mg/kg weight). Rats were killed at 6 hours, 12 hours, 24 hours, 48 hours, 96 hours, and 7 days. GI produced a marked increase in HIF-1-alpha expressions in the hippocampus at 6 hours and peaked at 48-96 hours. The expressions of survivin and caspase-3 were increased lightly in a similar time course. These molecular changes were accompanied by massive cell loss and apoptosis in the hippocampal regions. 2ME2 treatment reduced the expression of HIF-1-alpha, increased survivin expression, and decreased the expression of caspase-3. These results indicate that survivin and HIF-1-alpha were involved in the anti-apoptotic effect of 2ME2 treated following GI. 2ME2 may decrease the HIF-1-alpha expression, up-regulate the survivin expression, inhibit the expression of caspase-3, and finally reduce apoptosis after GI.

Variations in blood pressure and heart rate in conscious rats with cervical lymphatic blockade.

The possible effects of cervical lymphatic blockade (CLB) on a series of parameters in conscious freely moving rats were analysed. Blood pressure (BP) and heart rate (HR) for conscious male Sprague-Dawley rats at 1, 3, 7, 11, 15 and 21 days after a CLB or a sham operation were monitored continuously for 24 hours with a computerized recording system. Since BP and HR were subjected to spontaneous variations, blood pressure variability (BPV) and heart rate variability (HRV) were expressed as the standard deviation of beat-to-beat BP and HR values. The baroreflex sensitivities (BRS) were determined by measuring the heart period (HP = 60,000/HR) prolongation in response to the elevation in BP induced by an intravenous administration of phenylephrine at 1, 7, 15 and 21 days after the CLB or sham operation. Compared with those in sham-operated rats, the values of systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial blood pressure (MAP), HR and BRS in CLB rats were significantly lower, whereas the values of BPV and HRV were markedly raised in CLB rats at different time points. Furthermore, the impaired ultrastructure in the dorsomedial nucleus of the solitary tract (dmNTS) including degeneration, apoptosis and necrosis in neurons and gliacytes, were apparent from the 1st to 15th day but the changes were most significant at 7th day after CLB operation. Structural changes appeared to be closely related to functional changes of the dmNTS at each time point. Thus, in CLB conscious rats, a significant decline in blood pressure accompanied by dysfunction in its regulation might be due to the impaired structure in the dmNTS.

Inhibition of beta-amyloid precursor protein gene in SK-N-SH cells by piperlonguminine/dihydropiperlonguminine components separated from Chinese herbal medicine Futokadsura stem.

Alzheimer disease (AD) is a progressive neurodegenerative disease characterized by progressive cognitive and memory decline. Amyloid precursor protein (APP) is a transmembrane protein, it has been known to play an important role in AD pathogenesis. Previous studies have shown that a Chinese herb Futokadsura stem can selectively inhibit the expression of amyloid precursor protein (APP) gene. We want to find the effective components in Futokadsura stem which have the inhibitory effect. Futokadsura stem was separated and purified with chemical methods, and then different separation components were added on SK-N-SH cells in different concentrations. Using MTT methods, we detected proliferation activity of SK-N-SH cells which were treated with different separation components of Futokadsura stem. Using RT-PCR, Western blot methods, we detected APP gene expression in SK-N-SH cells after they are treated with different Futokadsura stem separation components. We found that piperlonguminine/dihydropiperlonguminine components (1:0.8) separated from Futokadsura stem acetic ether extracts could selectively inhibit the expression of APP gene in SK-N-SH cells in mRNA and protein levels. This inhibition effect is concentration-dependent. Under experimental concentrations, the components did not affect the proliferation activity of SK-N-SH cells. These data suggest that piperlonguminine/dihydropiperlonguminine components are the effective components in Futokadsura stem which can inhibit the expression of APP gene.

Ischemic preconditioning relieves ischemia/reperfusion injury of hippocampus neurons in rat by inhibiting p53 and bax expressions.

OBJECTIVE: To examine whether ischemic preconditioning (IPC) can protect neuron against delayed death in CA1 subfield of hippocampus following reperfusion of a lethal ischemia in rats, and explore the role of p53 and bax in this process. METHODS: We examined the effect of IPC on delayed neuron death, neuron apoptosis, expressions of p53 and bax gene in the CA1 area of hippocampus in the rats using HE staining, flow cytometry, RT-PCR, and immunohistochemistry technique. RESULTS: IPC enhanced the quantity of survival cells in the CA1 region of hippocampus (216 +/- 9 cells/0.72 mm2 vs. 30 +/- 5 cells/0.72 mm2, P < 0.01) , decreased the percentages of apoptotic neurons of hippocampus caused by ischemia/reperfusion (2.06% +/- 0.21% vs. 4.27% +/- 0.08%, P < 0.01 ), and weakened the expressions of p53 and bax gene of hippocampus compared with ischemia/reperfusion without IPC. CONCLUSION: IPC can protect the neurons in the CA1 region of hippocampus against apoptosis caused by ischemia/reperfusion, and this process may be related to the reduced expressions of p53 and bax.

Antihypertensive effect of total flavonoid fraction of Astragalus complanatus in hypertensive rats.

The purpose of the present study was to quantify the antihypertensive effect of the total flavonoid (TF), extracted from the seed of Astragalus complanatus R. Brown, and to observe its effect on the renin-angiotensin system (RAS) in both renal hypertensive rats (RHR) and spontaneously hypertensive rats (SHR). RHR were created by the two-kidney one clip (2K1C) method. Systolic blood pressure was measured in conscious rats by the tail-cuff method. Plasma angiotensin II (AngII) and plasma renin activity (PRA) were measured with radioimmunoassay at 60 min after drug administration. The effects of TF on cardiac hemodynamics were also recorded in anesthetized RHR and SHR. TF was given by oral administration in low dose (100 mg/kg) and high dose (200 mg/kg) respectively. Compared to pre-administration control, TF induced an obvious decrease in systolic blood pressure in conscious normotensive Wistar rat, RHR and SHR. In the three groups the systolic blood pressure reached the lowest value at 60 min after TF. TF also induced a significant decrease in blood pressure in anesthetized RHR and SHR. At 60 min after treatment of TF, mean arterial pressure in high dose group (200 mg/kg) was decreased by 17% in RHR and by 17% in SHR respectively (P < 0.01). The depressor effect of TF lasted for at least 60 min. Cardiac output, heart rate and +/- dp/dtmax did not change. Conversely, total peripheral resistance was significantly decreased. The decrease in plasma AngII was found in both RHR and SHR. On the contrary, PRA increased at the same time. These findings suggested that TF is effective in reducing blood pressure in both RHR and SHR. The antihypertensive action of TF was attributed to a decrease in TPR secondary to a decrease in plasma concentration of AngII caused by TF.

Drainage of macromolecules from the Caudato-Putamen of rat brain.

To study the drainage of interstitial fluid and macromolecules from the brain parenchyma, an improved method was developed to inject tracers including Chinese ink in group I and phycoerythrin (PE) in group II into the right caudato-putamen of rat brain. Rats were sacrificed on the 1st, 3rd, 7th, 14th, 21st day after injection in group I and at the 0.5, 1, 2, 5, 24 hour in group II. Distribution of tracers was observed by electron microscopy and fluorescence confocal microscopy. The results showed that tracers distributed diffusely in the white matter at all time points whereas they spread selectively along perivascular spaces in the gray matter by 7 days (d) in group I and 5 hours (h) in group II. Chinese ink was ingested by perivascular phagocytes by 7 d after ink injection. The endothelial cells of capillaries in the gray matter had fluorescence staining in cytoplasm and no staining in nuclei by 24 h after PE injection. Animals in group II were stained with tracers in lateral ventricles, bilateral cervical lymph nodes, and the wall of carotid arteries. These results demonstrated that [1] the macromolecules could be cleared from the caudato-putamen through extracellular space of the neuropil in the white matter and perivascular space in the gray matter, [2] perivascular phagocytes and endothelial cells of capillaries played important roles in clearing macromolecules from the perivascular space, and [3] cervical lymph nodes were involved in draining macromolecules from the brain parenchyma.

[The changes of bcl-2, bax expression and neuron apoptosis in the hippocampus after the blockade of cervical lymphatics of rats].

To investigate the changes in bcl-2, bax expression and neuron apoptosis in the hippocampus after the blockade of cervical lymphatics, the model of lymphostatic encephalopathy was established by occluding and removing both the superficial and deep cervical lymph nodes in rats. The animals were sacrificed at 1, 2, 3, 5, 7 and 14 d after operation. H and E staining was used to observe the structure of brain tissues and TUNEL staining was used to detect in situ cell apoptosis in the hippocampus. The expression of bcl-2 and bax in the hippocampus were examined by RT-PCR. The results showed that cerebroedema appeared at day 2 and was most serious at day 5 after the blockade of cervical lymphatics. The number of TUNEL positive cells began to increase at day 2 and reached the maximum at day 5. The expression of bax began to increase at day 1 and reached the maximum at day 2. The expression of bcl-2 began to decrease at day 1 and dropped to the minimum at day 5. The items mentioned above recovered to control level at day 14. These results suggest that lymphostatic encephalopathy following the blockade of cervical lymphatics result in changes in bcl-2 and bax expression in the hippocampus and that apoptosis is the main form of neuron death.

Arecoline excites the colonic smooth muscle motility via M3 receptor in rabbits.

Arecoline is an effective component of areca (betel nuts, a Chinese medicine named pinang or binglang). The purpose of this study was to investigate the effect of arecoline on the motility of distal colon in rabbits and its mechanisms involved. Strips of colonic smooth muscle were suspended in organ baths containing Krebs solution, and their isometric contractions were examined. The response of smooth muscle to arecoline in colonic strips was recorded. The effects of atropine, gallamine and 1,1-dimethyl-4-diphenylacetoxypiperidiniumiodide (4-DAMP) on arecoline-induced contraction were also observed. Arecoline (1 nM - 1 microM) produced a concentration-dependent contraction in both the longitudinal and the circular smooth muscle of rabbit colon. Atropine (10 microM) abolished the arecoline (80 nM)--induced contraction. M3 receptor antagonist, 4 - DAMP (0.4 microM), abolished the arecoline (80 nM)--related response, whereas M2 receptor antagonist, gallamine (0.4 microM), did not affect the effect of arecoline. These results suggest that arecoline excites the colonic motility via M3 receptor in rabbits.

Effect of oxytocin on contraction of rabbit proximal colon in vitro.

AIM: To investigate the effects of oxytocin (OT) on isolated rabbit proximal colon and its mechanism. METHODS: Both longitudinal muscle (LM) and circular muscle (CM) were suspended in a tissue chamber containing 5 mL Krebs solution (37 degrees ), bubbled continuously with 950 mL x L(-1) O(2) and 50 mL x L(-1) CO(2). Isometric spontaneous contractile responses to oxytocin or other drugs were recorded in circular and longitudinal muscle strips. RESULTS: OT (0.1 U x L(-1)) failed to elicit significant effects on the contractile activity of proximal colonic smooth muscle strips (P>0.05). OT (1 to 10 U x L(-1)) decreased the mean contractile amplitude and the contractile frequency of CM and LM. Hexamethonium (10 micromol x L(-1)) partly blocked the inhibition of oxytocin (1 U x L(-1)) on the contractile frenquency of CM. N(omega))-nitro-L-arginine-methylester (L-NAME, 1 micromol x L (-1)), progesterone (32 micromol x L(-1)) and estrogen (2.6 micromol x L(-1)) had no effects on OT-induced responses. CONCLUSION: OT inhibits the motility of proximal colon in rabbits. The action is partly relevant with N receptor, but irrelevant with that of NO, progesterone or estrogen.

Effects of progesterone on gastric emptying and intestinal transit in male rats.

AIM: To study the dose-dependent of progesterone (P) effect and the interaction between the oxytocin (OT) and P on gastrointestinal motility. METHODS: In order to monitor the gastric emptying and intestinal transit, the SD male rats were intubated via a catheter with normal saline (3 ml/kg) containing Na(2)(51)CrO(4) (0.5 microCi/ml) and 10% charcoal. OT was dissolved into normal saline and P was dissolved into 75% alcohol. RESULTS: Low does of P (1 mg/kg, i.p.) enhanced the gastric emptying (75+/-3%, P<0.05) and high dose of P (5 mg/kg, i.p.) inhibit it (42+/-11.2%, P<0.01). P (1 mg/kg) increased the intestinal transit (4.2+/-0.3, P<0.05) while the higher dose (10-20 mg/kg) had no effect. OT (0.8 mg/kg, i.p.) inhibited the gastric emptying (23.5+/-9.8%, P<0.01). The inhibitory effects of P(20 mg/kg) (32+/-9.7%, P<0.05) and OT (0.8 mg/kg) on gastric emptying enhanced each other when the two chemicals were administrated simultaneously (17+/-9.4%, P<0.01). CONCLUSION: Low dose of P increased GI motility while high dose of P decreased it. During the later period of pregnancy, elevated plasma level of OT may also participate in the gastrointestinal inhibition.

Effect of areca on contraction of colonic muscle strips in rats.

AIM: To investigate the effects of areca on the contractile activity of isolated colonic muscle strips in rats and mechanism involved. METHODS: Each strip (LMPC, longitudinal muscle of proximal colon; CMPC, circular muscle of proximal colon; LMDC, longitudinal muscle of distal colon; CMDC, circular muscle of distal colon.) was suspended in a tissue chamber containing 5 mL Krebs solution (37 degrees C), bubbled continuously with 950 mL.L(-1) O(2) and 50 mL.L(-1) CO(2). The mean contractile amplitude (A), the resting tension (T), and the contractile frequency (F) were simultaneously recorded on recorders. RESULTS: Areca dose dependently increased the mean contractile amplitude, the resting tension of proximal and distal colonic smooth muscle strips in rats (P<0.05). It also partly increased the contractile frequency of colonic smooth muscle strips in rats (P<0.05). The effects were partly inhibited by atropine (the resting tension of LMPC decreased from 0.44 +/- 0.12 to 0.17 +/- 0.03; the resting tension of LMDC decreased from 0.71 +/- 0.14 to 0.03 +/- 0.01; the mean contractile amplitude of LMPC increased from -45.8 +/- 7.2 to -30.5 +/- 2.9; the motility index of CMDC decreased from 86.6 +/- 17.3 to 32.8 +/- 9.3; P<0.05 vs areca), but the effects were not inhibited by hexamethonium (P>0.05). CONCLUSION: Areca stimulated the motility of isolated colonic smooth muscle strips in rats. The stimulation of areca might be relevant with M receptor partly.

[Depressive effect of total flavonoid fraction of Asttragalus complanatus R. Br and its influence upon hemodynamics in SHR].

OBJECTIVE: To investigate effects of total flavonoid fraction of Asttragalus complanatus on blood pressure in conscious spontaneously hypertensive rats(SHR) and hemodynamics in anesthetized SHR. METHOD: Blood pressure was recorded with tail-culf method and hemodynamics was recorded with polygraph after administration of total flavonoid fraction of Asttragalus complanatus. RESULT: Total flavonoid fraction of Asttragalus complanatus (100, 200 mg.kg-1) could decrease blood pressure of conscious SHR(decreasing 7.1%, P < 0.05 and 9.3%, P < 0.01 respectively). Meanwhile, there was no significant change in heart rate(HR). R Br (200 mg.kg-1) could decrease total peripheral resistance (TPR) (decreasing 20%, P < 0.05) but did not influence the cardiac output(CO) and heart rate in anesthetized SHR. CONCLUSION: Total flavonoid fraction of Asttragalus complanatus possesses obvious hypotensive effect, mainly by means of decreasing the TPR.