Neuroprotective effect of Shenqi Fuzheng injection pretreatment in aged rats with cerebral ischemia/reperfusion injury.

Shenqi Fuzheng injection is extracted from the Chinese herbs Radix Astragali and Radix Codonopsis. The aim of the present study was to investigate the neuroprotective effects of Shenqi Fuzheng injection in cerebral ischemia and reperfusion. Aged rats (20-22 months) were divided into three groups: sham, model, and treatment. Shenqi Fuzheng injection or saline (40 mL/kg) was injected into the tail vein daily for 1 week, after which a cerebral ischemia/reperfusion injury model was established. Compared with model rats that received saline, rats in the treatment group had smaller infarct volumes, lower brain water and malondialdehyde content, lower brain Ca(2+) levels, lower activities of serum lactate dehydrogenase and creatine kinase, and higher superoxide dismutase activity. In addition, the treatment group showed less damage to the brain tissue ultrastructure and better neurological function. Our findings indicate that Shenqi Fuzheng injection exerts neuroprotective effects in aged rats with cerebral ischemia/reperfusion injury, and that the underlying mechanism relies on oxygen free radical scavenging and inhibition of brain Ca(2+) accumulation.

Inhibition of the K+ channel K(Ca)3.1 reduces TGF-ß1-induced premature senescence, myofibroblast phenotype transition and proliferation of mesangial cells.

OBJECTIVE: K(Ca)3.1 channel participates in many important cellular functions. This study planned to investigate the potential involvement of K(Ca)3.1 channel in premature senescence, myofibroblast phenotype transition and proliferation of mesangial cells. METHODS & MATERIALS: Rat mesangial cells were cultured together with TGF-ß1 (2 ng/ml) and TGF-ß1 (2 ng/ml) + TRAM-34 (16 nM) separately for specified times from 0 min to 60 min. The cells without treatment served as controls. The location of K(Ca)3.1 channels in mesangial cells was determined with Confocal laser microscope, the cell cycle of mesangial cells was assessed with flow cytometry, the protein and mRNA expression of K(Ca)3.1, α-smooth muscle actin (α-SMA) and fibroblast-specific protein-1 (FSP-1) were detected with Western blot and RT-PCR. One-way analysis of variance (ANOVA) and Student-Newman-Keuls-q test (SNK-q) were used to do statistical analysis. Statistical significance was considered at P<0.05. RESULTS: Kca3.1 channels were located in the cell membranes and/or in the cytoplasm of mesangial cells. The percentage of cells in G0-G1 phase and the expression of K(ca)3.1, α-SMA and FSP-1 were elevated under the induction of TGF-ß1 when compared to the control and decreased under the induction of TGF-ß1+TRAM-34 when compared to the TGF-ß1 induced (P<0.05 or P<0.01). CONCLUSION: Targeted disruption of K(Ca)3.1 inhibits TGF-ß1-induced premature aging, myofibroblast-like phenotype transdifferentiation and proliferation of mesangial cells.

Premature senescence and cellular phenotype transformation of mesangial cells induced by TGF-B1.

BACKGROUND: Transforming growth factor-ß1 (TGF-ß1) is a polypeptide member of the transforming growth factor ß superfamily of cytokines and performs many cellular functions. Its overexpression may lead to renal fibrosis. AIM: This study planed to investigate the effects of TGF-ß1 on the cell cycle and phenotype of mesangial cells. METHODS: Rat mesangial cells were cultured together with different concentrations (0, 1, 2, 5, and 10 ng/mL) of TGF-ß1 for specified times from 0 min to 72 h. 0 ng/mL TGF-ß1 and 0 min served as controls. Cell cycles were assessed by flow cytometry and α-smooth muscle actin expression (α-SMA) protein expression by western blot analysis. All data were presented as Mean ± SD. Statistical analysis was performed by using one-way analysis of variance and correlation analysis. Results were considered significant at p < 0.05. RESULTS: After 15 min of co-culture with different concentrations of TGF-ß1, the percentage of mesangial cells in G0/G1 phase was significantly elevated compared to the control (p < 0.05). 12 h co-culture induced cell hyperplasia, 24 h co-culture obvious up-regulation of α-SMA (p < 0.01) and one or two cells' myofibroblast phenotype transition, and 36 h co-culture several cells' phenotype transition. Correlation analysis prompted that the TGF-ß1-induced premature aging was time-dependent (p < 0.01). CONCLUSION: TGF-ß1 may induce mesangial cells' premature senescence and myofibroblast-like phenotype transformation time-dependently, which may contribute to the development of early stage of glomerulosclerosis.

Uremic anorexia and gastrointestinal motility dysfunction correlate with the changes of ghrelin system in hypothalamus.

AIM: Ghrelin can act as a signal for meal initiation and play a role in the regulation of gastrointestinal (GI) motility via hypothalamic circuit. This study investigated the correlation between changes of hypothalamic ghrelin system and GI motility dysfunction and anorexia in rats with chronic renal failure (CRF). METHODS: Sprague-Dawley (SD) rats (male/female 1:1, 180 ± 20 g) were randomly classified into a CRF group and control group (n = 8 per group). 5/6 nephrectomy was used to construct the CRF model. When plasma creatinine concentration (PCr) and blood urea nitrogen (BUN) in the CRF group were twice higher than the normal, food intake (g/24 h) and gastrointestinal interdigestive myoelectric complex (IMC) were detected. Then all rats were killed for assessment of the mRNA expression of ghrelin and growth hormone secretagogue receptor (GHS-R) in hypothalamus using reverse transcription-polymerase chain reaction. Analysis of variance, Student-Newman-Keuls-q-test and Correlation Analysis were used to do statistical analysis. P < 0.05 was considered as statistically significant. RESULTS: Compared to the control group, the CRF group was obviously decreased in the food intake (g/24 h), the phase III duration and amplitude and the ghrelin and GHS-R expression in the hypothalamus (P < 0.05). There was a positive correlation between them (P < 0.05). CONCLUSION: Changes of ghrelin and GHS-R in the hypothalamus correlate with gastrointestinal motility dysfunction and anorexia in rats with CRF.

Uremic anorexia and ghrelin expression in the amygdala.

BACKGROUND/AIMS: Ghrelin can act as a signal for mealtime hunger and meal initiation. Amygdala is indispensable in appetitive behavior motivated by learned emotions. This study was to investigate the alteration of ghrelin in the amygdala of rats with chronic renal failure (CRF) and its relation with uremic anorexia. METHODS: SD rats were randomly classified into CRF group and control group (n=16 per group). The CRF model was constructed using 5/6 nephrectomy. When plasma creatinine (PCr) and blood urea nitrogen (BUN) in the CRF group were twice more than the normal level, food intake (g/24h) was measured and then all rats were killed for detection of ghrelin protein expression in the amygdala using immunohistochemical analysis and mRNA expression using RT-PCT. Statistics was conducted with one-way analysis of variance, Student-Newman-Keuls-q test and correlation analysis. RESULTS: By the 8th week after the surgery, the BUN and PCr of CRF rats exceeded double the normal level, and their food intake was obviously decreased compared with the controls (P<0.05). The protein and mRNA expression of ghrelin in the amygdala of CRF group were significantly reduced, and there was a positive correlation between this reduction and the decrease in food intake (P<0.05). CONCLUSION: The reduction of amygdala's ghrelin in CRF rats may be associated with uremic anorexia.

Chronic renal failure impacts the expression of ghrelin and its receptor in hypothalamus and hippocampus.

BACKGROUND/AIMS: Ghrelin plays a central role in the regulation of gastrointestinal (GI) motility. This study aimed to investigate the expression of ghrelin and growth hormone secretagogue receptor (GHSR) in the central nervous system of rats with chronic renal failure (CRF). METHODS: Sprague-Dawley rats (male, 180 ± 20 g, n = 24) were treated by 5/6 nephrectomy to construct CRF model. As their plasma creatinine concentration and blood urea nitrogen were maintained more than double the normal level for 2 weeks, they were killed for assessing the expression of ghrelin and GHSR in hypothalamus and hippocampus using immunohistochemistry and real-time polymerase chain reaction (RT-PCR). The rats (male, 180 ± 20 g, n = 24) treated by Sham operation served as a control. One-way analysis of variance and Student-Newman-Keuls q test were used to analyze group difference and a p-value of <0.05 was considered as statistically significant. RESULTS: Compared with the controls, the ghrelin and GHSR expression was obviously increased in the hippocampus (p < 0.05) but decreased in the hypothalamus of rats with CRF (p < 0.05). CONCLUSIONS: CRF was found to impact the expression of ghrelin and GHSR in hypothalamus and hippocampus. This might be associated with the CRF-induced GI motility dysfunction.

[Fas and TNFR1 expressions after cerebral ischemia and reperfusion in rats: association with cell apoptosis and the effects of Bcl-2 overexpression].

OBJECTIVE: To investigate the effect of Bcl-2 overexpression on Fas and TNFR1-mediated apoptosis and its possible mechanism in rat hippocampus following global ischemia/reperfusion (IR). METHODS: Ninety healthy male SD rats were randomly divided into sham operated group, IR group and Bcl-2 overexpression group (BT group). Rat model of global IR was established by the 4-V0 method. The expressions of Bcl-2, Fas and TNFR1 and the cell apoptosis in the CA1 and CA3 regions were examined by HE staining, immunohistochemistry and TUNEL method. RESULTS: In IR group, the neurons in the CA1 region showed an obvious reduction in number with disordered arrangement and interstitial edema 48 h after global IR. Such changes were not obvious in BT group. Immunohistochemistry showed that Fas expression in the CA1 region reached the peak level at 6 h in IR group with a greater expression intensity than that in BT group (P<0.05). TNFR1 was expressed at a higher level in IR group than in BT group (P<0.05), reaching the peak level at 24 h. In the sham group, the expression of Fas and TNFR1 was not detected the in CA1 and CA3 regions. Global IR caused increased cell apoptosis in the CA1 and CA3 regions, starting at 6 h and reached peak at 24 to 48 h. The cell apoptosis was less obvious in BT group (P<0.05). CONCLUSION: Fas and TNFR1 are expressed in the CA1 and CA3 regions after global IR in rats, suggesting the involvement of death receptor in cerebral IR injury. Bcl-2 overexpression decreases the expression of Fas and TNFR1 and cell apoptosis after global IR, thus offering protective effect against cerebral IR injury.

Relationship between transmembrane signal transduction pathway and DNA repair and the mechanism after global cerebral ischemia-reperfusion in rats.

OBJECTIVE: To investigate the protein levels of phospho-ERK and phospho-APE/Ref-1 in hippocampal neurons after global cerebral ischemia reperfusion in rats, and observe the relationship between transmembrane signal transduction and repair of DNA damage. The role of ERK signal transduction pathway following global cerebral ischemia reperfusion in rats is further discussed. METHODS: Ninety healthy male SD rats were divided into 3 groups randomly: Sham group (S group), Ischemia reperfusion group (IR group) and Pd98059 pretreatment/ischemia reperfusion group (PD group). Global cerebral ischemia reperfusion model was established by four-vessel occlusion (4-VO) method, and reperfusion was performed 5 minutes following ischemia. Protein levels of phospho-ERK and phospho-APE/Ref-1 were detected using immunohistochemical method at 2 h, 6 h, 12 h, 24 h, 48 h and 72 h after reperfusion, and neuron apoptosis was observed by HE and TUNEL staining. RESULTS: In CA1 region of IR group, TUNEL positive cells began to appear at 6 h after IR, and reached the apex during 24 h to 48 h. However, TUNEL positive was most strongly exhibited in PD group. In IR group, phospho-ERK was obviously detected in CA3 region at 2 h after IR, and its level was gradually decreased from 6 h until totally absent at 48 h. Besides, phospho-ERK expression in PD group was weaker than that in IR group. For phospho-APE/Ref-1, its expression began to appear in CA1 region in IR group at 2 h after IR, with no obvious changes during 2 h to 12 h. Phospho-APE/Ref-1 expression began to decrease at 24 h and this decrease continued thereafter. Expression level of phospho-APE/Ref-1 in PD group was lower than that in IR group. Results showed the concurrence of decreased phospho-ERK expression level and increased neuron apoptosis after cerebral ischemia reperfusion, the former of which was consistent with the decrease of phospho-APE/Ref-1 expression. Also, the greater the inhibition of ERK phosphorylation was, the greater decrease of APE/Ref-1 expression occurred. CONCLUSION: Activation of ERK signal transduction pathway increased the expression of phospho-APE/Ref-1, and thus faciliated the repair of DNA damage. So, activation of ERK signal transduction pathway may protect neurons from apoptosis after cerebral ischemia reperfusion.