XingNaoJing injection ameliorates cerebral ischaemia/reperfusion injury via SIRT1-mediated inflammatory response inhibition.

Context: XingNaoJing injection (XNJ), extracted from a traditional compound Chinese medicine Angong niuhuang pill, is well known for treating stroke in the clinic, but the specific effects and mechanisms remain unclear.Objective: We investigated the mechanistic basis for the protective effect of XNJ on cerebral ischaemia/reperfusion (I/R) injury.Materials and methods: Five groups of 10 SD rats underwent 2 h of middle cerebral artery occlusion (MCAO) followed by 24 h reperfusion. XNJ at 10 and 15 mL/kg was intraperitoneally administered 24 h before ischaemia and at the onset of reperfusion respectively. The silent information regulator 1 (SIRT1) inhibitor EX527 was intracerebroventricularly injected 0.5 h before reperfusion. Cerebral infarction size, neurological scores, morphological changes, and expression levels of inflammatory mediators and SIRT1 were measured. Furthermore, human brain microvascular endothelial cells (HBMECs) were subjected to 3 h oxygen and glucose deprivation (OGD) followed by 24 h reoxygenation to mimic cerebral I/R in vitro. EX527 pre-treatment occurred 1 h before OGD. SIRT1 and inflammatory mediator levels were analyzed.Results: Both XNJ doses significantly decreased cerebral infarct area (40.11% vs. 19.66% and 9.87%) and improved neurological scores and morphological changes. Inflammatory mediator levels were remarkably decreased in both model systems after XNJ treatment. XNJ also enhanced SIRT1 expression. Notably, the SIRT1 inhibitor EX527 attenuated the XNJ-mediated decrease in inflammation in vivo and in vitro.Conclusions: XNJ improved cerebral I/R injury through inhibiting the inflammatory response via the SIRT1 pathway, which may be a useful target in treating cerebral I/R injury.

XingNaoJing injections protect against cerebral ischemia/reperfusion injury and alleviate blood-brain barrier disruption in rats, through an underlying mechanism of NLRP3 inflammasomes suppression.

The aim of this study was to explore the neuroprotective effect and mechanism of XingNaoJing injections (XNJ) on cerebral ischemia injury and blood-brain barrier (BBB) disruption. Middle cerebral artery occlusion (MCAO) method was applicated to establish the model of cerebral ischemia/reperfusion (I/R) injury in rats. BBB permeability after I/R injury was assessed with the leaking amount of Evans Blue and the expression of occludin and ZO-1. The expression of NOD-like receptor family, pyrin domain containing (NLRP3) was checked to explore the inhibition of inflammation by XNJ. The results showed that XNJ could significantly increase the survival percent, decrease the infarct area and ameliorate neurological deficits and brain damage after I/R injury. Leaking amount of Evans Blue was reduced by XNJ, and the expression of tight junction protein, occludin and ZO-1 was also up-regulated by XNJ, which showed a role of protection on BBB disruption. The expression of NLRP3 was inhibited after exposure of XNJ, which was associated with inhibition of the inflammatory response. In summary, XNJ could suppress NLRP3 inflammasomes and improve BBB disruption and brain damage in rats after cerebral I/R injury, which provided a beneficial insight to further explore XNJ.

Xingnaojing Injection Protects against Cerebral Ischemia Reperfusion Injury via PI3K/Akt-Mediated eNOS Phosphorylation.

Xingnaojing (XNJ) injection, derived from traditional Chinese medicine formulation, has a protective effect against stroke, but the underlying mechanism is unclear, which severely limited its clinical application. This research aims to elucidate the role and mechanism of XNJ in reducing cerebral ischemic reperfusion (I/R) injury. Rats received 2 h cerebral ischemia followed by reperfusion of 24 h and were intraperitoneally given 5, 10, or 15 ml/kg XNJ 24 h before ischemia and at the onset of reperfusion, respectively. TTC staining, HE staining, and neurological score were implied to evaluate the effectiveness of XNJ. The protein expressions of PI3K/Akt and eNOS signaling were measured. Experiments were further performed in human brain microvascular endothelial cells (HBMECs) to investigate the protective mechanisms of XNJ. HBMECs were subjected to 3 h oxygen and glucose deprivation following 24 h of reoxygenation (OGD) to mimic cerebral I/R in vitro. PI3K inhibitor LY294002 was added with or without the preconditioning of XNJ. Multiple methods including western blot, immunofluorescence, DAPI staining, JC-1, and flow cytometry were carried out to evaluate the effect of XNJ on HBMECs. XNJ could improve rat cerebral ischemic injury and OGD induced HBMECs apoptosis. In vivo and in vitro researches indicated that the mechanism might be relevant to the activation of PI3K/Akt/eNOS signaling.

Triptolide induces autophagy and apoptosis through ERK activation in human breast cancer MCF-7 cells.

To investigate the effects of triptolide (TPI) on proliferation, autophagy and death in human breast cancer MCF-7 cells, and to elucidate the associated molecular mechanisms, intracellular alterations were analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry assays. The results of the MTT assay revealed that TPI significantly reduced the MCF-7 cell survival rate when the concentration was >10 nmol/l. TPI activated a caspase cascade reaction by regulating Bcl-2-associated X protein (Bax), caspase-3 and B-cell lymphoma 2 expression, and promoted programmed cell death via the mitochondrial pathway. The results demonstrated that TPI significantly reduced the cell proliferation rate and viability in a time- and dose-dependent manner, which was confirmed by western blotting and immunofluorescent staining. TPI induced autophagy and influenced p38 mitogen-activated protein kinases, extracellular signal-regulated kinase (Erk)1/2, and mammalian target of rapamycin (mTOR) phosphorylation, which resulted in apoptosis. When cells were treated with a combination of TPI and the Erk1/2 inhibitor U0126, the downregulation of P62 and upregulation of Bax were inhibited, which demonstrated that the inhibition of Erk1/2 reversed the autophagy changes induced by TPI. The results indicated that Erk1/2 activation may be a novel mechanism by which TPI induces autophagy and apoptosis in MCF-7 breast cancer cells. In conclusion, TPI affects the proliferation and apoptosis of MCF-7 cells, potentially via autophagy and p38/Erk/mTOR phosphorylation. The present study offers a novel view of the mechanisms by which TPI regulates cell death.

Antioxidation of Cerium Oxide Nanoparticles to Several Series of Oxidative Damage Related to Type II Diabetes Mellitus In Vitro.

BACKGROUND It is well known that cerium oxide nanoparticles (CeNPs) have intense antioxidant activity. The antioxidant property of CeNPs are widely used in different areas of research, but little is known about the oxidative damage of Cu2+ associated with Type II diabetes mellitus (T2DM). MATERIAL AND METHODS In our research, the function of CeNPs was tested for its protection of ß-cells from the damage of Cu2+ or H2O2. We detected hydroxyl radicals using terephthalic acid assay, hydrogen peroxide using Amplex Ultra Red assay, and cell viability using MTT reduction. RESULTS We found that CeNPs can persistently inhibit Cu2+/H2O2 evoked hydroxyl radicals and hydrogen peroxide in oxidative stress of ß-cells. CONCLUSIONS CeNPs will be useful in developing strategies for the prevention of T2DM.

[Effect of preventive moxibustion on heat shock proteins and stress hormones in natural menopausal rats].

OBJECTIVE: To investigate the effect of preventive moxibustion at the acupoint of 'Guanyuan" (CV 4) on the stress-related hormones, proteins and genes in natural menopausal rats, and explore its protective mechanism. METHODS: One hundred and twenty eight healthy female SD rats were used in this study, in which 16 rats aged 4 months were used as normal control group, the rest 10-month-old rats with disorder of estrus cycle were randomly divided into 7 groups, including 4 control groups at the age of 10, 12, 14 and 16 months, and 3 preventive moxibustion groups at the age of 12, 14 and 16 months, 16 cases in each group. The 10-month-old rats were treated with preventive moxibustion at the acupoint of "Guanyuan" (CV 4), twice a week for 8 weeks consistently, then observed on the following 12, 14 and 16 months. Its effects on the level of heat shock protein 70 (HSP 70), heat shock protein 70 mRNA (HSP 70 mRNA), corticotropin-releasing hormone (CRH), beta-endorphin (beta-EP), adrenocorticotrophic hormone (ACTH) in the different nuclei of hypothalamus were examined with radio-immunity, immunohistochemistry or in situ hybridization methods. RESULTS: In the control groups, the expression of HSP 70 and HSP 70 mRNA was increased in paraventricular, arcuate and supraoptic nucleus, the level of CRH, beta-EP was showed with declining trend in volatility, while the content of ACTH was increased. In comparison to the same-aged control groups, the preventive moxibustion groups were showed that the expression of HSP 70 in the 14-month-old group was lower and the expression of HSP 70 mRNA in the 12 and 16-month-old group was significantly higher in the paraventricular nucleus (all P < 0.05), the expression of HSP 70 in the 12 and 14-month-old groups and HSP 70 mRNA in the 12-month-old group was significantly increased in the arcuate nucleus (P < 0.05, P < 0.01), while the expression of HSP 70 in the 14-month-old group and HSP 70 mRNA in the 12 and 16-month-old group was significantly decreased in the supraoptic nucleus (all P < 0.05), the content of CRH and ACTH decreased significantly in 14-month group (P < 0.05, P < 0.01), but the content of beta-EP obviously went up in the 12-month-old group (P < 0.05). CONCLUSION: Preventive moxibustion at the acupoint of "Guanyuan" (CV 4) can regulate the expression of stress proteins, genes and hormones in the hypothalamus for the natural menopausal rats, which might be playing a protective role.

[Observation on the protective effect of preventive acupuncture and moxibustion on uterus in ovariectomized rats].

OBJECTIVE: To observe the effect of preventive acupuncture and moxibustion of "Guanyuan" (CV 4) on uterus in ovariectomized (OVX) rats. METHODS: A total of 80 female SD rats were randomly divided into 5 groups: normal control, sham-operation (sham), model, preventive acupuncture (PA) and preventive moxibustion (PM) groups, with 16 cases in each. PA (with the needle retained for 20 min after insertion) and PM (one moxa-cone/rat) were applied to "Guanyuan" (CV 4) separately before ovariectomy, two times a week, 4 weeks altogether. Then, ovariectomy was performed on rats of model, PA and PM groups. Uterus tissue was taken under anesthesia for homogenate (10 rats/group) or sectioning (5-6 microm, 6 rats/group). Uterus estradiol (E2), progestone (P) were detected with radioimmunoassay; superoxide dismutase (SOD), nitric oxide synthase (NOS), and malondialdehyde (MDA) were assayed with immunoturdidimetry. Partial uterus slices were stained with H & E method for observing morphological changes, or stained with immunohistochemical method or with in situ hybridization method for displaying estrogen receptor (ER)-alpha and ER-alpha mRNA expression separately. RESULTS: After ovariectomy, the rat's uterus presented obvious squamous metaplasia, hyperplasy, and thickening of the endomembrane, decrease in glands and blood vessels and increase in fibrous connective tissue, etc; while the situation was evidently better in PA and PM groups. In comparison with normal control group, E2, P contents and SOD, NOS activities of uterus tissue in model group decreased significantly (P < 0.01), while MDA in model group increased evidently (P < 0.01). Compared with model group, uterus P content in PA and PM groups increased obviously (P < 0.05), while uterus MDA decreased apparently (P < 0.01). Compared with normal control group, the expression of ER-alpha and ER-alpha mRNA in model group was downregulated markedly (P < 0.01); after PA and PM, both ER-alpha and ER-alpha mRNA expression increased obviously (P < 0.01). No significant differences were found between PA and PM groups in the above mentioned 7 indexes (P > 0.05). CONCLUSION: Preventive acupuncture and moxibustion of CV4 can postpone the structural degeneration of uterus in OVX rats, which may be related to their effects in modulating the secretion of uterus E2 and P, upregulating the expression of ER-a and ER-alpha mRNA, and improving the anti-oxidative ability.

[Experimental study on changes of expressions of ER-alpha and ER-alpha mRNA in different hypothalamic nuclei and the effect of "preventive acupuncture and moxibustion" in ovariectomy rats].

OBJECTIVE: To explore the mechanism of "preventive acupuncture and moxibustion" for regulating hypothalamic function in ovariectomy rats. METHODS: Thirty female SD rats, aged 3.5 months, were randomly divided into normal group, sham operation group, ovariectomy model group, preventive acupuncture group and preventive moxibustion group. After "preventive acupuncture and moxibustion" were previously given to the preventive acupuncture group and the preventive moxibustion group for one month, respectively. At the same time, ovarietomy was made in the preventive acupuncture group, the preventive moxibustion group and the ovariectomy model group. For the sham operation group, only a little fat around the ovary was removed without ovariectomy, and nothing was not applied to the normal group. Immunohistochemical and in situs hybridization methods were used to investigate changes of expressions of ER-alpha and ER-alpha mRNA in paraventricular nucleus, supraoptic nucleus and arcuate nucleus. RESULTS: In the model group the expressions of ER-alpha and ER-alpha mRNA in paraventricular nucleus and the expression of ER-alpha mRNA in supraoptic nucleus increased significantly (P<0.01), but the expression of ER-alpha dropped significantly in both supraoptic nucleus and arcuate nucleus (P<0.01). After preconditioning with acupuncture or moxibustion, the expression of ER-alpha mRNA in paraventricular nucleus significantly reduced (P<0.05), and the expression of ER-alpha had no significant change (P>0.05); the expression of ER-alpha in supraoptic nucleus and arcuate nucleus significantly increased (P<0.01 or P<0.05), and the expression of ER-alpha mRNA in paraventricular nucleus had no significant change (P>0.05). CONCLUSION: "Preventive acupuncture and moxibustion" at "Guanyuan" (CV 4) have an effect on the expressions of ER-alpha and ER-alpha mRNA in different hypothalamic nuclei in ovariectomy rats, which possibly is one of the ways for modulating the hypothalamic activity.

[Effects of electroacupuncture on CRH, IL-2 and beta-EP contents in the hypothalamus in the rat of adjuvant arthritis].

OBJECTIVE: To explore Mechanisms of electroacupuncture in anti-inflammation and immunoregulation, and to compare differences among "Dazhui" (GV 14), "Mingmen "(GV 4) and non-acupoint in these actions. METHODS: Adjuvant arthritis rat model was made. The rats were divided into 5 groups: normal group, model group, "Dazhui" (GV 14) electroacupuncture group, "Mingmen" (GV 4) electroacupuncture group and non-acupoint electroacupuncture group. Effects of electroacupuncture on local joint inflammation and corticotropin-releasing hormone (CRH), beta-endorphin (betaEP) and interleukin-2 (IL-2) contents in the hypothalamus were observed and differences of the actions among different points were compared. RESULTS: CRH content in the hypothalamus in the "Dazhui" (GV 14) electroacupuncture group decreased as compared with the model group (P < 0.05); there was no significant change in beta-EP and IL-2 contents of the hypothalamus in the 3 electroacupuncture groups as compared with the model group (P > 0.05); there was positive correlation between CRH and IL-2 contents, and between IL-2 and beta-EP contents in the hypothalamus. The swelling rate of the toe in the "Dazhui" (GV 14) group and the "Mingmen" (GV 4) group was lower than that in the non-acupoint group (P < 0 05). CONCLUSION: Electroacupuncture exerts anti-inflammation and immunoregulation possibly via regulation of CRH, IL-2 and beta-EP each other, and the anti-inflammation action of "Dazhui" (GV 14) and "Mingmen" (GV 4) is stronger than that of non-acupoint.