Does Serum Vitamin D Level Affect COVID-19 Infection and Its Severity?-A Case-Control Study.

BACKGROUND: As effective medication to treat COVID-19 is currently unavailable, preventive remedies may be particularly important. OBJECTIVE: To examine the relationship between serum 25-hydroxy vitamin D (25(OH)D) level and COVID-19 infection, its severity, and its clinical case characteristics. METHODS: This case-control study compared serum 25(OH)D levels and rates of vitamin D deficiency (VDD) between 80 healthy controls and 62 patients diagnosed with COVID-19 and admitted to Guangxi People's Hospital, China, 2/16/2020-3/16/2020. Cases were categorized into asymptomatic, mild/moderate, and severe/critical disease. Logistic regression analysis was conducted to examine the associations between 25(OH)D level, or VDD, and case status/severity of COVID-19 while controlling for demographics and comorbidities. A threshold level of vitamin D for conveying COVID-19 risk was estimated. RESULTS: Severe/critical COVID-19 cases were significantly older and had higher percentages of comorbidity (renal failure) compared to mild cases. The serum 25(OH)D concentration in COVID-19 patient was much lower than that in healthy control. And 25(OH)D level was the lowest in severe/critical cases, compared with mild cases. In further, significantly higher rates of VDD were found in COVID-19 cases (41.9%) compared to healthy controls (11.1%). And VDD was the greatest in severe/critical cases (80%), compared with mild cases (36%). These statistically significant associations remained even after controlling for demographics and comorbidities. A potential threshold of 25(OH)D (41.19 nmol/L) to protect against COVID-19 was identified. CONCLUSION: Elderly and people with comorbidities were susceptible to severe COVID-19 infection. VDD was a risk factor for COVID-19, especially for severe/critical cases. While further confirmation is needed, vitamin D supplementation may have prevention or treatment potential for COVID-19 disease.

BH3 mimetics derived from Bim-BH3 domain core region show PTP1B inhibitory activity.

A series of our previously described BH3 peptide mimetics derived from Bim-BH3 domain core region were found to exhibit weak to potent PTP1B binding affinity and inhibitory activities via target-based drug screening. Among these compounds, a 12-aa Bim-BH3 core sequence peptide conjugated to palmitic acid (SM-6) displayed good PTP1B binding affinity (KD = 8.38 nmol/L), inhibitory activity (IC50 = 1.20 µmol/L) and selectivity against other PTPs (TCPTP, LAR, SHP-1 and SHP-2). Furthermore, SM-6 promoted HepG2 cell glucose uptake and inhibited the expression of PTP1B, indicating that SM-6 could improve the insulin resistance effect in the insulin-resistant HepG2 cell model. These results may indicate a new direction for the application of BH3 peptide mimetics and promising PTP1B peptide inhibitors could be designed and developed based on SM-6.

Ghrelin stimulates growth hormone release from the pituitary via hypothalamic growth hormone-releasing hormone neurons in the cichlid, Oreochromis niloticus.

Ghrelin, a gut-brain peptide hormone, is implicated in a multiplicity of biological functions, including energy homeostasis and reproduction. Neuronal systems that are involved in energy homeostasis as well as reproduction traverse the hypothalamus; however, the mechanism by which they control energy homeostasis is not fully understood. The present study analyzes the anatomical relationship of neurons expressing gonadotropin-releasing hormone (GnRH), neuropeptide Y (NPY) and growth hormone-releasing hormone (GHRH) in a cichlid, tilapia (Oreochromis niloticus). Additionally, we examine in vivo effects of ghrelin on these hypothalamic neurons and plasma growth hormone (GH) and insulin-like growth factor-1 (IGF-1) levels. Double-immunofluorescence showed neuronal fiber associations between GnRH, NPY and GHRH in the brain and pituitary. Intracerebroventricular injection of ghrelin had no effect on numbers, soma size, or optical density of GnRH and NPY neurons, whereas the number of GHRH neurons was significantly decreased in the animals injected with ghrelin when compared to controls, which may indicate administered ghrelin promoted GHRH release. Plasma GH and pituitary GH mRNA levels were significantly increased in the animals injected with ghrelin. These results suggest that central administration of ghrelin primarily act on hypothalamic GHRH neurons to stimulate GH release from the pituitary in the tilapia.

Molecular identification of the Dyn/Kor system and its potential role in the reproductive axis of goldfish.

To ascertain the significance of the dynorphin/kappa-opioid receptor (Dyn/Kor) system in fish reproduction, prodynorphin (pdyn) cDNA was cloned from goldfish. Two Dyn peptides (DynA and DynB) are present in the goldfish prodynorphin precursor. Both DynA and DynB are biologically active as they are able to functionally interact with the goldfish Kor expressed in cultured eukaryotic cells to suppress forskolin-induced CRE promoter activity. RT-PCR analysis showed that pdyn is widely expressed in brain regions, with the highest expression in hypothalamus. During ovarian development, hypothalamic pdyn and kor mRNA levels are lower in the early vitellogenic stage. Then the biological effects of Dyn peptides on salmon gonadotropin releasing hormone (sgnrh), luteinizing hormone beta (lhb) and follicle stimulating hormone beta (fshb) mRNA synthesis were further investigated in goldfish. Intraperitoneal injections of DynA and DynB significantly reduced hypothalamic sgnrh and pituitary lhb and fshb mRNA levels in male goldfish, but these two peptides only down-regulated sgnrh and lhb mRNA expression in female goldfish. In vitro studies revealed that DynA also decreased lhb mRNA levels in primary cultures of pituitary cells, indicating that this peptide can exert its actions at the pituitary level. Our findings suggest that the Dyn/Kor system plays a negative role in regulating the reproductive axis in goldfish.

Distinct functions of neuromedin u and neuromedin s in orange-spotted grouper.

Neuromedin U (NMU) and neuromedin S (NMS) play inhibitory roles in the regulation of food intake and energy homeostasis in mammals. However, their functions are not clearly established in teleost fish. In the present study, nmu and nms homologs were identified in several fish species. Subsequently, their cDNA sequences were cloned from the orange-spotted grouper (Epinephelus coioides). Sequence analysis showed that the orange-spotted grouper Nmu proprotein contains a 21-amino acid mature Nmu peptide (Nmu-21). The Nms proprotein lost the typical mature Nms peptide, but it retains a putative 34-amino acid peptide (Nmsrp). In situ hybridization revealed that nmu- and nms-expressing cells are mainly localized in the hypothalamic regions associated with appetite regulation. Food deprivation decreased the hypothalamic nmu mRNA levels but induced an increase of nms mRNA levels. Periprandial expression analysis showed that hypothalamic expression of nmu increased significantly at 3 h post-feeding, while nms expression was elevated at the normal feeding time. I.p. injection of synthetic Nmu-21 peptide suppressed the hypothalamic neuropeptide y (npy) expression, while Nmsrp administration significantly increased the expression of npy and orexin in orange-spotted grouper. Furthermore, the mRNA levels of LH beta subunit (lhß) and gh in the pituitary were significantly down-regulated after Nmu-21 peptide administration, while Nmsrp was able to significantly stimulate the expression of FSH beta subunit (fshß), prolactin (prl), and somatolaction (sl). Our results indicate that nmu and nms possess distinct neuroendocrine functions and pituitary functions in the orange spotted grouper.

Day-night and reproductive cycle profiles of melatonin receptor, kiss, and gnrh expression in orange-spotted grouper (Epinephelus coioides).

It is suggested that the MT1 melatonin receptor mediates the effects of melatonin on reproduction in rodents. Three melatonin receptor types, MT1, MT2, and Mel1c, have been identified in fish. To understand the potential roles of each type of melatonin receptor on reproduction, we explored the day-night and reproductive cycle profiles of melatonin receptor, kiss, and gnrh expression in the orange-spotted grouper (Epinephelus coioides). cDNAs encoding melatonin receptors (MT1, MT2, and Mel1c) were first isolated from the brain of the orange-spotted grouper. Quantitative real-time PCR analysis demonstrated that the expression levels of MT1 and MT2 were higher in most of the brain areas and pituitary, while mel1c mRNA was mainly distributed in some peripheral tissues and the pituitary. The expression levels of MT1 were much higher than those of MT2 and mel1c in most of the brain regions, and the day-night expression variations of MT1 were counter to those of kiss2 and gnrh1. Reproductive cycle variations in MT1 daytime expression were different from those for kiss2 and gnrh1, and contrary to ovarian fecundity. Our results suggest that MT1 may modulate gnrh1 expression through kiss2, or may directly influence it. Together, these signal cascades may regulate the seasonal breeding of the orange-spotted grouper. As the day-length variations were consistent with the ovarian fecundity variation observed during the reproductive cycle, we infer that photoperiod affects ovarian development of the orange-spotted grouper through MT1.

A novel neuropeptide in suppressing luteinizing hormone release in goldfish, Carassius auratus.

The fish reproductive axis is regulated by many neuroendocrine factors. However, factors involved in the suppression of this axis are largely uncharacterized. In this study, we describe a novel neuropeptide derived from the spexin precursor acting as a negative factor to suppress the reproductive axis in teleost. The cDNA sequences of the spexin precursors have been cloned from both zebrafish and goldfish. A 14-aa mature peptide with the C-terminal amidated (spexin-14a: NWTPQAMLYLKGTQ-NH2) is conceivably generated by processing of the spexin precursors in both species. Spexin is mainly expressed in the brain and ovary of zebrafish and spexin-14a-ir cells are located in several brain regions of goldfish. Functionally, goldfish spexin-14a could significantly suppress luteinizing hormone (LH) release in cultured goldfish pituitary cells. Moreover, intraperitoneal injection of spexin-14a could effectively suppress serum LH level. The mRNA expression of spexin is lower in the breeding season and hypothalamic expression of spexin is regulated by gonadal hormones. These results constitute the first report on the novel role of spexin in the negative regulation of the reproductive axis in teleost.

Evidences for the regulation of GnRH and GTH expression by GnIH in the goldfish, Carassius auratus.

Gonadotrophin-inhibitory hormone (GnIH) plays an important role in regulating of reproduction in teleosts. To clarify the mode of action of GnIH on the synthesis of gonadotropin releasing hormone (GnRH) and gonadotrophin (GtH), three GnIHR cDNAs were cloned from the goldfish brain. In situ hybridization results showed that GnIHRs were localized to the hypothalamus and pituitary. In the hypothalamus, GnIHRs were found in the NPP, NPO and NLT, whereas sGnRH neurons were reported to be located, and potentially regulated by GnIH. In the pituitary, only two GnIHRs were observed and they were localized to the PI instead of the adenohypophysis where GtH-expressing cells are localized, suggesting indirect regulation of GtH by GnIH. In vivo, intraperitoneal (i.p.) injections of synthetic goldfish GnIH-II peptide and GnIH-III peptide significantly decreased sGnRH and FSHß mRNA levels. Only GnIH-II decreased LHß mRNA levels significantly. In vitro, both GnIH-II and GnIH-III showed no effect on GtH synthesis, but an inhibition of GnRH-stimulated LHß and FSHß synthesis was observed when GnIH-III was applied to primary pituitary cells in culture. Thus, GnIH could contribute to the regulation of gonadotropin in the brain and pituitary in teleosts.

Structural and functional characterization of neuropeptide Y in a primitive teleost, the Japanese eel (Anguilla japonica).

In the present study, the first full-length cDNA encoding Neuropeptide Y (NPY) was cloned from the brain of Japanese eel (Anguilla japonica). The open reading frame of Japanese eel NPY gene is 294 bp in length, encoding a precursor protein of 97 amino acids, which contains a 36-amino-acid mature peptide. Sequence analysis showed that the Japanese eel NPY peptide is similar to that of other species. Real-time PCR revealed that NPY in Japanese eel is mainly expressed in the brain, especially in the hypothalamus and the optic tectum thalamus. The effect of a negative energy balance on NPY gene expression was examined subsequently. The mRNA level of NPY in the hypothalamus and the optic tectum thalamus showed a pronounced increase after 4 days of food deprivation. The biological activities of Japanese eel NPY were further investigated in vivo and in vitro. Intraperitoneal injection of the NPY peptide into Japanese eel could potently elevate the expression of the mammalian gonadotropin-releasing hormone (mGnRH) in hypothalamus and the follicle-stimulating hormone beta (FSHß), the luteinizing hormone beta (LHß) and growth hormone (GH) in pituitary. In static incubation studies, the stimulatory effects of NPY on mGnRH expression in hypothalamic fragments and on FSHß, LHß and GH expression in pituitary cells were also observed. However, in vivo and in vitro studies showed that NPY exhibits an inhibitory action on the expression of thyroid-stimulating hormone beta (TSHß) in pituitary. The results indicate that NPY is involved in the regulation of multiple physiological processes in Japanese eel.

Molecular cloning and functional characterization of the first non-mammalian 26RFa/QRFP orthologue in Goldfish, Carassius auratus.

In this study, we used data mining approach to predict 26RFa/QRFP precursors from fish, amphibian, reptile and avian species and subsequently cloned a 26RFa/QRFP precursor cDNA from goldfish brain based on the predicted sequences information. The goldfish 26RFa/QRFP precursor cDNA encoded a propeptide of 168 amino acids (aa) with predicted signal peptide of 30 aa at N-terminal and putative mature peptides, including 26RFa (26 aa) and 7RFa (7 aa) located at the C-terminal. Multiple sequence alignment showed almost all of the 26RFa/QRFP mature peptides possessed KGGFXFRF-amide motifs (X=G, S, A or N) at their C-terminus, and the last three residues FRF were fully conserved across vertebrates, indicating that the evolutionary pressure has exerted to conserve several C-terminal amino acid residues among the known and predicted 26RFa/QRFP precursors. Real-time PCR revealed that 26RFa/QRFP gene was expressed abundantly in goldfish hypothalamus, optic tectum-thalamus and testis. The regulation of goldfish hypothalamic 26RFa/QRFP gene expression by negative energy balance and putative role of goldfish 26RFa/QRFP in the control of luteinizing hormone (LH) release were studied. Hypothalamic 26RFa/QRFP gene expression was pronouncedly increased at 4 days after food deprivation. Furthermore, intraperitoneal (IP) injection of synthesized goldfish 26RFa/QRFP at a dose of 1 microg/g bodyweight significantly increased serum LH levels at 1h. However, LH levels were not significantly changed by IP injection of goldfish 26RFa/QRFP at lower dosage or at other time points (3 and 6 h), or by incubation of goldfish primary cell cultures. These results suggested that goldfish 26RFa/QRFP shared some similar features with its mammalian counterparts and partly exerted the regulatory function in energy homeostasis and hypothalamic-pituitary-gonadal (HPG) axis as observed in mammalian species.

[Effects of electroacupuncture on the expression of epidermal growth factor receptor and glial fibrillary acidic protein after spinal cord injury in rats].

OBJECTIVE: To study the molecular mechanism of electroacupuncture (EA) in the treatment of spinal cord injury (SCI) in rats. METHODS: Forty-five male SD rats were randomized into control, model and EA groups with 15 cases in each group which was further divided into 3 subgroups (3 d, 7 d and 14 d) at average. SCI (T10) model was duplicated by using modified Allen's method. EA (2 Hz, 2-6 mA) was applied to bilateral "Jiaji" [EX-B 2, superior and inferior to the injured locus (T10)] for 30 min, continuously for 3 days, 7 days and 14 days respectively in different subgroups. Changes of SCI rats' behavior (hind-limb motor) were detected by using Basso Beattie Bresnahan (BBB) locomotor scoring scale. The immuno-reaction (IR) activity of epidermal growth factor receptor (EGFR) and glial fibrillary acidic protein (GFAP) in gray matter of the injured cord was determined using immunohistochemical technique on day 3, 7 and 14 separately. RESULTS: Compared with control group, BBB scores of model and EA groups were significantly lower in the 3 subgroups (P < 0.01); while in comparison with the 3 subgroups of model group, BBB scores of the corresponding time in EA group were significantly higher (P < 0.01). Compared with control group, IR-positive cells of both EGFR and GFAP in model and EA groups increased remarkably at the 3 time-points in number (P < 0.01); while those of EGFR and GFAP of EA group were significantly fewer than those of model group at the 3 time-points (P < 0.01). CONCLUSION: EA can effectively improve SCI rats' hind-limb locomotor, which may be closely related to its functions in suppressing the expression of EGFR and GFAP in the injured spinal cord and in promoting nerve axon regeneration.

Electroacupuncture regulates TRPM7 expression through the trkA/PI3K pathway after cerebral ischemia-reperfusion in rats.

Recently, it was demonstrated that TRPM7 is an essential mediator of anoxia-induced neuronal death. Meanwhile, nerve growth factor (NGF) is known to have survival and neuroprotective effects by interacting with the high affinity neurotrophin receptor, tropomyosin-related kinase A (trkA). In the present study, we found that electroacupuncture (EA) treatment could up-regulate trkA expression after focal cerebral ischemia in rats. At the same time, EA therapy obviously decreased the high expression of TRPM7 induced by ischemia. Using K252a to inhibit trkA, we found that the EA-mediated down-regulation of TRPM7 was significantly suppressed in rats subjected to cerebral ischemia. TrkA can utilize two distinct signaling pathways: the phosphatidylinositol 3-kinase (PI3K) pathway and the extracellular signal-related kinase (ERK) pathway. We found that the effect of EA on TRPM7 was also inhibited by a PI3K inhibitor, while an ERK inhibitor had no effect. Taken together, our findings suggest that EA can reverse the ischemia-induced increase of TRPM7 levels through the trkA-PI3K pathway.

Effect of electroacupuncture on reperfusion ventricular arrhythmia in rat.

Protective effect and mechanism of electroacupuncture (EA) on acute reperfusion ventricular arrhthmia was investigated. Ventricular arrhythmia was induced by occlusion of the proximal left anterior descend (LAD) branch of coronary artery for 5 min and followed with 15 min reperfusion. EA on acupoint "Neiguan", "Jianshi" was performed at 30 min before ligation and continued another 5 min during ischemia. Isoprenaline (20, 30 and 50 microg/kg) or atropine (1 mg/ kg) was intravenously injected at 5 min before ischemia. The results showed that EA significantly decreased the incidence of ischemia/reperfusion (I/R) induced ventricular tachycardia (VT), ventricular fibrillation (VF) and mortality as compared to I/R group. Atropine partially suppressed the EA's effect of antiarrhythmia; Isoprenaline increased the incidence and severity of reperfusion arrhythmia, which was inhibited by EA, but this inhibition of EA was blocked with increasing dose of isoprenaline. The results indicated that EA treatment could prevent the occurrence of reperfusion ventricular arrhythmia in rats with myocardial ischemia, and its mechanism might be related to the regulation of EA on the beta-adrenoceptors and M-cholinergic receptor activation in myocardium.

Electroacupuncture regulates NMDA receptor NR1 subunit expression via PI3-K pathway in a rat model of cerebral ischemia-reperfusion.

Cell survival is regulated by the balance between death and survival signals. Previous studies have shown that the N-methyl-d-aspartate receptors (NMDARs) are responsible for the glutamate-induced excitotoxicity in the postischemic brain. Meanwhile, nerve growth factor (NGF) is critically involved in cell survival and neuroprotective effects via the extracellular signal-related kinase (ERK) pathway or the phosphatidylinositol 3-kinase (PI3-K) pathway mediated by the high affinity NGF receptor, tropomyosin-related kinase A (TrkA). Clinically, electroacupuncture (EA) has been shown to produce beneficial effects on stroke patients. However, the detailed mechanisms mediating the beneficial effects of EA on stroke are still unknown. In the present study, we found that EA treatment reversed the high expression of NR1 subunit and up-regulated the level of TrkA in a rat model of middle cerebral artery occlusion. Using protein kinase inhibitors of specific intracellular signaling pathways, we found that the neuroprotective effects of EA appear to be mediated by stimulation of the PI3-K pathway, but not ERK pathway. These findings may provide important experimental evidence for the clinical application of EA treatment for stroke patients.

Effect of electroacupuncture on TRPM7 mRNA expression after cerebral ischemia/reperfusion in rats via TrkA pathway.

The effect of electroacupuncture (EA) on TRPM7 mRNA expression of focal cerebral ischemia in rats and further the role of EA in the relationship between TRPM7 and trkA pathway was investigated. Thirty SD rats were randomly divided into 5 groups : normal group, ischemia/reperfusion group, EA treated group (ischemic rats with EA treatment), TE infusion group (ischemic rats with EA treatment and TE buffer infusion), AS-ODN group (ischemic rats with EA treatment and antisense trkA oligonucleotide infusion). The stroke animal model was established by the modified method of middle cerebral artery occlusion. Antisense trkA oligonucleotide that blocked NGFs effects was injected into cerebroventricle before EA. The TRPM7 mRNA was detected by RT-PCR method. The results showed that there were low TRPM7 mRNA levels in cortex and hippocampus in normal group. Compared with normal group, TRPM7 mRNA expression was increased significantly in ischemia/reperfusion group (P<0.05). A significant reduction in the expression of TR-PM7 mRNA was found in EA treated group in contrast to ischemia/reperfusion group (P<0.05). The expression of TRPM7 mRNA in AS-ODN group was remarkably increased compared with EA treated group and TE infusion group (P<0.05). The results indicated that TRPM7 channels in the ischemic cortex and hippocampus in rats might play a key role in ischemic brain injury. EA could reverse the overexpression of TRPM7 in cerebral ischemia/reperfusion rats. And the inhibitory effect of EA on TRPM7 channels might be through trkA pathway.

Effect of Bizhongxiao Decotion (BZXD) on Some Cytokines in Plasma of Rats with CII-induced Rheumatoid Arthritis.

OBJECTIVE: To investigate the influence of bizhongxiao decoction (BZXD) which is a Traditional Chinese medicine for RA including, on the plasma TNF-α and IL-1ß in rats with CII-induced arthritis (CIA) and explore the protective mechanism of BZXD in the treatment of rheumatoid arthritis. METHODS: 75 SD rats were divided into four groups randomly. Normal control group (n=5) not be treated any more. The CIA rat was established by subcutaneous injection with bovine II collagen (B II C) and complete Freund, s adjuvant (CFA) after 7d breeding. The CIA rats were divided into the CIA group (n=16), BZXD group (n=29) treated with BZXD and the MTX group (n=25) treated with methotrexate. All rats were killed after various intervals (25, 30, 35, 40, or 45d). At the end of each time interval, we collected the blood of each rat. To detect TNF-α and IL-1ß in plasma with radio-immunity kit. RESULTS: BIIC and CFA can be used to copying CIA model. The incidence of arthritis was 88%. The plasma TNF-α and IL-1ß levels of CIA group, BZXD group and MTX group were notably higher than those of normal control group (p<0.05), moreover, the CIA group was higher than those of the MTX group and BZXD group at various interval (p<0.01). TNF-α and IL-1ß rose step by step in CIA group but decreased in BZXD group and MTX group gradually. Moreover, in BZXD group were lower than those in MTX group (p<0.05). CONCLUSION: TNF-α and IL-1ß play a very important role in the formation and development of RA. BZXD can notably decrease the plasma TNF-α and IL-1ß levels, which was better than MTX.

Effects of electroacupuncture on the pain threshold and the NMDA R1 mRNA in DRG on neuropathic pain rats.

To observe the effect of multiple electroacupuncture (EA) on the pain threshold and the regulation of N-methyl-D-aspartate (NMDA) receptor in dorsal root ganglia (DRG) of neuropathic pain rats. Rats were prepared with a unilateral chronic constriction injury (CCI) to the sciatic nerve. EA was done in acupoints "Huan Tiao" and "Yang Ling Quan" for 30 min every day and the thermal thresholds were detected after EA at 3, 5, 7, 10, 14 days after operation. On day 14 after nerve injury, the in situ hybridization method was used to investigate the change of NMDA R1 mRNA in L4-L5 DRG. The thermal threshold reduced significantly from day 3 after operation in CCI rats. After multiple EA treatment, the ipsilateral thermal hyperalgesia relieved gradually and the thermal threshold had no difference with control side after day 5 (P > 0.05). From Day 7 after operation, the thermal threshold at each time point were significantly different compared with CCI group respectively (P > 0.05). Moreover the EA had accumulative effect. On Day 14 after operation, the NMDAR1 mRNA positive neurons and the mean optic density in ipsilateral L4-5 DRG were less than that of control side (P < 0.05), mainly in medium and small neurons. After EA treatment, the NMDAR1 mRNA positive neurons in ipsilateral DRG had no considerable difference comparing with those of control side, significantly increased comparing with CCI group (P < 0.05). It's concluded that the NMDA receptors in DRG relate closely with the generation and development of neuropathic pain. The multiple EA treatment can attenuate the thermal hyperlagesia of neuropathic pain rats and regulate the NMDA receptor.

Influence of electroacupuncture on the mRNA of heat shock protein 70 and 90 in brain after cerebral ischemia/reperfusion of rats.

To explore the anti-apoptotic role of electroacupuncture (EA) and its molecular mechanisms after cerebral ischemia/reperfusion (IR) of rats, by using animal model of middle cerebral artery occlusion (MCAO), the changes of the cleavage of PARP were observed by Western blot and the mRNA of heat shock protein (Hsp) 70 and Hsp90 beta detected by competitive RT-PCR after cerebral IR and EA treatment. The results were as follows: (1) The cleavage of PARP was increased in ischemic hippocampus, and EA treatment could attenuate the level of the cleavage remarkably; (2) The mRNA expression of Hsp70 was increased in the ischemic cortex and hippocampus and was further increased after EA treatment; (3) The mRNA expression of Hsp90 beta was decreased in ischemic cortex and hippocampus and the decrease was relatively slight after EA treatment. The above results demonstrated EA treatment could protect neurons from apoptosis after cerebral IR. One of the molecular mechanisms was the promotion of the inducible expression of Hsp70 and the improvement of the inhibition of the expression of Hsp90.

Effect of ginsenoside Re on cardiomyocyte apoptosis and expression of Bcl-2/Bax gene after ischemia and reperfusion in rats.

To observe the effect of ginsenoside Re on cardiomyocyte apoptosis and Bcl-2/Bax gene expression after ischemia (30 min) and reperfusion (6 h) in rats and to elucidate the possible mechanisms of ginsenoside Re on inhibition of cardiomyocyte apoptosis, the ischemia/reperfusion heart model was established by ligating the left anterior descending branch of coronary artery in Wistar rats. The apoptotic cardiomyocytes were confirmed by transmission electron microscopy and counted by in situ nick end labeling (TUNEL) method and light microscopy. The mRNA and protein expression of Bcl-2 and Bax genes were studied by in situ hybridization and immunohistochemical staining. Mean optical density (OD) value of the positive fields of mRNA and protein expression was quantitatively examined by image analysis system. The results were as follows: (1) The apoptotic cardiomyocytes were found in ischemic fields in the ischemia/reperfusion group and weren't observed in the sham-operation group by transmission electron microscopy; (2) The numbers of the apoptotic cells were 134.45 +/- 45.61/field in the ischemia/reperfusion group, and 90.66 +/- 19.22/field in the ginsenoside Re-treated group. The differences was significant between two groups (P < 0.01); (3) Gene expression of Bcl-2 and Bax were increased significantly in the ischemia/reperfusion group and ginsenoside Re-treated group when compared with the sham-operation group. There was no significant difference in the gene expression of Bcl-2 between the ginsenoside Re-treated group and ischemia/reperfusion group (P > 0.05), but gene expression of Bax was decreased significantly in the ginsenoside Re-treated group as compared with the ischemia/reperfusion group (P < 0.01). The ratio of Bcl-2/Bax was increased significantly in the ginsenoside Re-treated group when compared with the ischemia/reperfusion group and sham-operation group. These findings suggest that myocardial ischemia-reperfusion can induce cardiomyocyte apoptosis, and ginsenoside Re can significantly inhibit cardiomyocyte apoptosis induced by ischemia-reperfusion in rats. It is concluded that ginsenoside Re inhibits cardiomyocyte apoptosis by inhibiting expression of pro-apoptotic Bax gene and raising the ratio of Bcl-2/Bax.