[Effect of electroacupuncture pretreatment on ferroptosis in neurons of rats with cerebral ischemia-reperfusion injury].

OBJECTIVE: To observe the effect of electroacupuncture（EA）preconditioning on ferroptosis in rats with cerebral ischemia-reperfusion injury （CIRI）, so as to explore the neuroprotective mechanism of EA preconditioning. METHODS: Male SD rats were randomly divided into sham operation, model, EA, inhibitor and inducer groups with 20 rats in each group. The CIRI model was established by modified Zea Longa occlusion of the middle cerebral artery. Before modeling, EA treatment （2 Hz/15 Hz, 1-2 mA） was applied to "Baihui"（GV20）, "Fengfu"（GV16） and "Dazhui"（GV14） for rats of the EA group, 20 min a day for 7 consecutive days. Rats of the inhibitor group were intraperitoneally injected with ferristatin-1（25 mg/kg）at a slow and uniform rate. Rats of the inducer group were intraperitoneally injected with Erastin（100 mg/kg） after 7 days of EA preconditioning, once every 2 h for a total of 4 times. The CIRI models were prepared 2 d later after the above interventions finished by thread-occlusion. The degree of neurological impairment was evaluated by modified Zea Longa score. The percentage of infarct size was calculated by TTC staining. The ultrastructure of neurons in hippocampus was observed by transmission electron microscope. The contents of ferrous ion （Fe2+）, malondialdehyde （MDA） and glutathione （GSH） in cerebral tissue and reactive oxygen species （ROS） in serum were determined by biochemical method. The changes of mitochondrial membrane potential in rats brain tissues were detected by flow cytometry. The mRNA and protein expression levels of glutathione peroxidase 4 （GPX4）, acyl-CoA synthetase long-chain family member 4 （ACSL4）, transferrin receptor （TFRC）, 15-lipoxygenase （15-LOX） and cyclooxygenase-2 （COX-2） in the ischemic hippocampal region of CIRI rats were detected by real-time quantitative PCR and Western blot, respectively. RESULTS: Compared with the sham operation group, the neurological impairment score, the percentage of cerebral infarction area, the contents of MDA and Fe2+ in cerebral tissue as well as ROS in serum, the protein and mRNA expression levels of ACSL4, TFRC, 15-LOX, COX-2 in hippocampal tissue were increased （P<0.01）, while the content of GSH in cerebral tissue, the protein and mRNA expression levels of GPX4 in hippocampal tissue were decreased （P<0.01）, and mitochondria in brain tissue were significantly damaged （P<0.01） in the model group. Compared with the model group, the above indexes were all reversed （P<0.05, P<0.01） in the EA group and inhibitor group. Compared with the EA group, the neurological impairment score, the percentage of cerebral infarction area, the contents of MDA and Fe2+ in cerebral tissue as well as ROS in serum, the protein and mRNA expression le-vels of ACSL4, TFRC, 15-LOX, COX-2 in hippocampal tissue were increased （P<0.05, P<0.01）, while the content of GSH in cerebral tissue, the protein and mRNA expression levels of GPX4 in hippocampal tissue were decreased （P<0.01, P<0.05）, and mitochondria in brain tissue were significantly damaged （P<0.05） in the inducer group. CONCLUSION: EA preconditioning has neuroprotective effect on CIRI rats, which may be related to inhibiting ACSL4/TFRC/15-LOX/COX-2 expression and increasing GSH/GPX4 expression.

[Effect of Tongdu Tiaoshen electroacupuncture pretreatment on PPARγ-mediated pyroptosis of cerebral cortex in rats with cerebral ischemia reperfusion injury].

OBJECTIVE: To observe the effect of Tongdu Tiaoshen (promoting the circulation of the governor vessel and regulating the spirit) electroacupuncture (EA) pretreatment on pyroptosis mediated by peroxisome proliferators-activated receptor γ (PPARγ) of the cerebral cortex in rats with cerebral ischemia reperfusion injury (CIRI) and explore the potential mechanism of EA for the prevention and treatment of CIRI. METHODS: A total of 110 clean-grade male SD rats were randomly divided into a sham-operation group, a model group, an EA group, an EA + inhibitor group and an agonist group, 22 rats in each group. In the EA group, before modeling, EA was applied to "Baihui" (GV 20), "Fengfu" (GV 16) and "Dazhui" (GV 14), with disperse-dense wave, 2 Hz/5 Hz in frequency, 1 to 2 mA in intensity, lasting 20 min; once a day, consecutively for 7 days. On the base of the intervention as the EA group, on the day 7, the intraperitoneal injection with the PPARγ inhibitor, GW9662 (10 mg/kg) was delivered in the EA + inhibitor group. In the agonist group, on the day 7, the PPARγ agonist, pioglitazone hydrochloride (10 mg/kg) was injected intraperitoneally. At the end of intervention, except the sham-operation group, the modified thread embolization method was adopted to establish the right CIRI model in the rats of the other groups. Using the score of the modified neurological severity score (mNSS), the neurological defect condition of rats was evaluated. TTC staining was adopted to detect the relative cerebral infarction volume of rat, TUNEL staining was used to detect apoptosis of cerebral cortical nerve cells and the transmission electron microscope was used to observe pyroptosis of cerebral cortical neural cells. The positive expression of PPARγ and nucleotide-binding to oligomerization domain-like receptor protein 3 (NLRP3) in the cerebral cortex was detected with the immunofluorescence staining. The protein expression of PPARγ, NLRP3, cysteinyl aspartate specific protease-1 (caspase-1), gasdermin D (GSDMD) and GSDMD-N terminal (GSDMD-N) in the cerebral cortex was detected with Western blot. Using the quantitative real-time fluorescence-PCR, the mRNA expression of PPARγ, NLRP3, caspase-1 and GSDMD of the cerebral cortex was detected. The contents of interleukin (IL)-1ß and IL-18 in the cerebral cortex of rats were determined by ELISA. RESULTS: Compared with the sham-operation group, the mNSS, the relative cerebral infarction volume and the TUNEL positive cells rate were increased (P<0.01), pyroptosis was severe, the protein and mRNA expression levels of PPARγ, NLRP3, caspase-1 and GSDMD were elevated (P<0.01); and the protein expression of GSDMD-N and contents of IL-1ß and IL-18 were increased (P<0.01) in the model group. When compared with the model group, the mNSS, the relative cerebral infarction volume and the TUNEL positive cells rate were decreased (P<0.01), pyroptosis was alleviated, the protein and mRNA expression levels of PPARγ were increased (P<0.01), the protein and mRNA expression levels of NLRP3, caspase-1 and GSDMD were decreased (P<0.01), the protein expression of GSDMD-N was reduced (P<0.01); and the contents of IL-1ß and IL-18 were lower (P<0.01) in the EA group and the agonist group; while, in the EA + inhibitor group, the protein expression of PPARγ was increased (P<0.01), the protein and mRNA expression levels of NLRP3 and GSDMD were decreased (P<0.01, P<0.05), the mRNA expression of caspase-1 was reduced (P<0.01); and the contents of IL-1ß and IL-18 were lower (P<0.01). When compared with the EA + inhibitor group, the mNSS, the relative cerebral infarction volume and the TUNEL positive cells rate were decreased (P<0.05, P<0.01), pyroptosis was alleviated, the protein and mRNA expression levels of PPARγ were increased (P<0.01), the protein and mRNA expression levels of NLRP3, caspase-1 and GSDMD were decreased (P<0.01), the protein expression of GSDMD-N was reduced (P<0.01); and the contents of IL-1ß and IL-18 were declined (P<0.01) in the EA group. Compared with the agonist group, in the EA group, the relative cerebral infarction volume and the TUNEL positive cells rate were increased (P<0.05, P<0.01), the mRNA expression of PPARγ was decreased (P<0.01) and the protein expression of GSDMD-N was elevated (P<0.05); and the contents of IL-1ß and IL-18 were higher (P<0.01). CONCLUSION: Tongdu Tiaoshen EA pretreatment can attenuate the neurological impairment in the rats with CIRI, and the underlying mechanism is related to the up-regulation of PPARγ inducing the inhibition of NLRP3 in the cerebral cortex of rats so that pyroptosis is affected.

[Research on building method of spleen kidney Yang deficiency diarrhea rats model].

Spleen kidney Yang deficiency (SKYD) diarrhea is a common syndrome in tranditional Chinese medicine (TCM). Until now, there is not an ideal SKYD diarrhea rat model for the research. In this study, we compared single factor way (method I, injecting hydrocortisone and gavaging Sennae Folium) with compound factors way(method II, gavaging adenine, improper diet, exhaustion, and gavaging Sennae Folium) on establishing SKYD diarrhea rat model. After modelling, diarrhea index, D-xylose excretory rate, NOS/cGMP signal transduction system, organ index and histopathology examination were used to evaluate the two ways. The results showed that, compared with health group, all the assessment criterias of method I and method II had significant differences (P < 0.01, 0.05). In addition, the index such as diarrhea index, NOS/cGMP signal transduction system, organ index (kidney, testis and thymus) and histopathology examination had significant differences (P < 0.01, 0.05) between method I and method II. In conclusion, the compound factors modelling method better conforms to the symptom of diarrhoea model caused by SKYD. This new modelling method provides a basis for studying on TCM astringents warming and tonifying the spleen and kidney, relieving diarrhea.

[Purgative activity difference and mechanism of raw and processed Rhei Radix et rhizoma].

OBJECTIVE: To investigate the purgative activity difference and mechanism of raw and processed Rhei Radix et Rhizoma. METHODS: Mail mice were divided into 9 groups:normal group (saline), model group, Tongbianling group (1 g/kg), raw Rhei Radix et Rhizoma groups (5.00, 2.50 and 1.25 g/kg), processed Rhei Radix et Rhizoma groups (5.00, 2.50 and 1.25 g/kg). After oral administration for 5 days, the diarrhea experiment and intestinal propelling test were carried out to evaluate the purgative effect difference of raw and processed Rhei Radix et Rhizoma. Wistar rats were divided into normal group (normal saline), model group, Tong-bianling group (0.5 g/kg), raw Rhei Radix et Rhizoma group (1.25 g/kg), processed Rhei Radix et Rhizoma group (1.25 g/kg). The changes of levels of gastin( GAS), motilin( MTL), vasoactive peptide (VIP), neurotensin (NT), somatostatin ( SS), acetylcholinesterase (AchE), substance P(SP) and endothelin (ET-1)in blood of the rats and gastin (GAS), motilin( MTL), vasoactive peptide (VIP) and neurotensin (NT) in colon of the rats were detected. RESULT: The levels of GAS, MTL, VIP, NT, SS, AchE, SP and ET-1 in serum of raw Rhei Radix et Rhizoma group, and GAS, MTL and VIP in colon of raw Rhei Radix et Rhizoma group were different obviously comparing with the processed one (P < 0.01). CONCLUSION: The raw Rhei Radix et Rhizoma and the processed one had obvious differences in regulating intestinal gastrointestinal hormone and neurotransmitter. This effect may be the reason or one of the reasons for purgative activity difference between raw and processed Rhei Radix et Rhizoma.

[Microbial biomass and its correlations with carbon, nitrogen, and phosphorus in the sediments of Taihu Lake].

To explore the responses and feedbacks of the microbes in the sediments of Taihu Lake to the sediment nutrients, an investigation was made on the microbial biomass carbon (MB(C)), microbial biomass nitrogen (MB(N)), microbial biomass phosphorus (MB(P)), and their correlations with the total organic carbon (TOC), total nitrogen (TN), and total phosphorus (TP) in the sediments. The microbial biomass in the sediments was 184.66 mg x kg(-1), being higher at the lakeside than in the mid-lake region. The MB(C) was higher in the western coastal region, Zhushan Bay, and Meiliang Bay, with an average of 127.57 mg x kg(-1), MB(N) was higher in Meiliang Bay, Gonghu Bay, mid-lake region close to Meiliang Bay and Gonghu Bay, and eastern costal region, with an average of 19.25 mg x kg(-1), and MB(P) was higher in the eastern region and parts of the mid-lake region, with an average was 19.09 mg x kg(-1). The TOC high value zone (> or = 2.30 g x kg(-1)) was mainly in Zhushan Bay, western coastal region, Meiliang Bay, and Gonghu Bay, with an average of 1.59 g x kg(-1), TN high value zone (> or = 0.30 g x kg(-1)) was mainly in the Gonghu Bay, Meiliang Bay, Zhushan Bay, and western costal region, with an average of 0.21 g x kg(-1), and TP high value zone (> or = 1.20 g x kg(-1)) was mainly in the eastern coastal region and parts of the mid-lake region, with an average of 0.55 g x kg(-1). The TOC/TN ratio in the sediments was 7-19, with an average of 8.97, which showed that the organic substances in the sediments had obvious dual sources, among which, terrestrial organisms were mainly in the west side of the lake. The microbial biomass in the sediments was significantly positively correlated with sediment TOC and TN but had less correlation with sediment TP, and the MB(C)/MB(N) was significantly correlated with sediment TOC/TN, suggesting that the microbes in the sediments of Taihu Lake were mainly affected by the sediment TOC and TN, and the changes of the TOC/TN had significant effects on the microbial community structure.

Halomonas daqingensis sp. nov., a moderately halophilic bacterium isolated from an oilfield soil.

A Gram-negative, moderately halophilic, short rod-shaped, aerobic bacterium with peritrichous flagellae, strain DQD2-30(T), was isolated from a soil sample contaminated with crude oil from the Daqing oilfield in Heilongjiang Province, north-eastern China. The novel strain was capable of growth at NaCl concentrations of 1-15 % (w/v) [optimum at 5-10 % (w/v)]. Phylogenetic analyses based on 16S rRNA gene sequences showed that the novel strain belonged to the genus Halomonas in the class Gammaproteobacteria; the highest 16S rRNA gene sequence similarities were with Halomonas desiderata DSM 9502(T) (98.8 %), Halomonas campisalis A4(T) (96.6 %) and Halomonas gudaonensis CGMCC 1.6133(T) (95.1 %). The major cellular fatty acids of strain DQD2-30(T) were C(18 : 1)omega7c (43.97 %), C(19 : 0 )cyclo omega8c (23.37 %) and C(16 : 0) (14.83 %). The predominant respiratory lipoquinone was ubiquinone with nine isoprene units (Q9). The DNA G+C content was 67.0 mol%. The DNA-DNA hybridization values of strain DQD2-30(T) with the most closely related species of the genus Halomonas were 51.8 %, 28.4 % and 23.5 % for H. desiderata, H. campisalis and H. gudaonensis, respectively. Based on these analyses, strain DQD2-30(T )(=CGMCC 1.6443(T)=LMG 23896(T)) is proposed to represent the type strain of a novel species, Halomonas daqingensis sp. nov.

[Comparison study on total flavonoid content and anti-free redical activity of the leaves of bamboo, phyllostachys nigra, and Ginkgo bilabo].

OBJECTIVE: To investigate the differences of total flavonoid (TF) content and antifree radical activity between the-leaves of bamboo and Gingo biloba, as well as their seasonal changes. METHOD: Spectrophotometery and Chemiluminescence methods were adopted to determine TF and half inhibiting concentration (IC50) on active oxygen free radicals of the leaves of bamboo, phyllostachys nigra (Lodd. ex. Lindl.) Munro, and Ginkgo biloba. Two kinds of leaves were picked in the same plot at the same time monthly. RESULT: The TF of bamboo leaf varied in the range of 0.67%-1.71% (in dry basis of leaf, below as same) throughout a year, the minimum apparing in June and the maximum in July, then going down obviously, and remaining at a much high lever during November to next April. However, the TF of Ginkgo bilabo leaf varied in 1.48%-2.49% during whole growing period, early April to late November. It ascended with the growth of leaf, reaching the top during June and July, the going down slowly, and finally another peak appeared before defoliation. The average IC50 values on O2-. and .OH of bamboo leaf were at 11.0 micrograms.mL-1 and 5.3 mg.mL-1, and Ginkgo biloba at 19.0 micrograms.mL-1 and 3.6 mg.mL-1, respectively. CONCLUSION: The TF content and anti-free radical activity the bamboo leaf are comparable with the leaf of ginkgo biloba, which is a kind of potential resources for natural antioxidant and free radical scavenger.