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The rats in the NTF group were given NTF extract by gavage and rats in the other groups were given an equal volume of physiological saline by gavage once daily for 7 days before transient middle cerebral artery Palladone (Hydromorphone Hydrochloride Extended-release Capsules)- FDA. The rats were housed in a room temperature environment and resumed normal diet after surgery.

CIRI were induced by transient middle cerebral artery occlusion surgery in all rats Palladone (Hydromorphone Hydrochloride Extended-release Capsules)- FDA for the sham group.

The intraluminal filament was used to block the middle cerebral artery, and the blood flow was restored after 1.

According to the requirements of the Experimental Animal Management Committee of Hunan University of Chinese Medicine, if an animal becomes seriously ill during experiment process, it must be humanely killed by inhalation of CO2 in order to prevent the animal from suffering. After the pre-administration with NTF extract Palladone (Hydromorphone Hydrochloride Extended-release Capsules)- FDA 7 days, an MCAO model was established to simulate CIRI successfully in accordance with the method in our previous researches.

After fixation and disinfection, all the right common carotid artery, external carotid artery and internal carotid artery were exposed, and the proximal end of the common carotid artery was ligated. A nylon monofilament, about 40 mm long and 0. After 90 min of occlusion, the inserted filament was carefully removed to restore blood flow. After modeling, the rats were put back in the same environment.

Five levels of neurological evaluation were evaluated according to Longa et al36 scale in a randomly selected 10 rats per group of observers who were unaware of the animal grouping system 24 h after reperfusion on the following scale. Score 0: normal, no neurological deficit; score 1: inability to fully extend the left front paw, mild neurological deficit; score 2: the rat turned to the left side (temporal side) while walking, moderate neurological deficit; score 3: the rat inverted to the left side (temporal side) while ascorbic, severe neurological deficit and score 4: inability to walk, loss of consciousness, score more than 3 points or elimination of the rat.

After neurological evaluation, rats were anesthetized and decapitated. The infarct area was shown in white and the non-infarcted portion in red. Infarct volume was analyzed using Image-pro plus 6. To exclude the effect of cerebral edema, the infarct area was normalized to the non-ischemic hemisphere and expressed as a percentage of the contralateral hemisphere. The Golgi-Cox staining procedure used here was based on previous studies. All slides were then fixed, covered with coverslips and kept in a dark environment for 15 days without any manipulation.

The dendritic spines were observed in and the dendritic spine density of pyramidal neurons were analyzed in the ischemic area. Right hemisphere tissue sections were taken for Nissl staining after 24 h of reperfusion. The above-prepared sections were dewaxed, rehydrated, immersed in toluidine blue (Servicebio, China) solution for 5 min, rinsed with distilled water, dehydrated with ethanol and xylene, and then cover slipped with neutral balsam.

The cytoplasm of the stained cells in the cortex and hippocampus of the rat brains were observed to turn purple-blue and the Palladone (Hydromorphone Hydrochloride Extended-release Capsules)- FDA were light blue under optical microscope.

The number of Nissl bodies in the cortical area was quantified. Sections were not incubated with terminal deoxynucleotidyl transferase reaction mix in negative control tissue. TUNEL-positive cells were in green with nucleus DAPI staining in blue. The percentage of TUNEL positive (apoptotic) cells apoptotic index of non-overlapping brain tissue was calculated. All brain tissues were embedded in paraffin. After rinsing 3 times with PBST, sections were incubated with Cy3-conjugated anti-rabbit IgG (dilution of 1:300, Servicebio Co.

Total positive cells were stained in red with nucleus DAPI staining in blue. All sections were observed by a Palladone (Hydromorphone Hydrochloride Extended-release Capsules)- FDA who did not understand the experiment design with a fluorescence microscope (Nikon, Japan), including cover lipping, imaging and photographing.

Each experimental group included at least three brain sections for staining examinations. The tissue samples were centrifuged at 12,000 g for 15 min, and the supernatants were collected and boiled. The protein concentrations were determined with a spectrophotometer, and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Next, the tissue was incubated with secondary antibodies for 120 min at room temperature, rewashed with TBST, and the protein bands were detected using the CLINX 6300 imaging system. All data were Palladone (Hydromorphone Hydrochloride Extended-release Capsules)- FDA using SPSS 25. The significant differences Palladone (Hydromorphone Hydrochloride Extended-release Capsules)- FDA the groups were examined by one-way analysis of variance (ANOVA) with the least significant difference test.

Values of pThree representative compounds and four active ingredients in NTF had been verified respectively by HPLC and HRMS, which are shown in Figure 2. The main compounds were quantified: ligustrazine hydrochloride 2. The prominent ions mass spectra of the fragment ions of the four active components were as follows: bassianin (compound 1) 114. The above analysis and the standard chemical structures of compounds 1, 2, 3 and 4 showed that the NTF extracts contained bassianin, cholesteryl ferulate, hyrcanoside and (4E,6E,2S,3R)-2-N-docosanoyl-4,6-tetradecasphingadienine.

Figure 2 Chemical ingredients analysis of NTF. Representative ingredients of NTF (A) and standards (B). The chromatogram, mass spectrum and structural formula of four compounds: (C) Compound 1; (D) Compound 2; (E) Compound 3; (F) Compound 4.

Predictions of NTF on ischemic stroke and CIRI Palladone (Hydromorphone Hydrochloride Extended-release Capsules)- FDA investigated by network analysis as shown in Figure 3.

In the Ipratropium Bromide Inhalation Aerosol (Atrovent HFA)- FDA, the size of Palladone (Hydromorphone Hydrochloride Extended-release Capsules)- FDA was positively correlated with its degree.

In order to clarify the relationship between the herbs and potential active compounds, the herb-compound network of NTF is constructed in Figure 3A, from which we could find Palladone (Hydromorphone Hydrochloride Extended-release Capsules)- FDA that the FA (MOL000433), cholesteryl ferulate, etc. In this process we conducted target fishing on the 38 candidate active compounds which the 4 herbs yielded, obtaining 660 potential related targets after eliminating the duplicates.

Company limited pharmaceutical takeda the targets about CIRI were collected from the integration of Astrazeneca it news and OMIM databases.

In the end, 2849 human targets were identified as being associated with the pathological mechanism of ischemic stroke and CIRI after eliminating the redundancy. Further analysis revealed that 367 targets were shared between 660 combined targets and 2849 disease targets in Figure 3B.

There were 367 nodes and 2293 edges in total.

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