Exploring the neuroprotection of the combination of astragaloside A, chlorogenic acid and scutellarin in treating chronic cerebral ischemia via network analysis and experimental validation.

Chronic cerebral ischemia (CCI) primarily causes cognitive dysfunction and other neurological impairments, yet there remains a lack of ideal therapeutic medications. The preparation combination of Astragalus membranaceus (Fisch.) Bunge and Erigeron breviscapus (Vant.) Hand.-Mazz have been utilized to ameliorate neurological dysfunction following cerebral ischemia, but material basis of its synergy remains unclear. The principal active ingredients and their optimal proportions in this combination have been identified through the oxygen and glucose deprivation (OGD) cell model, including astragaloside A, chlorogenic acid and scutellarin (ACS), and its efficacy in enhancing the survival of OGD PC12 cells surpasses that of the combination preparation. Nevertheless, mechanism of ACS against CCI remains elusive. In this study, 63 potential targets of ACS against CCI injury were obtained by network pharmacology, among which AKT1, CASP3 and TNF are the core targets. Subsequent analysis utilizing KEGG and GO suggested that PI3K/AKT pathway may play a crucial role for ACS in ameliorating CCI injury. Then, a right unilateral common carotid artery occlusion (rUCCAO) mouse model and an OGD PC12 cell model were established to replicate the pathological processes of CCI in vivo and in vitro. These models were utilized to explore the anti-CCI effects of ACS and its regulatory mechanisms, particularly focusing on PI3K/AKT pathway. The results showed that ACS facilitated the restoration of cerebral blood flow in CCI mice, enhanced the function of the central cholinergic nervous system, protected against ischemic nerve cell and mitochondrial damage, and improved cognitive function and other neurological impairments. Additionally, ACS upregulated the expression of p-PI3K, p-AKT, p-GSK3ß and Bcl-2, and diminished the expression of Cyto-c, cleaved Caspase-3 and Bax significantly. However, the PI3K inhibitor (LY294002) partially reversed the downregulation of Bax, Cyto-c and cleaved Caspase-3 expression as well as the upregulation of p-AKT/AKT, p-GSK3ß/GSK3ß, and Bcl-2/Bax ratios. These findings suggest that ACS against neuronal damage in cerebral ischemia may be closely related to the activation of PI3K/AKT pathway. These results declared first time ACS may become an ideal candidate drug against CCI due to its neuroprotective effects, which are mediated by the activated PI3K/AKT pathway mitigates mitochondrial damage and prevents cell apoptosis.

Xiaoxuming decoction cutting formula reduces LPS-stimulated inflammation in BV-2 cells by regulating miR-9-5p in microglia exosomes.

The Background: Stroke is one of the leading causes of morbidity and mortality, and the inflammatory mechanism plays a crucial role in stroke-related brain injury and post-ischemic tissue damage. Xiaoxuming decoction (XXMD) is the first prescription for the treatment of "zhongfeng" (a broad concept referring to stroke) in the Tang and Song Dynasties of China and has a significant position in the history of stroke treatment. Through the study of ancient medical records and modern clinical evidence, it is evident that XXMD has significant efficacy in the treatment of stroke and its sequelae, and its pharmacological mechanism may be related to post-stroke inflammation. However, XXMD contains 12 medicinal herbs with complex composition, and therefore, a simplified version of XXMD, called Xiaoxuming decoction cutting (XXMD-C), was derived based on the anti-inflammatory effects of the individual herbs. Therefore, it is necessary to explore and confirm the anti-inflammatory mechanism of XXMD-C. Aim of the study: Based on the previous experiments of our research group, it was found that both XXMD and XXMD-C have anti-inflammatory effects on LPS-induced microglia, and XXMD-C has a better anti-inflammatory effect. Since miRNAs in exosomes also participate in the occurrence and development of cardiovascular diseases, and traditional Chinese medicine can regulate exosomal miRNAs through intervention, this study aims to explore the anti-inflammatory mechanism of XXMD-C in the treatment of post-stroke inflammation through transcriptome sequencing, providing a basis for the application of XXMD-C. Materials and methods: XXMD-C was extracted using water and filtered through a 0.22 µm membrane filter. The main chemical components of the medicinal herbs in XXMD-C were rapidly qualitatively analyzed using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). Cell viability was determined using the CCK-8 assay, and an LPS-induced BV-2 cell inflammation model was established. The expression of inflammatory cytokines was detected using ELISA and Western blot (WB). Extracellular vesicles were extracted using ultracentrifugation, and identified using transmission electron microscopy (TEM), nanoparticle tracking analysis, and WB. Differential miRNAs were screened using smallRNA-seq sequencing, and validated using RT-PCR and Western blot. Results: The UPLC-Q-TOF-MS analysis revealed that representative components including ephedrine, pseudoephedrine, cinnamaldehyde, baicalin, baicalein, wogonin, and ginsenoside Rg1 were detected in XXMD-C. The results of ELISA and WB assays showed that XXMD-C had a therapeutic effect on LPS-induced inflammation in BV-2 cells. TEM, nanoparticle tracking analysis, and WB results demonstrated the successful extraction of extracellular vesicles using high-speed centrifugation. Differential miRNA analysis by smallRNA-seq identified miR-9-5p, which was validated by RT-PCR and WB. Inhibition of miR-9-5p was found to downregulate the expression of inflammatory factors including IL-1ß, IL-6, iNOS, and TNF-α. Conclusion: The study found that XXMD-C has anti-neuroinflammatory effects. Through smallRNA-seq sequencing of extracellular vesicles, miR-9-5p was identified as a key miRNA in the mechanism of XXMD-C for treating neuroinflammation, and its in vivo anti-inflammatory mechanism deserves further investigation.

Exploring the Two Herb Combination Strategy to Treat Injured PC12 Cells.

In view of the advantages of the combination of traditional Chinese medicine (TCM) in the treatment of cerebral ischemia, we studied the differences in the efficacy and mechanism between the preparation combination and the component combination in order to explore the two herb combination strategy to treat injured PC12 cells. Cobalt chloride (CoCl2) combined with a glucose-free medium was employed to induce oxidative damage of PC12 cells. Then, the optimal combination of Astragalus mongholicus (Ast) and Erigeron breviscapus (Eri) injection was selected and combined following uniform design methods after screening their safe and effective concentration on PC12 cells. Further, the component combination screened comprises 10 µM astragaloside A, 40 µM scutellarin, and 75 µM chlorogenic acid in two herbs. Then, MTT, Annexin V-FITC/PI, immunofluorescence, and Western blot analysis were used to evaluate the efficacy and mechanism of the preparation combination and the component combination on injured PC12 cells. The results showed that the optimal preparation combination for cell pro-survival was Ast injection and Eri capsule with a concentration of 6:1.8 (µM). The component combination (10 µM astragaloside A, 40 µM scutellarin, and 75 µM chlorogenic acid) was more effective than the preparation combination. Both combinations remarkably reduced apoptotic rate, the fluorescence intensity of caspase-3, and intracellular reactive oxygen species (ROS) level; meanwhile, they upregulated the expression levels of p-Akt/Akt, Bcl-2/Bax, and Nrf2. These effects were more evident in the component combination. In conclusion, both combinations can inhibit the injury induced by CoCl2 combined with a glucose-free medium on PC12 cells, thus promoting cell survival. However, the efficiency of the component combination over the preparation combination may be due to its stronger regulation of the PI3K/Akt/Nrf2 signaling pathway related to oxidative stress and apoptosis.

N-linoleyltyrosine protects neurons against Aß1-40-induced cell toxicity via autophagy involving the CB2/AMPK/mTOR/ULK1 pathway.

Beta-amyloid protein (Aß) is one of the most important pathogenic factors of Alzheimer's disease (AD). N-linoleyltyrosine (NITyr) was synthesized in our laboratory and exerted neuroprotective effects in APP/PS1 transgenic mice in previous reports. In this study, the neuroprotective effects and mechanisms of NITyr were evaluated in Aß1-40-treated primary cortical neurons for the first time in vitro. NITyr treatment attenuated cytotoxicity induced by Aß1-40, and the best effect of NITyr was observed at 1 µmol/L. NITyr treatment increased the BDNF protein expression and the ratio of p-CREB/CREB, but weakened the Caspase-3 protein expression. Meanwhile, NITyr enhanced the expressions of autophagy-related proteins (LC3-II, Beclin-1, ATG5 and ATG13). The autophagy inhibitor 3-methyladenine (3MA) reversed the effects of NITyr on cell viability and the protein expressions of neuron-related proteins, including BDNF, p-CREB and Caspase-3. The CB2 receptor antagonist AM630 weakened the neuroprotective effects of NITyr and the autophagy-related protein expression (LC3-II, Beclin-1, ATG5 and ATG13). Moreover, NITyr significantly increased the expressions of p-AMPK, p-mTOR and p-ULK1, but not p-p38. AM630 ablated the above phenomenon. Therefore, NITyr protected the neurons against Aß1-40-induced cytotoxicity by inducing autophagy, which involved the CB2/AMPK/mTOR/ULK1 pathway.

Calycosin suppresses colorectal cancer progression by targeting ERß, upregulating PTEN, and inhibiting PI3K/Akt signal pathway.

High intake of phytoestrogen has been reported to be associated with the prevention of colorectal cancer (CRC). Calycosin belongs to the phytoestrogen that has been shown to suppress CRC cells in our previous study. However, its anticancer activity and molecular mechanisms have not been elucidated. In this study, we analyzed the effect of calycosin on the viability and apoptosis of human CRC HCT116 and SW480 cells via MTT assay, flow cytometry assay, and caspase-3/7 activity assay. The protein expressions of estrogen receptor ß (ERß), PTEN, and PI3K/Akt signal pathways were determined by Western blot analysis. And then, the alterations of biological behavior in CRC cells transfected with ERß siRNA were analyzed. Mouse xenograft models were further performed to detect the antitumor effect in vivo. The results show that calycosin reduces CRC cell viability, induces cell apoptosis, and suppresses xenograft tumor growth. The protein expressions of ERß and PTEN are significantly upregulated following calycosin treatment, whereas p-AKT/AKT ratio and Bcl-2 level are downregulated. Suppressing ERß with siRNA partially attenuates the reduction in viability and apoptosis induced by calycosin. Our results indicate that calycosin shows inhibitory effects on CRC cells, which might be obtained by targeting ERß, upregulating PTEN, and inhibiting the PI3K/Akt signal pathway.

Drug Release from Disulfide-Linked Prodrugs: Role of Thiol Agents.

The disulfide bond (SS) has been widely used in prodrugs for the redox-responsive drug release, but its drug release mechanism and rate were seldom compared in different thiol agents. Herein, self-assembling nanoaggregates (NAs) formed by camptothecin (CPT)-oleic acid (OA) prodrugs linked by two frequently used SS linkers (ETCSS and ACSS) were used for such comparative investigation. It is found that the cleavage of ETCSS was directly coupled with CPT release, whereas the breakage of ACSS resulted in the generation of CPT intermediates, the chemical stability of which determined CPT release. In both cases, the redox-responsive drug release was highly dependent on the reactivity between SS and thiol agents, with an order of dithiothreitol > cysteine ≈ glutathione. Moreover, the presence of SS significantly accelerated the extracellular CPT release, which was around 3-4 fold higher than intracellular CPT release. Therefore, the in vitro cytotoxicity of SS-linked CPT-OA NAs could not be ascribed to the glutathione-trigged intracellular drug release but rather to the SS-accelerated extracellular CPT release. The above results would effectively guide the rational design and evaluation of SS-linked prodrug NAs for efficient drug delivery.

Indirect Competitive Determination of Tetracycline Residue in Honey Using an Ultrasensitive Gold-Nanoparticle-Linked Aptamer Assay.

Tetracycline residue in honey has become an increasingly important food safety problem. In this work, an ultrasensitive gold nanoparticles (AuNPs)-linked aptamer assay was developed to determine the tetracycline residue in honey. First, a tetracycline-bovine serum albumin conjugate coating was applied to a microplate. Then, with the incubation of AuNPs-linked aptamer, the fixed tetracycline in the microplate competed for the limited aptamer with the free tetracycline in the sample. Higher amounts of free tetracycline in the sample were associated with more competitive binding of aptamer-AuNPs, and the aptamer-AuNPs binding with tetracycline-BSA was lower. Finally, as a kind of nanozyme, AuNPs exhibited peroxidase activity and oxidized 3,3',5,5'-tetramethylbenzidine, transforming it from colorless to blue, and achieving the measurement at 652 nm. The analytical performance-including linearity, limit of detection, selectivity, precision, repeatability, and accuracy-has been investigated. It was successfully applied to the determination of tetracycline in honey samples with high accuracy and sensitivity.

Optimization and Evaluation of the Thermosensitive In Situ and Adhesive Gel for Rectal Delivery of Budesonide.

Budesonide is a glucocorticoid for the treatment of ulcerative colitis (UC). The current study aims to develop a thermosensitive in situ and adhesive gel for rectal delivery of budesonide. HPMC K4M was selected as the adhesive agent based on the adhesive force and the effect on gel performance. The formulation of gel was optimized by using the central composite design-response surface methodology (CCD-RSM); a mathematical model was successfully developed to predict desired formulations as well as to analyze relationships between the amount of Pluronic F-127, Pluronic F-68, and HPMC K4M and the performances of gel. Based on CCD-RSM, a thermosensitive in situ and adhesive gel consisting of 0.002% budesonide, 0.74% HPMC, 4.87% F-68, and 19.0% F-127 was developed. Furthermore, the in vivo behavior of gel was evaluated in Sprague-Dawley rats. In comparison with budesonide solution, rectal administration of budesonide gel at 0.1 mg/kg in rats showed relative bioavailability of 230% with significant increase in rectum uptake.

[Effect of caffeic acid, seopoletin and scutellarin on rat retinal neurons in vitro].

OBJECTIVE: To observe the effect of caffeic acid, seopoletin and scutellarin on rat retinal neurons in vitro and explore neuroprotection in glaucoma of Erigeron breviscapus. METHOD: The retinal of 18 post-natal 2-3 days Sprague-Dawley rats were dissociated into cell suspension with trypsin digestion. The cell suspension was implated in 96-well culture plates covered with hyaluronic acid and laminin in each well. After culturing for 3 days, caffeic acid, seopoletin and scutellarin were added to the cultures, continue to culture 2 days. Then, the A of living cells in each well was tested by MTT colorimetric microassay. Some of the 5-day culture cells were identified by Nissel technique. RESULT: Most of the living cells were retinal neurons by Nissel identification. The number of living cells increased significantly in high concentrations of caffeic acid, seopoletin and scutellarin compared with control group (P < 0.05, P < 0.01). CONCLUSION: caffeic acid, seopoletin and scutellarin can all promote retinal neurons to live in vitro, with caffeic acid being most effective.