Integrating network pharmacology and pharmacological evaluation to explore the protective mechanism of Ershiwuwei Zhenzhu pill in ischemic stroke.

ETHNOPHARMACOLOGICAL RELEVANCE: Ershiwuwei Zhenzhu Pill (EZP), a representative and classic formula in Tibetan medicine, is commonly used in the treatment of various cerebrovascular diseases, including ischemic stroke (IS). Nevertheless, their efficacy and potential mechanism in treating IS have yet to be investigated. AIM OF THE STUDY: This study aimed to investigate the potential mechanisms of EZP in the treatment of IS based on network pharmacology and experimental verification. MATERIALS AND METHODS: The chemical profile of EZP was characterized using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). The targets related to the compounds in EZP were predicted by the Swiss Target Prediction and Target Net platform, and targets of IS were collected from the Gene Cards and OMIM databases. Subsequently, a protein-protein interaction (PPI) network of targets was constructed and analyzed by the STRING database and Cytoscape software, version 3.7.1. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed, and an ingredient-target-pathway network was constructed. Ultimately, the middle cerebral artery occlusion (MCAO) model was established to evaluate the anti-IS effects of EZP by detecting the neurological deficit score, HE, Nissl and TCC staining, and inflammatory factors, and the expression of key protein targets was detected by western blotting. RESULTS: A total of 129 components were identified in EZP. Network pharmacology revealed 3136 compound targets and 2826 disease-related targets, and 412 overlapping proteins were obtained as potential therapeutic targets. The PPI network results showed that 6 key targets (AKT1, SRC, VEGFA, TP53, TNF and EGFR) were core targets of EZP in the treatment of IS. Western blotting demonstrated that the expression levels of AKT1, VEGFA, TP53, SRC, TNF and EGFR in the brain tissue of MCAO rats were significantly changed after treatment with EZP compared to the model group. CONCLUSIONS: EZP ameliorated IS in MCAO rats. The underlying mechanism might be associated with inhibiting inflammation and apoptosis, promoting angiogenesis and protecting neurons by regulating multiple targets and pathways.

[Research progress on precious Tibetan medicine formula in prevention and treatment of central nervous system diseases].

Precious Tibetan medicine formula is a characteristic type of medicine commonly used in the clinical treatment of central nervous system diseases. Through the summary of modern research on the precious Tibetan medicine formulas such as Ratnasampil, Ershiwuwei Zhenzhu Pills, Ershiwewei Shanhu Pills, and Ruyi Zhenbao Pills, it is found that they have obvious advantages in the treatment of stroke, Alzheimer's disease, epilepsy, angioneurotic headache, and vascular dementia. Modern pharmacological studies have shown that the mechanisms of precious Tibetan medicine formulas in improving central nervous system diseases are that they promote microcirculation of brain tissue, regulate the permeability of the blood-brain barrier, alleviate inflammation, relieve oxidative stress damage, and inhibit nerve cell apoptosis. This review summarizes the clinical and pharmacological studies on precious Tibetan medicine formulas in prevention and treatment of central nervous system diseases, aiming to provide a reference for future in-depth research and innovative discovery of Tibetan medicine against central nervous diseases.

[Mechanism of Tibetan medicine Ershiwuwei Songshi Pills in regulating intestinal flora and improving non-alcoholic steatohepatitis].

This study aimed to investigate the effect of Tibetan medicine Ershiwuwei Songshi Pills(ESP) on the intestinal flora of non-alcoholic steatohepatitis(NASH) mice. Forty-eight male C57 BL/6 mice were randomly divided into the control group, model(methionine-choline-deficient, MCD) group, high-(0.8 g·kg~(-1)), medium-(0.4 g·kg~(-1)), and low-dose(0.2 g·kg~(-1)) ESP groups, and pioglitazone(PGZ, 10 mg·kg~(-1)) group, with eight mice in each group. Mice in the control group were fed with normal diet, while those in the remaining five groups with MCD diet for five weeks for inducing NASH. During modeling, they were gavaged with the corresponding drugs. The changes in body mass, daily water intake, and daily food intake were recorded. At the end of the experiment, the liver tissues were collected and stained with hematoxylin-eosin(HE) for observing the pathological changes, followed by oil red O staining for observing fat accumulation in the liver. The levels of serum aspartate aminotransferase(AST) and alanine aminotransferase(ALT) and triglyceride(TG) in liver tissue were measured. The changes in intestinal flora of mice were determined using 16 S rRNA high-throughput sequencing technology. The results showed that compared with the model group, the high-, medium-and low-dose ESP groups and the PGZ group exhibited significantly lowered AST and ALT in serum and TG in liver tissues and alleviated hepatocellular steatosis and fat accumulation in the liver. As demonstrated by 16 S rRNA sequencing, the abundance index and diversity of intestinal flora decreased in the model group, while those increased in the ESP groups. Besides, the Firmicutes to Bacteroidetes ratio decreased at the phylum level. In the alteration of the composition of intestinal flora, ESP reduced the abundance of Erysipelotrichia and Faecalibaculum but increased the abundance of Desulfovibrionaceae, Rikenellaceae, Lachnospiraceae, and Ruminococcaceae. This study has revealed that ESP has a protective effect against NASH induced by MCD diet, which may be related to its regulation of the changes in intestinal flora, alteration of the composition of intestinal flora, and inhibition of the intestinal dysbiosis.

[Serum metabolomics of Tibetan medicine Ershiwuwei Songshi Pills against chronic cholestasis in mice].

A chronic cholestasis model was induced in mice by feeding a diet containing 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine(DDC). The effects of Ershiwuwei Songshi Pills(ESP) on endogenous metabolites in mice with chronic cholestasis were investigated by metabolomics analysis based on liquid chromatography-mass spectrometry(LC-MS). The results showed that ESP was effective in improving pathological injury and reducing serum levels of alanine aminotransferase(ALT), aspartate aminotransferase(AST), alkaline phosphatase(ALP), and total bile acid in the model mice. Meanwhile, 13 common differential metabolites were revealed in metabolomic screening between the model/control group and the model/ESP group, including uric acid, glycolaldehyde, kynurenine, flavin adenine dinucleotide, L-3-phenyllactic acid, I-urobilin, leukotriene D4(LTD4), taurocholic acid, trioxilin A3, D-inositol-1,4-diphosphate, PC [16:0/20:2(11Z,14Z)], PC[14:0/22:2(13Z,16Z)], and PC[20:4(5Z,8Z,11Z,14Z)/20:4(5Z,8Z,11Z,14Z)]. After ESP intervention, the levels of all 13 differential metabolites were significantly retraced, and pathway analysis showed that ESP achieved its therapeutic effect mainly by affecting arachidonic acid metabolism, glycerophospholipid metabolism, tryptophan metabolism, and primary bile acid biosynthesis. This study elucidated the mechanism of action of ESP against chronic cholestasis based on metabolites.

[Ershiwuwei Shanhu Pills regulate Akt/mTOR/GSK-3ß signaling pathway to alleviate Alzheimer's disease mice].

The present study investigated the mechanism of the Tibetan patent medicine Ershiwuwei Shanhu Pills(ESP) in alleviating Alzheimer's disease in mice via Akt/mTOR/GSK-3ß signaling pathway. BALB/c mice were randomly assigned into a blank control group, a model group, low(200 mg·kg~(-1)), medium(400 mg·kg~(-1)) and high(800 mg·kg~(-1)) dose groups of ESP, and donepezil hydrochloride group. Except the blank control group, the other groups were given 20 mg·kg~(-1) aluminum chloride by gavage and 120 mg·kg~(-1) D-galactose by intraperitoneal injection for 56 days to establish Alzheimer's disease model. Morris water maze was used to detect the learning and memory ability of mice. The level of p-tau protein in mouse hippocampus and the levels of superoxide dismutase(SOD), malondialdehyde(MDA), catalase(CAT), and total antioxidant capacity(T-AOC) in hippocampus and serum were detected. Hematoxylin-eosin staining and Nissl staining were performed for the pathological observation of whole brain in mice. TdT-mediated dUTP nick-end labeling(TUNEL) staining was employed for the observation of apoptosis in mouse cortex. Western blot was adopted to detect the protein levels of p-mTOR, p-Akt, and GSK-3ß in the hippocampus. Compared with the model group, the ESP groups showcased alleviated pathological damage of the whole brain, decreased TUNEL positive cells, reduced level of p-tau protein in hippocampus, and risen SOD, CAT, and T-AOC levels and declined MDA level in hippocampus and serum. Furthermore, the ESP groups had up-regulated protein levels of p-mTOR and p-Akt while down-regulated protein level of GSK-3ß in hippocampus. Therefore, ESP can alleviate the learning and memory decline and oxidative damage in mice with Alzheimer's disease induced by D-galactose combined with aluminum chloride, which may be related to Akt/mTOR/GSK-3ß signaling pathway.

[Protective effect and mechanism of Ershiwuwei Zhenzhu Pills on cerebral apoplexy rats].

To study the protective effect of Ershiwuwei Zhenzhu Pills on ischemic stroke rats. Ninety 4-weeks-old SPF male SD rats were randomly divided into 6 groups(n=15):sham operation group, model group, nimodipine group(12 mg·kg~(-1)), Ershiwuwei Zhenzhu Pills high-dose group(400 mg·kg~(-1)), Ershiwuwei Zhenzhu Pills medium-dose group(200 mg·kg~(-1)), Ershiwuwei Zhenzhu Pills low-dose group(100 mg·kg~(-1)).The permanent middle cerebral artery occlusion model(PMCAO) was established in the model group, nimodipine group, and Ershiwuwei Zhenzhu Pills groups by the improved thread plug method, while the sham operation group did not insert the thread plug.Nimodipine group and Ershiwuwei Zhenzhu Pills groups were given intragastric administration once a day for 24 days before the modeling operation, and once 1 hour before the modeling operation, while sham operation group and model group were given equal volumes of distilled water.The neuroethology of the surviving rats was measured; The volume of cerebral infarction in rats was measured by TTC method; The histopathology of rat brain was observed by HE method; The expression levels of tumor necrosis factor α(TNF-α),interleukin-1ß(IL-1ß),interleukin-6(IL-6),malondialdehyde(MDA),superoxide dismutase(SOD) and catalase(CAT) in serum were detected by ELISA;The mRNA expressions of Notch 1,Jagged 1,Hes 1 and Bcl-2 in rat brain were detected by RT-PCR;Western blot was used to detect the expression levels of caspase-3 protein in rat brain; the expression levels of vascular endothelial growth factor(VEGF) and CD34 positive cells in rat brain were detected by immunofluorescence.The low, medium and high dose groups of Ershiwuwei Zhenzhu Pills and nimodipine group could significantly reduce the neurobehavioral score and cerebral infarction volume of rats with permanent middle cerebral artery occlusion, reduce the morphological changes of nerve cells, decrease the expression of TNF-α,IL-1ß and IL-6 in rat serum, increase the activity of SOD and CAT,and reduce the level of MDA.Furthermore, the expression levels of Notch l, Jagged l, Hes l and Bcl-2 mRNA were significantly increased, and the expression level of caspase-3 protein was decreased.Meanwhile, the number of VEGF and CD34 positive cells increased in the treatment group.The differences were statistically significant. Ershiwuwei Zhenzhu Pills has a protective effect on ischemic stroke rats, and its mechanism may be related to anti-inflammation, anti-oxidation, promotion of nerve cell proliferation, inhibition nerve cell apoptosis and promotion of angiogenesis.

Comparison of the Effect and Complications of Sequential Bilateral Arthroplasty at Different Time Intervals and Simultaneous Bilateral Total Knee Arthroplasty: A Single-Centre Retrospective Cohort Study.

INTRODUCTION: To the authors' knowledge, there is no current consensus regarding the optimal interprocedural interval for patients who have undergone bilateral total knee arthroplasty (BTKA). The purpose of this study is to evaluate complication rates and functional outcome in patients who have undergone BTKA (simultaneous or sequential at different time intervals), and to determine an optimal time frame for the second knee. METHODS: Data from 315 patients who were able to tolerate simultaneous BTKA according to the anaesthesiologist's preoperative assessment between 2016 and 2020 were analysed retrospectively. According to the operative time interval, they were divided into simultaneous, ≤ 1-month sequential, 1- to 3-month sequential, and ≥ 3-month sequential BTKA groups. The primary outcomes were revision and readmission rates during the follow-up period, and the secondary outcomes were hospital length of stay (LOS), transfusion and postoperative complications. RESULTS: There was no difference in the implant survival or readmission rate between the groups (p > 0.05). Multivariable linear regression showed that interprocedural interval and body mass index (BMI) affected LOS; the LOS of simultaneous BTKA was the shortest (p < 0.05). BMI was associated with an increased LOS of 0.25 days (95% CI 0.02-0.48, p = 0.03). A modified Poisson regression model showed that the odds of blood transfusion were reduced in sequential BTKAs of any interval (p < 0.05), and preoperative haemoglobin (Hb) was also a risk factor (RR 0.96, 95% CI 0.95-0.98, p < 0.001). The interprocedural interval was not a risk factor for postoperative cardiovascular and cerebrovascular complications. CONCLUSION: For appropriate patients, simultaneous BTKA is beneficial. However, higher preoperative haemoglobin was required to mitigate the high blood transfusion rate associated with simultaneous surgeries. If suitable patients refuse simultaneous BTKA for other non-medical reasons, sequential BTKA with an interval greater than 1 month is recommended.

Pharyngeal perforation following laryngoscopy in a patient with dysphagia secondary to diffuse idiopathic skeletal hyperostosis: A case report.

RATIONALE: Dysphagia is a common presenting symptom in elderly people. Nevertheless, dysphagia resulting from diffuse idiopathic skeletal hyperostosis (DISH) of patients' cervical spine may be due to several factors. Despite computed tomography scan showing the size and shape of osteophytes, endoscopy may be necessary to exclude other intrinsic causes of dysphagia. Due to the anatomic variation of the pharynx secondary to DISH, patients undergoing endoscopy are at risk of perforation. Once perforation occurs, inappropriate treatments may finally lead to an irretrievable outcome. PATIENT CONCERNS: A 58-year-old male patient with a 20-day history of dysphagia initially visited an ear-nose-throat (ENT) doctor. He had no neck pain and no other history of cervical disease. DIAGNOSIS: This patient with dysphagia due to DISH of the cervical spine underwent laryngoscopy to exclude other causes. Pharyngeal perforation resulted as a complication of the procedure. INTERVENTIONS: The patient underwent laryngoscopy and biopsy by an ENT doctor to exclude intrinsic causes. After the procedure, a perforation was formed on the posterior wall of the pharynx. Conservative management, that is, 1 week of nothing per oral, and 1 month of antibiotics, was adopted. On the 30th day after the examination, the patient was voluntarily discharged from the hospital and recommended to take antibiotics orally. OUTCOMES: On the 56th day, the patient experienced fever and neck pain. Magnetic resonance imaging showed that the cervical vertebral bodies and spinal cord were infected. On the midday of the 60th day, the patient had a failed resuscitation and died. LESSONS: DISH involving the cervical spine is a complicated cause of dysphagia. Due to the anatomic variation of the pharynx secondary to DISH, patients undergoing endoscopy are at risk of perforation. If other intrinsic causes of dysphagia have to be excluded with the aid of endoscopy, plain films and computed tomography images should be read carefully first. To minimize the risk of perforation, it is necessary to perform endoscopy extremely carefully, especially biopsy. Once perforation occurs, operative treatment may be more appropriate and effective.

Overexpression of RBM10 induces osteosarcoma cell apoptosis and inhibits cell proliferation and migration.

Osteosarcoma is the most common malignant bone tumor with high incidence in adolescence and poor prognosis. RBM10, a member of RBPs, was reported to be a tumor suppressor in many kinds of cancers. However, the roles of RBM10 in osteosarcoma remain unknown. In this study, we found that overexpression of RBM10 decreased osteosarcoma cell proliferation and colony formation in soft agar, and inhibited osteosarcoma cell migration and invasion. Our results also revealed that RBM10 overexpression induced osteosarcoma cell apoptosis via the inhibition of Bcl-2, the activation of caspase-3, and the transcription and production of TNF-α. Our results indicated that RBM10 acts as a tumor suppressor in osteosarcoma. This could enable to define a new strategy for diagnosis and treatment of patients with osteosarcoma.

MicroRNA-100 resensitizes resistant chondrosarcoma cells to cisplatin through direct targeting of mTOR.

Chondrosarcomas are malignant cartilage-forming tumors of bone which exhibit resistance to both chemotherapy and radiation treatment. miRNAs have been well demonstrated to regulate gene expression and play essential roles in a variety of biological processes, including proliferation, differentiation, migration, cell cycling and apoptosis. In this study, we obtained evidence that miR-100 acts as a tumor suppressor in human chondrosarcomas. Interestingly, cisplatin resistant chondrosarcoma cells exhibit decreased expression of miR-100 compared with parental cells. In addition, we identified mTOR as a direct target of miR-100. Overexpression of miR-100 complementary pairs to the 3' untranslated region (UTR) of mTOR, resulted in sensitization of cisplatin resistant cells to cisplatin. Moreover, recovery of the mTOR pathway by overexpression of S6K desensitized the chondrosarcoma cells to cisplatin, suggesting the miR-100-mediated sensitization to cisplatin dependent on inhibition of mTOR. In summary, the present studies highlight miR-100 as a tumor suppressor in chondrosarcoma contributing to anti-chemoresistance. Overexpression of miR-100 might be exploited as a therapeutic strategy along with cisplatin-based combined chemotherapy for the treatment of clinical chondrosarcoma patients.