[Effect of acupuncture on HIF-1α/NLRP3 inflammatory signaling pathway in the rats with cerebral ischemia-reperfusion injury].

OBJECTIVE: To observe the effects of Xingnao Kaiqiao (regaining consciousness and opening orifices) acupuncture therapy on the expression of hypoxia-inducible factor 1α (HIF-1α) and Nod-like receptor protein 3 (NLRP3) in cerebral ischemia-reperfusion rats, and to explore the mechanism of acupuncture against cerebral ischemia-reperfusion injury. METHODS: Seventy-two male SD rats were randomly divided into a sham-operation group, a model group, an acupuncture group and a non-point acupuncture group, with 18 rats in each one. Using modified Longa thread embolization method, the rat model of acute focal cerebral ischemia was prepared; and after 2 h ischemia, the reperfusion was performed to prepared the model of cerebral ischemia-reperfusion. Immediately after reperfusion, Xingnao Kaiqiao acupuncture method was applied to bilateral "Neiguan" (PC 6) and "Shuigou" (GV 26) in the acupuncture group, while in the non-point acupuncture group, acupuncture was delivered at non-points and all of the needles were retained for 30 min in these two groups. The samples were collected 24 h after reperfusion in the rats of each group. Zea-Longa neurological deficit score was used to evaluate the degree of cerebral neurological impairment, TTC staining was adopted to observe the volume percentage of cerebral infarction, HE staining was provided to observe the morphological changes of brain, and Western blot was applied for detecting the expression of HIF-1α and NLRP3 proteins in the cerebral cortex on the right side. RESULTS: Compared with the sham-operation group, neurological deficit score and volume percentage of cerebral infarction were increased in the model group (P<0.01), and HIF-1α and NLRP3 protein expression was elevated (P<0.01). Compared with the model group, neurological deficit score and volume percentage of cerebral infarction were decreased (P<0.01), and HIF-1α and NLRP3 protein expression was lower (P<0.01) in the acupuncture group. There was no significant difference in above indexes in the non-point acupuncture group compared with the model group (P>0.05). Compared with the sham-operation group, the brain tissue of the rats in the model group and the non-point acupuncture group was loose and edema, and the nuclei were shriveled. The brain tissue morphology in the acupuncture group was similar to that of the sham-operation group. CONCLUSION: Acupuncture can alleviate cerebral ischemia-reperfusion injury, and its mechanism may be related to the regulation of HIF-1α/NLRP3 signaling pathway to attenuate inflammatory response.

Gut microbiota-derived melatonin from Puerariae Lobatae Radix-resistant starch supplementation attenuates ischemic stroke injury via a positive microbial co-occurrence pattern.

Ischemic stroke is closely associated with gut microbiota dysbiosis and intestinal barrier dysfunction. Prebiotic intervention could modulate the intestinal microbiota, thus considered a practical strategy for neurological disorders. Puerariae Lobatae Radix-resistant starch (PLR-RS) is a potential novel prebiotic; however, its role in ischemic stroke remains unknown. This study aimed to clarify the effects and underlying mechanisms of PLR-RS in ischemic stroke. Middle cerebral artery occlusion surgery was performed to establish a model of ischemic stroke in rats. After gavage for 14 days, PLR-RS attenuated ischemic stroke-induced brain impairment and gut barrier dysfunction. Moreover, PLR-RS rescued gut microbiota dysbiosis and enriched Akkermansia and Bifidobacterium. We transplanted the fecal microbiota from PLR-RS-treated rats into rats with ischemic stroke and found that the brain and colon damage were also ameliorated. Notably, we found that PLR-RS promoted the gut microbiota to produce a higher level of melatonin. Intriguingly, exogenous gavage of melatonin attenuated ischemic stroke injury. In particular, melatonin attenuated brain impairment via a positive co-occurrence pattern in the intestinal microecology. Specific beneficial bacteria served as leaders or keystone species to promoted gut homeostasis, such as Enterobacter, Bacteroidales_S24-7_group, Prevotella_9, Ruminococcaceae and Lachnospiraceae. Thus, this new underlying mechanism could explain that the therapeutic efficacy of PLR-RS on ischemic stroke at least partly attributed to gut microbiota-derived melatonin. In summary, improving intestinal microecology by prebiotic intervention and melatonin supplementation in the gut were found to be effective therapies for ischemic stroke.

[Acupuncture regulates autophagic flux to antagonize cerebral ischemic injury in rats].

OBJECTIVE: To observe the effect of acupuncture on the cerebral infarct volume and expressions of Beclin1, microtubule-associated protein 1 light chain 3 (LC3) and p62 proteins related to cell autophagy in rats with cerebral ischemia (CI), so as to explore its mechanisms underlying improvement of CI injury. METHODS: Male SD rats were randomized into 3 groups: sham operation, model and acupuncture which were further divided into 4 subgroups according to different ischemia time-points: 3, 6, 12 and 24 h (n=7 in each subgroup). The CI model was established by occlusion of the middle cerebral artery (MCAO) with surgical suture-embolus. For rats of the acupuncture group, acupuncture was applied to "Shuigou" (GV26) and bilateral "Neiguan" (PC6), and twirled for 1-3 min every time, 10 times altogether, and kept for 30 min. The neurological deficit score accoding to Longa's method was used for assessing the neurological function. The CI volume was measured after 2, 3, 5-triphenyltetrazolium chloride staining. The expression levels of autophagy-related proteins Beclin1，LC3 and p62 in the brain tissue were detected using Western blot. RESULTS: Compared with those of the sham operation group，the neurological deficit scores at 2, 3, 6， 12 and 24 h after CI, and the infarct volumes, the expression levels of Beclin1 and the ratios of LC3-Ⅱ/LC3-Ⅰ at 3, 6, 12 and 24 h were considerably increased (P<0.01, P<0.05), and the expression levels of p62 at 3, 6, 12 and 24 h were significantly decreased (P<0.01) in the model group. Relevant to the model group, acupuncture stimulation of GV26 and PC6 induced an obvious decrease in the neurological deficit scores at 6, 12 and 24 h, CI volumes at 3, 6, 12 and 24 h, and the expression levels of Beclin1 and the ratios of LC3-Ⅱ/LC3-Ⅰ both at 6 and 12 h (P<0.01, P<0.05), and an evident increase in the expression levels of p62 at 6, 12 and 24 h after CI (P<0.05, P<0.01). CONCLUSION: Acupuncture stimulation of GV26 and PC6 can reduce the CI volume and improve neurological function in CI rats， which may be related to its efficacy in down-regulating the expression of Beclin1 and the ratio of LC3-Ⅱ/LC3-Ⅰ, and up-regulating the expression of p62 in the ischemic brain tissue， thereby improving autophagy flux.

Developing an AI-assisted clinical decision support system to enhance in-patient holistic health care.

Holistic health care (HHC) is a synonym for complete patient care, and as such an efficient clinical decision support system (CDSS) for HHC is critical to support the judgement of physician's decision in response of patient's physical, emotional, social, economic, and spiritual needs. The field of artificial intelligence (AI) has evolved considerably in the past decades and many AI applications have been deployed in various contexts. Therefore, this study aims to propose an AI-assisted CDSS model that predicts patients in need of HHC and applies an improved recurrent neural network (RNN) model, long short-term memory (LSTM) for the prediction. The data sources include in-patient's comorbidity status and daily vital sign attributes such as blood pressure, heart rate, oxygen prescription, etc. A two-year dataset consisting of 121 thousand anonymized patient cases with 890 thousand physiological medical records was obtained from a medical center in Taiwan for system evaluation. Comparing with the rule-based expert system, the proposed AI-assisted CDSS improves sensitivity from 26.44% to 80.84% and specificity from 99.23% to 99.95%. The experimental results demonstrate that an AI-assisted CDSS could efficiently predict HHC patients.

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.

[Acupuncture intervention prolongates thrombolysis time window by regulating cerebral cortex angiogenesis-related genes and proteins in cerebral infarction rats].

OBJECTIVE: To observe the effect of acupuncture on the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and endostatin (Endostatin, ES) mRNAs and proteins (angiogenesis related factors) in the ischemic penumbra region in rats with cerebral infarction (CI), so as to explore its underlying mechanisms in prolonging the time window of thrombolysis therapy for CI. METHODS: A total of 48 male SD rats were randomly divided into sham operation, model, medication (6 h thrombolysis) and acupuncture (Acupunct)+medication groups (n=12 in each group). The CI model was established using modified auto-thrombus method. Six hours after thrombolysis, recombinant human tissue plasminogen activator (rt-PA,10 mg/kg) was given to rats of the thrombolysis group through tail vein. Acupuncture was applied at "Shuigou"(CV26) and bilateral "Neiguan" (PC6) 2 h after successful modeling, and the needles were retained for 30 minutes. Cerebral blood flow (CBF) was monitored during modeling in each group, and the neurological deficit score (0-7 points) was given 2 h and 24 h after successful modeling according to Bederson's methods. The cerebral infarction volume was observed after triphenyltetrazole chloride (TTC) staining. The protein and mRNA expression levels of VEGF, bFGF and ES in the ischemic penumbra region of the right cerebral cortex were detected by Western blot and real-time PCR, separately. RESULTS: The neurological deficit score at both 2 h and 24 h after modeling, percentage of cerebral infarction volume, and the expression levels of VEGF, bFGF and ES proteins and mRNAs in the model group were significantly higher than those of the sham operation group (P<0.01, P < 0.05). Compared with the model group, the neurological deficit score 24 h (not at 2 h) after modeling and percentage of cerebral infarction volume, and the expression levels of ES protein and mRNA in the Acupunct+medication group (not in the medication group) were notably lower (P<0.05, P<0.01), while the expression levels of VEGF and bFGF proteins and mRNAs in the Acupunct +medication group (not in the medication group) were considerably higher (P<0.01, P<0.05). No significant differences were found between medication and model groups in the CI percentage, VEGF, bFGF and ES proteins and mRNAs (P>0.05). The therapeutic effect of Acupunct +medication group was significantly superior to that of medication in lowering neurological deficit score, percentage of CI volume and expression of ES protein and mRNA and in up-regulating the expression of VEGF and bFGF proteins and mRNAs (P<0.05, P<0.01). CONCLUSION: Acupuncture and timely intervention can prolong the time window of thrombolysis in CI rats, which may be related to its function in up-regulating the expression of VEGF and bFGF mRNAs and proteins and in down-regulating the expression of ES mRNA and protein in ischemic cerebral cortex.

Comparative study on the intestinal absorption of three gastrodin analogues via the glucose transport pathway.

Gastrodin is the main active constituent of Tianma, a famous traditional Chinese herbal medicine. Our previous research has found that gastrodin is absorbed rapidly in the intestine by the sodium-dependent glucose transporter 1 (SGLT1). In the current report, gastrodin is the best glycoside compound absorbed via the glucose transport pathway. This study aimed to investigate the effect of the slight difference in chemical structure on the drug intestinal absorption via the glucose transport pathway. Traditional biopharmaceutical and computer-aided molecular docking methods were used to evaluate the intestinal absorption characteristics of three gastrodin analogues, namely, salicin, arbutin and 4-methoxyphenyl-ß-D-glucoside (4-MG). The oil-water partition coefficient (logP) experiments showed that the logP values of the gastrodin analogues followed the order: 4-MG > salicin > arbutin. In vitro Caco-2 cell transport experiments demonstrated that the apparent permeability coefficient (Papp) value of arbutin was higher than those of salicin and 4-MG. In situ single-pass intestinal perfusion experiments showed that the absorption of arbutin and 4-MG was better than that of salicin and that the absorption of the three compounds in the colon was lower than that in the small intestine. Quantitative real-time polymerase chain reaction results confirmed that the SGLT1 mRNA expression in the small intestine of rats was obviously higher than that in the colon of rats. In vivo pharmacokinetic experiments demonstrated that the oral bioavailability of salicin was lower than those of arbutin and 4-MG. In vitro and in vivo experiments showed that glucose or phlorizin (SGLT1 inhibitor) could decrease the intestinal absorption of the three compounds. Contrary to the above biopharmaceutical experiments, the computer-aided molecular docking test showed that the affinity of salicin to the vSGLT receptor was stronger than those of arbutin and 4-MG. In conclusion, the SGLT1 can facilitate the intestinal absorption of salicin, arbutin and 4-MG, and the slight difference in chemical structure can affect absorption.

[Large- scale prospective clinical study on prophylactic intervention of COVID-19 in community population using Huoxiang Zhengqi Oral Liquid and Jinhao Jiere Granules].

To scientifically evaluate the intervention effect of Chinese medicine preventive administration(combined use of Huo-xiang Zhengqi Oral Liquid and Jinhao Jiere Granules) on community population in the case of coronavirus disease 2019(COVID-19), a large cohort, prospective, randomized, and parallel-controlled clinical study was conducted. Total 22 065 subjects were included and randomly divided into 2 groups. The non-intervention group was given health guidance only, while the traditional Chinese medicine(TCM) intervention group was given two coordinated TCM in addition to health guidance. The medical instructions were as follows. Huoxiang Zhengqi Oral Liquid: oral before meals, 10 mL/time, 2 times/day, a course of 5 days. Jinhao Jiere Granules: dissolve in boiling water and take after meals, 8 g/time, 2 times/day, a course of 5 days, followed up for 14 days, respectively. The study found that with the intake of medication, the incidence rate of TCM intervention group was basically maintained at a low and continuous stable level(0.01%-0.02%), while the non-intervention group showed an overall trend of continuous growth(0.02%-0.18%) from 3 to 14 days. No suspected or confirmed COVID-19 case occurred in either group. There were 2 cases of colds in the TCM intervention group and 26 cases in the non-intervention group. The incidence of colds in the TCM intervention group was significantly lower(P<0.05) than that in the non-intervention group. In the population of 16-60 years old, the incidence rate of non-intervention and intervention groups were 0.01% and 0.25%, respectively. The difference of colds incidence between the two groups was statistically significant(P<0.05). In the population older than 60 years old, they were 0.04% and 0.21%, respectively. The incidence of colds in the non-intervention group was higher than that in the intervention group, but not reaching statistical difference. The protection rate of TCM for the whole population was 91.8%, especially for the population of age 16-60(95.0%). It was suggested that TCM intervention(combined use of Huoxiang Zhengqi Oral Liquid and Jinhao Jiere Granules) could effectively protect community residents against respiratory diseases, such as colds, which was worthy of promotion in the community. In addition, in terms of safety, the incidence of adverse events and adverse reactions in the TCM intervention group was relatively low, which was basically consistent with the drug instructions.

Metabolism and Toxicity of Emodin: Genome-Wide Association Studies Reveal Hepatocyte Nuclear Factor 4α Regulates UGT2B7 and Emodin Glucuronidation.

Emodin is the main toxic component in Chinese medicinal herbs such as rhubarb. Our previous studies demonstrated that genetic polymorphisms of UDP-glucuronosyltransferase 2B7 (UGT2B7) had an effect on the glucuronidation and detoxification of emodin. This study aimed to reveal the transcriptional regulation mechanism of UGT2B7 on emodin glucuronidation and its effect on toxicity. Emodin glucuronic activity and genome and transcriptome data were obtained from 36 clinical human kidney tissues. The genome-wide association studies (GWAS) identified that four single nucleotide polymorphisms (SNPs) (rs6093966, rs2868094, rs2071197, and rs6073433), which were located on the hepatocyte nuclear factor 4α (HNF4A) gene, were significantly associated with the emodin glucuronidation (p < 0.05). Notably, rs2071197 was significantly associated with the gene expression of HNF4A and UGT2B7 and the glucuronidation of emodin. The gene expression of HNF4A showed a high correlation with UGT2B7 (R2 = 0.721, p = 5.83 × 10-11). The luciferase activity was increased 7.68-fold in 293T cells and 2.03-fold in HepG2 cells, confirming a significant transcriptional activation of UGT2B7 promoter by HNF4A. The knockdown of HNF4A in HepG2 cells (36.6%) led to a significant decrease of UGT2B7 (19.8%) and higher cytotoxicity (p < 0.05). The overexpression of HNF4A in HepG2 cells (31.2%) led to a significant increase of UGT2B7 (24.4%) and improved cell viability (p < 0.05). Besides, HNF4A and UGT2B7 were both decreased in HepG2 cells and rats after treatment with emodin. In conclusion, emodin used long term or in high doses could inhibit the expression of HNF4A, thereby reducing the expression of UGT2B7 and causing hepatotoxicity.

Glucuronidation and its effect on the bioactivity of amentoflavone, a biflavonoid from Ginkgo biloba leaves.

OBJECTIVES: Ginkgo biloba leaves contain amentoflavone (AMF), a dietary flavonoid that possesses antioxidant and anticancer activity. Flavonoids are extensively subjected to glucuronidation. This study aimed to determine the metabolic profile of AMF and the effect of glucuronidation on AMF bioactivity. METHODS: A pharmacokinetic study was conducted to determine the plasma concentrations of AMF and its metabolites. The metabolic profile of AMF was elucidated using different species of microsomes. The antioxidant activity of AMF metabolites was determined using DPPH/ABTS radical and nitric oxide assays. The anticancer activity of AMF metabolites was evaluated in U87MG/U251 cells. KEY FINDINGS: Pharmacokinetic studies indicated that the oral bioavailability of AMF was 0.06 ± 0.04%, and the area under the curve of the glucuronidated AMF metabolites (410.938 ± 62.219 ng/ml h) was significantly higher than that of AMF (194.509 ± 16.915 ng/ml h). UGT1A1 and UGT1A3 greatly metabolized AMF. No significant difference was observed in the antioxidant activity between AMF and its metabolites. The anticancer activity of AMF metabolites significantly decreased. CONCLUSIONS: A low AMF bioavailability was due to extensive glucuronidation, which was mediated by UGT1A1 and UGT1A3. Glucuronidated AMF metabolites had the same antioxidant but had a lower anticancer activity than that of AMF.

Versatile cationic liposomes for RIP3 overexpression in colon cancer therapy and RIP3 downregulation in acute pancreatitis therapy.

Because the induction of strong host antitumor responses plays a very important role in antitumor therapy, identifying effective approaches to elicit immunogenic cell death could have important implications. RIP3-dependent necroptotic cancer cells have been reported to release damage-associated molecular patterns and enhance antitumor immunity. In this study, hyaluronic acid-conjugated cationic liposomes (DOTAP/DOPE/PEG-DSPE/CHOL) (HA-P-LP) were prepared as a vector for mRIP3-pDNA overexpression in tumours. Compared with standard cationic liposomes, this vector markedly increased cellular gene internalisation in vitro, enhanced the tumour-targeting effect in vivo and exhibited a significant antitumor effect in combination with adjuvant chloroquine. Considering the dramatic increase in RIP3 under the pathological condition of pancreatitis and the correlation between pancreatitis and necroptosis, non-HA-conjugated liposomes with the same formulation loaded with shRNA mRIP3-pDNA effectively controlled the disease by decreasing the serum amylase concentration and inflammatory cell infiltration. The versatile cationic liposomes loaded with plasmids with opposing functions in this study provide a new concept and method for both tumour therapy and pancreatitis therapy.

Transplantation of fecal microbiota rich in short chain fatty acids and butyric acid treat cerebral ischemic stroke by regulating gut microbiota.

The gut microbiota and its short chain fatty acid (SCFA) metabolites have been established to play an important protective role against neurodegenerative diseases. Our previous study demonstrated that cerebral ischemic stroke triggers dysfunctional gut microbiota and increased intestinal permeability. In this study, we aimed to clarify the mechanism by which gut microbiota and SCFAs can treat cerebral ischemic stroke in rat middle cerebral artery occlusion models and use the information to develop new therapies. Our results show that oral administration of non-absorbable antibiotics reduced neurological impairment and the cerebral infarct volume, relieved cerebral edemas, and decreased blood lipid levels by altering the gut microbiota. We also found that ischemic stroke decreased intestinal levels of SCFAs. And that transplanting fecal microbiota rich in these metabolites was an effective means of treating the condition. Compared with other SCFAs, butyric acid showed the highest negative correlation with ischemic stroke. Supplementation with butyric acid treated models of ischemic stroke effectively by remodeling the gut microbiota, enriching the beneficial Lactobacillus, and repairing the leaky gut. In conclusion, interfering with the gut microbiota by transplanting fecal bacteria rich in SCFAs and supplementing with butyric acid were found to be effective treatments for cerebral ischemic stroke.

A novel macrophage-mediated biomimetic delivery system with NIR-triggered release for prostate cancer therapy.

BACKGROUND: Macrophages with tumor-tropic migratory properties can serve as a cellular carrier to enhance the efficacy of anti neoplastic agents. However, limited drug loading (DL) and insufficient drug release at the tumor site remain the main obstacles in developing macrophage-based delivery systems. In this study, we constructed a biomimetic delivery system (BDS) by loading doxorubicin (DOX)-loaded reduced graphene oxide (rGO) into a mouse macrophage-like cell line (RAW264.7), hoping that the newly constructed BDS could perfectly combine the tumor-tropic ability of macrophages and the photothermal property of rGO. RESULTS: At the same DOX concentration, the macrophages could absorb more DOX/PEG-BPEI-rGO than free DOX. The tumor-tropic capacity of RAW264.7 cells towards RM-1 mouse prostate cancer cells did not undergo significant change after drug loading in vitro and in vivo. PEG-BPEI-rGO encapsulated in the macrophages could effectively convert the absorbed near-infrared light into heat energy, causing rapid release of DOX. The BDS showed excellent anti-tumor efficacy in vivo. CONCLUSIONS: The BDS that we developed in this study had the following characteristic features: active targeting of tumor cells, stimuli-release triggered by near-infrared laser (NIR), and effective combination of chemotherapy and photothermotherapy. Using the photothermal effect produced by PEG-BPEI-rGO and DOX released from the macrophages upon NIR irradiation, MAs-DOX/PEG-BPEI-rGO exhibited a significant inhibitory effect on tumor growth.

Puerariae Lobatae Radix with chuanxiong Rhizoma for treatment of cerebral ischemic stroke by remodeling gut microbiota to regulate the brain-gut barriers.

The combination of Puerariae Lobatae Radix (PLR) and Chuanxiong Rhizoma (CXR) is commonly used to treat cerebrovascular diseases. This work aimed to clarify the mechanisms of their action in treating cerebral ischemic stroke from the perspective of gut microecology. The PLR and CXR combination effectively improved the neurological function, reduced the cerebral infarction and relieved the complications of cerebral ischemic stroke, including dyslipidemia, increased blood viscosity and thrombotic risk. Cerebral ischemic stroke triggered gut microbial disturbances by enriching pathogens and opportunistic microorganisms, including Bacteroides, Escherichia_Shigella, Haemophilus, Eubacterium_nodatum_group, Collinsella, Enterococcus, Proteus, Alistipes, Klebsiella, Shuttleworthia and Faecalibacterium. Cerebral ischemic stroke also increased the intestinal permeability, disrupted the gut barrier and caused intestinal microbial translocation. Occludin, claudin-5 and ZO-1 levels in the brain-gut barriers showed a high positive correlation. However, the combination remodeled the gut microecology by modulating endogenous bacteria whose effects may mitigate cerebral damage, such as Alloprevotella, Ruminococcaceae, Oscillospira, Lachnospiraceae_NK4B4_group, Akkermansia and Megasphaera, protected the brain-gut barriers by increasing claudin-5 and ZO-1 levels; and weakened the gut microbiota translocation by decreasing diamine oxidase, lipopolysaccharide and d-lactate. Although nimodipine effectively reduced the cerebral infarction, it did not relieve the gut microbiota dysbiosis and instead aggravated the gut barrier disruption and microbiota translocation. In conclusion, cerebral ischemic stroke caused gut microbiota dysbiosis, increased intestinal permeability, disrupted the gut barrier and triggered gut microbiota translocation. The PLR and CXR combination was an effective treatment for cerebral ischemic stroke that relieved the gut microbiota dysbiosis and brain-gut barriers disruption.

Asperuloside and Asperulosidic Acid Exert an Anti-Inflammatory Effect via Suppression of the NF-κB and MAPK Signaling Pathways in LPS-Induced RAW 264.7 Macrophages.

Hedyotis diffusa is a folk herb that is used for treating inflammation-related diseases in Asia. Previous studies have found that iridoids in H. diffusa play an important role in its anti-inflammatory activity. This study aimed to investigate the anti-inflammatory effect and potential mechanism of five iridoids (asperuloside (ASP), asperulosidic acid (ASPA), desacetyl asperulosidic acid (DAA), scandoside methyl ester (SME), and E-6-O-p-coumaroyl scandoside methyl ester (CSME)) that are presented in H. diffusa using lipopolysaccharide (LPS)-induced RAW 264.7 cells. ASP and ASPA significantly decreased the production of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in parallel with the inhibition of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), TNF-α, and IL-6 mRNA expression in LPS-induced RAW 264.7 cells. ASP treatment suppressed the phosphorylation of the inhibitors of nuclear factor-kappaB alpha (IκB-α), p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). The inhibitory effect of ASPA was similar to that of ASP, except for p38 phosphorylation. In summary, the anti-inflammatory effects of ASP and ASPA are related to the inhibition of inflammatory cytokines and mediators via suppression of the NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways, which provides scientific evidence for the potential application of H. diffusa.

Involvement of UDP-glucuronosyltransferases in higenamine glucuronidation and the gender and species differences in liver.

OBJECTIVES: Higenamine (HG), an active ingredient of Aconite root in Chinese herbal medicine, is mainly metabolized by UDP-glucuronosyltransferases (UGT). However, the systematic glucuronidation of HG in humans remains unclear. The purpose of this study was to investigate the glucuronidation of HG. METHODS: 12 recombinant human UGT (rUGT) isozymes were used to characterize the HG glucuronidation. Liver microsomes from male and female mice, rats, guinea pigs, dogs, and humans were used to determine the species and gender differences using liquid chromatography-mass spectrometry. KEY FINDINGS: One monoglucuronide was detected in reactions catalyzed by rUGT1A6, rUGT1A8, rUGT1A9, also human and dog liver microsomes. UGT1A9 is the most important glucuronosyltransferase that metabolizes HG. Because carvacrol, a specific inhibitor of UGT1A9, can significantly decrease the glucuronidation of HG in Human liver microsomes and UGT1A9. HG metabolism by UGT1A9 described in Michaelis-Menten kinetics (Km=15.4 mM,Vmax=2.2 nmol/mg/min) and glucuronidation in liver microsomes were species dependent. Gender did not affect the kinetic parameters among species except in rats. CONCLUSIONS: UGT1A9 is a major isoenzyme responsible for the glucuronidation of HG in Human liver microsomes (HLMs). Dog may be an appropriate animal model to evaluate HG metabolism.

Regulation of DMT1 on autophagy and apoptosis in osteoblast.

Iron overload has recently been associated with the changes in the bone microstructure that occur in osteoporosis. However, the effect of iron overload on osteoblasts is unclear. The purpose of this study was to explore the function of divalent metal transporter 1 (DMT1) in the pathological processes of osteoporosis. Osteoblast hFOB1.19 cells were cultured in medium supplemented with different concentrations (0, 50, 100, 200, 300, 400, 500 µmol/L) of ferric ammonium citrate (FAC) as a donor of ferric ions. We used western blotting and immunofluorescence to determine the levels of DMT1 after treatment with FAC. Apoptosis was evaluated by detecting the levels of cleaved caspase 3, BCL2, and BAX with western blotting. Autophagy was evaluated by detecting the levels of LC3 with western blotting and immunofluorescence. Beclin-1 expression was also assessed with western blotting. The autophagy inhibitor 3-methyladenine was used to determine whether autophagy affects the apoptosis induced by FAC. Our results show that FAC increased the levels of DMT1, upregulated the expression of BCL2, and downregulated the apoptosis-related proteins cleaved caspase 3 and BAX. Both LC3I/LC3II levels and beclin-1 were also increased, indicating that FAC increases the accumulation of autophagosomes in hFOB1.19 cells. FAC-induced autophagy was increased by the apoptosis inhibitor 3-MA but was reduced in DMT1 shRNA hFOB1.19 cells. These results suggest that the increased expression of DMT1 induces iron overload and iron overload induces osteoblast autophagy and apoptosis, thus affecting the pathological processes of osteoporosis. Clarifying the mechanisms underlying the effects of DMT1 will allow the identification of novel targets for the prevention and treatment of osteoporosis.

Gut microbial diversity analysis using Illumina sequencing for functional dyspepsia with liver depression-spleen deficiency syndrome and the interventional Xiaoyaosan in a rat model.

AIM: To investigate gut microbial diversity and the interventional effect of Xiaoyaosan (XYS) in a rat model of functional dyspepsia (FD) with liver depression-spleen deficiency syndrome. METHODS: The FD with liver depression-spleen deficiency syndrome rat model was established through classic chronic mild unpredictable stimulation every day. XYS group rats received XYS 1 h before the stimulation. The models were assessed by parameters including state of the rat, weight, sucrose test result and open-field test result. After 3 wk, the stools of rats were collected and genomic DNA was extracted. PCR products of the V4 region of 16S rDNA were sequenced using a barcoded Illumina paired-end sequencing technique. The primary composition of the microbiome in the stool samples was determined and analyzed by cluster analysis. RESULTS: Rat models were successfully established, per data from rat state, weight and open-field test. The microbiomes contained 20 phyla from all samples. Firmicutes, Bacteroidetes, Proteobacteria, Cyanobacteria and Tenericutes were the most abundant taxonomic groups. The relative abundance of Firmicutes, Proteobacteria and Cyanobacteria in the model group was higher than that in the normal group. On the contrary, the relative abundance of Bacteroidetes in the model group was lower than that in the normal group. Upon XYS treatment, the relative abundance of all dysregulated phyla was restored to levels similar to those observed in the normal group. Abundance clustering heat map of phyla corroborated the taxonomic distribution. CONCLUSION: The microbiome relative abundance of FD rats with liver depression-spleen deficiency syndrome was significantly different from the normal cohort. XYS intervention may effectively adjust the gut dysbacteriosis in FD.

CNP signal pathway up-regulated in rectum of depressed rats and the interventional effect of Xiaoyaosan.

AIM: To investigate the distribution and expression of C-type natriuretic peptide (CNP)/natriuretic peptide receptor B (NPR-B) in the rectum of a rodent depression model and the interventional effect of Xiaoyaosan (XYS). METHODS: Male rats (n = 45) of clean grade (200 ± 20 g) were divided into five groups after one week of adaptive feeding: primary control, depression model, low dose XYS, middle dose XYS, and high dose XYS. The animal experiment continued for 3 wk. Primary controls were fed normally ad libitum. The rats of all other groups were raised in solitary and exposed to classic chronic mild unpredictable stimulation each day. XYS groups were perfused intragastrically with low dose, middle dose, and high dose XYS one hour before stimulation. Primary control and depression model groups were perfused intragastrically with normal saline under similar conditions as the XYS groups. Three weeks later, all rats were sacrificed, and the expression levels of CNP and NPR-B in rectum tissues were analyzed by immunohistochemistry, real-time polymerase chain reaction, and Western blotting. RESULTS: CNP and NPR-B were both expressed in the rectum tissues of all rats. However, the expression levels of CNP and NPR-B at both gene and protein levels in the depression model group were significantly higher when compared to the primary control group (n = 9; P < 0.01). XYS intervention markedly inhibited the expression levels of CNP and NPR-B in depressed rats. The expression levels of CNP and NPR-B in the high dose XYS group did not significantly differ from the expression levels in the primary control group. Additionally, the high and middle dose XYS groups (but not the low dose group) significantly exhibited lower CNP and NPR-B expression levels in the rectum tissues of the respectively treated rats compared to the untreated depression model cohort (n = 9; P < 0.01). CONCLUSION: The CNP/NPR-B pathway is upregulated in the rectum of depressed rats and may be one mechanism for depression-associated digestive disorders. XYS antagonizes this pathway at least partially.

Preparation and evaluation of sustained-release solid dispersions co-loading gastrodin with borneol as an oral brain-targeting enhancer.

Borneol is a traditional Chinese medicine that can promote drug absorption from the gastrointestinal tract and distribution to the brain. However, stomach irritation may occur when high doses of borneol are used. In the present work, gastrodin, the main bioactive ingredient of the traditional Chinese drug "Tianma" (Rhizoma Gastrodiae) was used as a model drug to explore reasonable application of borneol. Sustained-release solid dispersions (SRSDs) for co-loading gastrodin and borneol were prepared using ethylcellulose as a sustained release matrix and hydroxy-propyl methylcellulose as a retarder. The dispersion state of drug within the SRSDs was analyzed by using scanning electron microscopy, differential scanning calorimetry, and powder X-ray diffractometry. The results indicated that both gastrodin and borneol were molecularly dispersed in an amorphous form. Assay of in vitro drug release demonstrated that the dissolution profiles of gastrodin and borneol from the SRSDs both fitted the Higuchi model. Subsequently, gastric mucosa irritation and the brain targeting of the SRSDs were evaluated. Compared with the free mixture of gastrodin and borneol, brain targeting of SRSDs was slightly weaker (brain targeting index: 1.83 vs. 2.09), but stomach irritation obviously reduced. Sustained-release technology can be used to reduce stomach irritation caused by borneol while preserving sufficient transport capacity for oral brain-targeting drug delivery.