Decrease of morphine-CPP by sinomenine via mediation of tyrosine hydroxylase, NMDA receptor subunit 2B and opioid receptor in the zebrafish brain.

To study the effects of sinomenine on conditioned place preference (CPP) zebrafish induced by morphine and expression levels of intracephalic tyrosine hydroxylase (TH), NMDA receptor subunit 2B (NR2B), µ-opioid receptor (zfmor) and δ-opioid receptors (zfdor1 and zfdor2), morphine (40mg/kg) was administrated to zebrafish and the effect of CPP was detected in these zebrafish treated with sinomenine. The expression of TH and NR2B was detected by immunohistochemistry; and the mRNA expression of opioid receptors zfmor, zfdor1 and zfdor2 in the zebrafish brain was assayed by RT-qPCR. In the CPP test, morphine induced significant behavioral alteration, while pretreatment with sinomenine or methadone, resulted in decreased activity time in the morphine-paired compartment significantly. Morphine also increased the integral optical density value of TH- and NR2B-positive cells in the zebrafish brain, and reduced the amount of opioid receptors. However, the compound sinomenine could attenuate these effects. These findings demonstrate that sinomenine (80mg/kg) decreased the CPP effects of zebrafish induced by morphine significantly, downregulated expression of TH and NR2B, and upregulated µ-opioid (zfmor) and δ-opioid (zfdor1 and zfdor2) receptor expression in the CPP zebrafish brains.

Expression of miR-133a-5p and ROCK2 in Heart in Methamphetamine-Induced Rats and Intervention of Rhynchophylline.

INTRODUCTION: Rhynchophylline, as a traditional Chinese medicine, was used for the treatment of drug addiction. OBJECTIVE: To investigate miRNAs expression profile in the rat hearts of methamphetamine dependence and the intervention mechanisms of rhynchophylline. MATERIALS AND METHODS: This study detected the expression profile of miRNAs in the methamphetamine-induced rat hearts by microarray and verified the expression of miR-133a-5P and Rho-associated, coiled-coil containing protein kinase 2 (ROCK2) protein. RESULTS: After conditioned place preference training, methamphetamine significantly increased the time spent in the drug-paired compartment, while rhynchophylline and MK-801 could reduce the time. Cluster analysis results of miRNAs showed that compared with the control group, the expression of miR-133a-5p in methamphetamine-induced rat hearts was decreased significantly; rhynchophylline could significantly increase the expression of miR-133a-5p. The result was verified by real-time polymerase chain reaction. The results of target gene predictive software and related research showed that ROCK2 protein may be the target gene of miR-133a-5p. The immunohistochemistry results of heart tissues showed that the expression of ROCK2 protein was significantly upregulated in the methamphetamine group and downregulate in the rhynchophylline group; the difference between the MK-801 group and the methamphetamine group was not significant. The result of western blot was consistent with the immunohistochemistry. CONCLUSION: The active ingredient of Chinese herbal medicine rhynchophylline can effectively inhibit the formation of methamphetamine-dependent conditional place preference (CPP) effect in rats to some extent. MiR-133a-5p may participate in the cardioprotective effects of CPP rats by targeting ROCK2.

Use of an UHPLC-MS/MS Method for Determination of Kuraridin and Characterization of Its Metabolites in Rat Plasma after Oral Administration.

Kuraridin is an active natural prenylated flavonoid ingredient originating from the well-known traditional Chinese medicine Sophora flavescens Ait., that possesses various bioactivities, such as antitumor activity, PLCγ1 inhibitory activity, glycosidase inhibitory activity, etc. However, there is no report on the plasma metabolic profile and pharmacokinetic study of kuraridin. The current study was designed to use an ultra-performance liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS) method for the quantification and characterization metabolites in rat plasma after oral administration of kuraridin. A liquid-liquid extraction method with ethyl acetate-acetonitrile (1:3) was used to extract the kuraridin from rat plasma samples. The chromatographic separation was carried out on a Hypersil GOLD UHPLC C18 column equipped with a C18 guard cartridge using a gradient elution with organic solvent-water as mobile phase. Based on comparing the retention times with reference standards or on the basis of MS2 fragmentation behaviors, a total of 19 metabolites were identified or tentatively characterized from rat plasma. Under the optimized conditions, the method showed good linearity (r² > 0.99) over the ranges of 1-500 ng/mL for kuraridin. The inter- and intra-day precisions were less than 8.95%, and the accuracy was in the range of -6.27-6.48%. The recovery of kuraridin ranged from 90.1% to 100.4%. The developed UHPLC-MS/MS method was thus successfully applied in the qualitative of metabolites and quantitative analysis of kuraridin in rat plasma.

Sinomenine Protects Against Morphine Dependence through the NMDAR1/CAMKII/CREB Pathway: A Possible Role of Astrocyte-Derived Exosomes.

Sinomenine is a nonaddictive alkaloid used to prevent morphine dependence, even thoughits mechanism isnot fully understood. Astrocytes aggravate the pathological process in their neighboring cellsthrough exosomes in central nervous system diseases. However, the effect of sinomenine on astrocyte-derived exosomes for the amelioration of morphine dependence has not been reported yet. In this study, we found that sinomenine prevented the morphine-induced conditionedplace preference in mice. Sinomenine reduced the levels of cAMP and intracellular Ca2+ in morphine-treated SH-SY5Y cells. Moreover, sinomenine inhibited the expressions of p-NMDAR1/NMDAR1, p-CAMKII/CAMKII, and p-CREB/CREB in the hippocampusof morphine-dependent mice and SH-SY5Y cells. Furthermore, we found that sinomenine inhibitedthe morphine-induced activation of astrocytesin vivo and in vitro. Afterwards, exosomes were isolated from cultured primary astrocytes treated with phosphate buffer saline (PBS, ctl-exo), morphine (mor-exo), or morphine and sinomenine (Sino-exo). Subsequently, morphine-treated SH-SY5Y cells were treated with ctl-exo, mor-exo, and Sino-exo. Results showed that Sino-exo reduced the level of cAMP, intracellular Ca2+, and the expression of p-CAMKII/CAMKII and p-CREB/CREB in morphine-treated SH-SY5Y cells. In conclusion, we demonstrated that sinomenine exhibited protective effects against morphine dependencein vivo and in vitro through theNMDAR1/CAMKII/CREB pathway. Sinomenine-induced alterationof the function of astrocyte-derived exosomes may contribute to the antidependence effects of sinomenine in morphine dependence.

Effect of Sinomenine on the Morphine-Dependence and Related Neural Mechanisms in Mice.

Evidence suggests that the dopamine receptor rate-limiting enzyme, tyrosine hydroxylase (TH), and the glutamate receptor, N-methyl-D-aspartate receptor 2B (NR2B), contribute to morphine dependence. Previous studies show that chronic exposure to morphine changes the expression of opioid receptors. In this study, we focus on the effects of sinomenine on morphine-dependent mice and its related neural mechanisms. Conditioned place preference (CPP) mouse model was established using morphine (9 mg/kg, s.c.), and their expression levels of TH and NR2B were observed by immunohistochemistry. Moreover, their mu opioid receptor (MOR) and delta opioid receptor (DOR) contents were assessed using quantitative reverse transcription polymerase chain reaction. Results showed that high sinomenine dose (80 mg/kg) effectively attenuated the behavior of CPP mice and reversed increased expression levels of TH and NR2B induced by morphine. Moreover, compared with the morphine group, sinomenine up-regulated the content of MOR to a normal level but did not significantly affect the DOR expression. In summary, these data indicate that sinomenine can inhibit morphine dependence by increasing the expression levels of TH, NR2B, and MOR in the mouse brain; however, DOR may not contribute to this effect.

Simultaneous determination of wogonin, oroxylin a, schisandrin, paeoniflorin and emodin in rat serum by HPLC-MS/MS and application to pharmacokinetic studies.

Wogonin and oroxylin A in Scutellariae Radix, schisandrin in Chinensis Fructus, paeoniflorin in Moutan Cortex and emodin in Polygoni Cuspidate Rhizome et Radix are anti-inflammatory active compounds. A method for simultaneous determination of the five compounds in rat was developed and validated using high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). The separation was performed on a Symmetry C18 column (4.6 × 50 mm, 3.5 µm) with acetonitrile and 0.1% formic acid aqueous solution as the mobile phases. The detection was performed using multiple-reaction monitoring with electrospray ionization source in positive-negative ion mode. The calibration curves showed good linearity (r ≥ 0.9955). The lower limit of quantification (LLOQ) was 5 ng/mL for wogonin and schisandrin, 10 ng/mL for oroxylin A and emodin, and 15 ng/mL for paeoniflorin, respectively. The relative standard deviations of intraday and interday precisions were <11.49 and 14.28%, respectively. The extraction recoveries and matrix effects were acceptable. The analytes were stable under the experiment conditions. The validated method has been successfully applied to pharmacokinetic studies of the five compounds in rats after oral administration of Hu-gan-kan-kang-yuan capsule. This paper would be a valuable reference for pharmacokinetic studies of Chinese medicine preparations containing the five compounds.

Developing an Absorption-Based Quality Control Method for Hu-Gan-Kang-Yuan Capsules by UFLC-QTOF-MS/MS Screening and HPLC-DAD Quantitative Determination.

Traditional Chinese Medicine Preparations (TCMPs) contain massive numbers of ingredients responsible for their multiple efficacies. An absorption-based quality control method for complicated TCMPs using Hu-gan-kang-yuan Capsule (HGKYC) as an example was developed. To select proper chemical markers for quality control of HGKYC, an ultra-fast liquid chromatography (UFLC) coupled with electrospray ionization quadrupole time-off light mass spectrometry (UFLC-QTOF-MS/MS) method was used for the rapid separation and structural identification of the constituents in the HGKYC extract and the rat serum after oral administration of HGKYC. As a result, one hundred and seven prototype constituents including flavonoids, organic acid, phenylpropanoids, anthraquinones, saponins, alkaloids, terpenes, phenols and amino acids in HGKYC extract, and 43 compounds found in rat serum after oral administration of HGKYC were unambiguously identified or tentatively characterized by comparing retention times and MS information with those of authentic standards or available literature references. Finally, a simple, low-cost and effective method of simultaneous determination for baicalein, wogonin, paeonol and emodin in HGKYC was developed using high performance liquid chromatography coupled with a diode array detector. In conclusion, an absorption-based quality control pattern was developed and successfully used for evaluating HGKYC.

Protective effects of Sapindus mukorossi Gaertn against fatty liver disease induced by high fat diet in rats.

OBJECTIVES: Study the effects of alcohol extract of Sapindus mukorossi Gaertn (AESM) on the metabolism of blood fat, morphology of fenestrated liver sinusoidal endothelial cells (LSEC), and the ultrastructure of liver cells of the rats with non-alcoholic fatty liver disease (NAFLD). METHODS: Divide SD rats into control group, model group, simvastatin (7.2 mg/kg) group, and S.mukorossi Gaertn group with high dosage (0.5 g/kg), moderate dosage (0.1 g/kg), and low dosage (0.05 g/kg). After feeding with fat-rich nutrients for 3 weeks and establishing the model of hepatic adipose, conduct intragastric administration and provide the rats with fat-rich nutrients at the same time. At the 43rd day, take blood sample and measure aminotransferase and different indexes of blood fat; take hepatic tissue for pathological section, and observe the hepatic morphological patterns under light microscope; obtain and fix the hepatic tissue after injecting perfusate into the body, and observe the changes of fenestrated LSEC under scanning electron microscope; observe the ultrastructure of liver cells under transmission electron microscope. RESULTS: High-dosage alcohol extracts of S.mukorossi Gaertn can alleviate the AST, ALT, TC, TG, LDL, γ-GT, and ALP level, as well as raise the HDL and APN level in the serum of NAFLD-rat model. In addition, through the observation from light microscope and electron microscopes, the morphology of the hepatic tissue and liver cells as well as the recovery of the fenestrated LSEC in the treatment group has become normal. CONCLUSIONS: Alcohol extracts of S.mukorossi Gaertn can regulate the level of blood fat and improve the pathological changes of the hepatic tissues in NAFLD-rat model, which demonstrates the effects of down-regulating fat level and protecting liver.

Effect of rhynchophylline on conditioned place preference on expression of NR2B in methamphetamine-dependent mice.

OBJECTIVE: To study the effect of rhynchophylline on N-methyl d-aspartate receptor subtype 2B subunit in hippocampus of Methamphetamine-induced conditioned place preference (CPP) mice. METHODS: Place preference mice models were established by methamphetamine; the expression of NR2B was observed by immunohistochemistry technique and Western blot. RESULTS: Methamphetamine (4mg/kg)-induced place preference mice model was successfully established; ketamine (15mg/kg), rhynchophylline (40mg/kg) and rhynchophylline (80mg/kg) can eliminate place preference; Immunohistochemistry showed that the number of NR2B-positive neurons in hippocampus was increased in the methamphetamine model group, whereas less NR2B-positive neurons were found in the ketamine group, low and high dosage rhynchophylline group. Western blot showed that the expression of NR2B protein was significantly increased in the model group, whereas less expression was found in the ketamine group, low and high dosage rhynchophylline group. CONCLUSIONS: NR2B plays an important role in the formation of methamphetamine-induced place preference in mice. Rhynchophylline reversed the expression of NR2B in the hippocampus demonstrates the potential effect of mediates methamphetamine induced rewarding effect.

Neuroprotective effects of bilobalide on cerebral ischemia and reperfusion injury are associated with inhibition of pro-inflammatory mediator production and down-regulation of JNK1/2 and p38 MAPK activation.

BACKGROUND: Mitogen-activated protein kinase (MAPK) signaling pathways are implicated in inflammatory and apoptotic processes of cerebral ischemia and reperfusion (I/R) injury. Hence, MAPK pathways represent a promising therapeutic target. Exploring the full potential of inhibitors of MAPK pathways is a useful therapeutic strategy for ischemic stroke. Bilobalide, a predominant sesquiterpene trilactone constituent of Ginkgo biloba leaves, has been shown to exert powerful neuroprotective properties, which are closely related to both anti-inflammatory and anti-apoptotic pathways. We investigated the neuroprotective roles of bilobalide in the models of middle cerebral artery occlusion and reperfusion (MCAO/R) and oxygen-glucose deprivation and reoxygenation (OGD/R) of cerebral I/R injury. Moreover, we attempted to confirm the hypothesis that its protection effect is via modulation of pro-inflammatory mediators and MAPK pathways. METHODS: Male Sprague-Dawley rats were subjected to MCAO for 2 h followed by reperfusion for 24 h. Bilobalide was administered intraperitoneally 60 min before induction of middle cerebral artery occlusion (MCAO). After reperfusion, neurological deficit scores, infarct volume, infarct weight, and brain edema were assessed. Ischemic penumbrae of the cerebral cortex were harvested to determine superoxide dismutase (SOD), malondialdehyde (MDA), nitric oxide, TNF-α, interleukin 1ß (IL-1ß), p-ERK1/2, p-JNK1/2, and p-p38 MAPK concentration. Similarly, the influence of bilobalide on the expression of nitric oxide, TNF-α, IL-1ß, p-ERK1/2, p-JNK1/2, and p-p38 MAPK was also observed in an OGD/R in vitro model of I/R injury. RESULTS: Pretreatment with bilobalide (5, 10 mg/kg) significantly decreased neurological deficit scores, infarct volume, infarct weight, brain edema, and concentrations of MDA, nitric oxide, TNF-α, IL-1ß, and increased SOD activity. Furthermore, bilobalide (5, 10 mg/kg) pretreatment significantly down-regulated both p-JNK1/2 and p-p38 MAPK expression, whereas they had no effect on p-ERK1/2 expression in the ischemic penumbra. Supporting these observations in vivo, pretreatment with bilobalide (50, 100 µM) significantly down-regulated nitric oxide, TNF-α, IL-1ß, p-JNK1/2, and p-p38 MAPK expression, but did not change p-ERK1/2 expression in rat cortical neurons after OGD/R injury. CONCLUSIONS: These data indicate that the neuroprotective effects of bilobalide on cerebral I/R injury are associated with its inhibition of pro-inflammatory mediator production and down-regulation of JNK1/2 and p38 MAPK activation.

Polysaccharide of Alocasia cucullata Exerts Antitumor Effect by Regulating Bcl-2, Caspase-3 and ERK1/2 Expressions during Long-Time Administration.

OBJECTIVE: To study the in vitro and in vivo antitumor effects of the polysaccharide of Alocasia cucullata (PAC) and the underlying mechanism. METHODS: B16F10 and 4T1 cells were cultured with PAC of 40 µg/mL, and PAC was withdrawn after 40 days of administration. The cell viability was detected by cell counting kit-8. The expression of Bcl-2 and Caspase-3 proteins were detected by Western blot and the expressions of ERK1/2 mRNA were detected by quantitative real-time polymerase chain reaction (qRT-PCR). A mouse melanoma model was established to study the effect of PAC during long-time administration. Mice were divided into 3 treatment groups: control group treated with saline water, positive control group (LNT group) treated with lentinan at 100 mg/(kg·d), and PAC group treated with PAC at 120 mg/(kg·d). The pathological changes of tumor tissues were observed by hematoxylin-eosin staining. The apoptosis of tumor tissues was detected by TUNEL staining. Bcl-2 and Caspase-3 protein expressions were detected by immunohistochemistry, and the expressions of ERK1/2, JNK1 and p38 mRNA were detected by qRT-PCR. RESULTS: In vitro, no strong inhibitory effects of PAC were found in various tumor cells after 48 or 72 h of administration. Interestingly however, after 40 days of cultivation under PAC, an inhibitory effect on B16F10 cells was found. Correspondingly, the long-time administration of PAC led to downregulation of Bcl-2 protein (P<0.05), up-regulation of Caspase-3 protein (P<0.05) and ERK1 mRNA (P<0.05) in B16F10 cells. The above results were verified by in vivo experiments. In addition, viability of B16F10 cells under long-time administration culture in vitro decreased after drug withdrawal, and similar results were also observed in 4T1 cells. CONCLUSIONS: Long-time administration of PAC can significantly inhibit viability and promote apoptosis of tumor cells, and had obvious antitumor effect in tumor-bearing mice.

Host miR-129-5p reverses effects of ginsenoside Rg1 on morphine reward possibly mediated by changes in B. vulgatus and serotonin metabolism in hippocampus.

Morphine addiction is closely associated with dysbiosis of the gut microbiota. miRNAs play a crucial role in regulating intestinal bacterial growth and are involved in the development of disease. Ginsenoside Rg1 exhibits an anti-addiction effect and significantly improves intestinal microbiota disorders. In pseudo-germfree mice, supplementation with Bacteroides vulgatus (B. vulgatus) synergistically enhanced Rg1 to alleviate morphine addiction. However, it is currently unknown the relationship between fecal miRNAs in morphine-exposed mice and their potential modulation of gut microbiome, as well as their role in mediating the resistance of ginsenoside Rg1 to drug addiction. Here, we studied the fecal miRNA abundance in mice treated with morphine to explore the different miRNAs expressed, their association with B. vulgatus and their role in the amelioration of morphine reward by ginsenoside Rg1. Our results indicated ginsenoside Rg1 attenuated the significant increase in miR-129-5p expression observed in the feces of morphine-treated mice. The miR-129-5p, specifically, inhibited the growth of B. vulgatus by modulating the transcript of the site-tag BVU_RS11835 and increased the levels of 5-hydroxytryptophan and indole-3-carboxaldehyde in vitro. Subsequently, we noticed that oral administration of synthetic miR-129-5p increased 5-HT levels in the hippocampus and inhibited the reversal effect of ginsenoside Rg1 both on the relative abundance of B. vulgatus in the feces and CPP effect induced by morphine exposure. In short, Ginsenoside Rg1 might play an indirect role in remodeling the B. vulgatus against morphine reward by suppressing miR-129-5p expression. These results highlight the role of miR-129-5p and B. vulgatus in morphine reward and the anti-morphine addiction of ginsenoside Rg1.

Rhynchophylline inhibits methamphetamine dependence via modulating the miR-181a-5p/GABRA1 axis.

ETHNOPHARMACOLOGICAL RELEVANCE: Uncaria rhynchophylla (Miq.) Miq. ex Havil. is a plant species that is routinely devoted in traditional Chinese medicine to treat central nervous system disorders. Rhynchophylline (Rhy), a predominant alkaloid isolated from Uncaria rhynchophylla (Miq.) Miq. ex Havil., has been demonstrated to reverse methamphetamine-induced (METH-induced) conditioned place preference (CPP) effects in mice, rats and zebrafish. The precise mechanism is still poorly understood, thus further research is necessary. AIM OF STUDY: This study aimed to investigate the role of miRNAs in the inhibitory effect of Rhy on METH dependence. MATERIALS AND METHODS: A rat CPP paradigm and a PC12 cell addiction model were established. Microarray assays were used to screen and identify the candidate miRNA. Behavioral assessment, real-time PCR, dual-luciferase reporter assay, western blotting, stereotaxic injection of antagomir/agomir and cell transfection experiments were performed to elucidate the effect of the candidate miRNA and intervention mechanism of Rhy on METH dependence. RESULTS: Rhy successfully reversed METH-induced CPP effect and the upregulated miR-181a-5p expression in METH-dependent rat hippocampus and PC12 cells. Moreover, suppression of miR-181a-5p by antagomir 181a reversed METH-induced CPP effect. Meanwhile, overexpression of miR-181a-5p by agomir 181a in combination with low-dose METH (0.5 mg/kg) elicited a significant CPP effect, which was blocked by Rhy through inhibiting miR-181a-5p. Finally, the result demonstrated that miR-181a-5p exerted its regulatory role by targeting γ-aminobutyric acid A receptor α1 (GABRA1) both in vivo and in vitro. CONCLUSION: This finding reveals that Rhy inhibits METH dependence via modulating the miR-181a-5p/GABRA1 axis, which may be a promising target for treatment of METH dependence.

[Comparative study on macroscopic and microscopic identification of Celastrus aculeatus and Kadsura heteroclite].

OBJECTIVE: To compare and identify Celastrus aculeatus and Kadsura heteroclita with pharmacognosy methods for analyzing the quality of the crude drug. METHODS: Pharmacognosy study on Celastrus aculeatus and Kadsura heteroclite was carried out through plant identification, crude drug identification and microscopic characteristics identification. The characteristics of Celastrus aculeatus and Kadsura heteroclite were compared. RESULTS: There were significant differences between Celastrus aculeatus and Kadsura heteroclite in appearance of cork, attachments on internal surface of cork,shape of leave edge, number of lateral vein, type of stoma and vessel, and the crystals, the stone cells and the fibers in the same part of both drugs. CONCLUSION: The pharmacognosy characteristics of both crude drugs can be used for identification and quality control on Celastrus aculeatus and Kadsura heteroclite.

[Experimental research on prevention and treatment of fatty liver rats with Abrus cantoniensis].

OBJECTIVE: To investigate the effects of Abrus cantoniensis (AC) on blood lipid metabolism, pathomorphological change of the liver and fenestrae of liver sinus endothelial cell (LSEC) in fatty liver disease rats. METHODS: SD rats were divided into 7 groups: blank control group,fatty liver model group, simvastatin group (7.2 mg/kg), Gynostemma pentaphyllum group (16.2 mg/kg), high dose (40 g/kg), middle dose (20 g/kg) and low dose (10 g/kg) of AC groups. All rats except blank control group were fed with high fat diet for the first 3 weeks, then treated with different conditions as previously mentioned for the next 3 weeks while keep on feeding with high fat diet. At the 43rd day,the abdominal aortic blood was collected for measuring the serum concentration of AST, ALT, TC, TG, HDL-C, LDL-C, and liver tissues were taken to make pathological sections for observation by optical microscope or were prepared for scanning electronic microscope. RESULTS: The levels of AST, ALT, TC, TG, LDL-C were obviously decreased while HDL-C were increased in fatty liver rats by AC high dose. Meanwhile the cell morphology of liver tissues and the fenestraes of LSEC were improved as well. CONCLUSION: AC can ameliorate the levels of blood lipid in fatty liver rats and improve the pathological change of liver tissues. To some extent AC has the function of prevention and treatment of fatty liver.

Effects of Pereskia aculeate Miller Petroleum Ether Extract on Complete Freund's Adjuvant-Induced Rheumatoid Arthritis in Rats and its Potential Molecular Mechanisms.

Objective: To investigate the therapeutic effect of petroleum ether extract of P. aculeate Miller (PEEP) on rheumatoid arthritis (RA). Methods: In vitro: The Cell Counting Kit-8 (CCK-8) was used to detect cell activity and select the optimal concentration of the extract; the effective site was screened by nitric oxide (NO) colorimetric method and Q-PCR method; the expression of p38, p-p38, p-MK2, and Tristetraprolin (TTP) in RAW 264.7 cells were detected by Western blot. In vivo: The rat model was established by complete Freund's adjuvant (CFA). The different doses of PEEP on CFA rats were observed with life status, paw swelling, spleen index, X-ray, Hematoxylin eosin (HE) staining; the secretion of Tumor necrosis factor α (TNF-α), interleukin-6 (IL-6) and Prostaglandin E2 (PGE2) were detected by Enzyme linked immunosorbent assay (ELISA); the expressions of p38, p-p38, p-MK2, and TTP in the ankle joints of CFA rats were detected by Western blot. Result: In vitro: PEEP, Ethyl Acetate Extract of P. aculeate Miller (EEEP), N-butanol Extract of P. aculeate Miller (BEEP) have no toxic effects on RAW264.7 macrophages. PEEP, EEEP, and BEEP reduce the secretion of NO in RAW264.7 cells induced by lipopolysaccharide (LPS), only PEEP significantly inhibited the mRNA expression levels of inflammatory factors TNF-α and IL-6; PEEP-dependently reduce the secretion of TNF-α and IL-6, decrease the expression of p-p38 and p-MK2, and the level of TTP phosphorylation in LPS-induced RAW264.7 cells. In vivo: PEEP improve the living conditions of CFA rats, reduce foot swelling, spleen index, bone surface erosion and joint space narrowing; reduce the formation of synovial cells, inflammatory cells and pannus in the foot and ankle joints. PEEP reduce the secretion of TNF-α, IL-6, PGE2 in rat serum, downregulate the expression of p-p38 and p-MK2 in the ankle joint, and reduce the phosphorylation of TTP. Conclusion: PEEP improve the living conditions of CFA rats, reduce the degree of foot swelling, protect immune organs, reduce inflammatory cell infiltration, cartilage damage, pannus formation, reduce inflammation and RA damage. The mechanism through regulating the signal pathway of p38 mitogen-activated protein kinase (p38/MAPK), which reduces the release of TNF-α, IL-6, and PGE2 in the serum.

Ginsenoside Rg1 mitigates morphine dependence via regulation of gut microbiota, tryptophan metabolism, and serotonergic system function.

BACKGROUND: Morphine dependence, a devastating neuropsychiatric condition, may be closely associated with gut microbiota dysbiosis. Ginsenoside Rg1 (Rg1), an active ingredient extracted from the roots of Panax ginseng C.A. Meyer, has potential health-promoting effects on the nervous system. However, its role in substance use disorders remains unclear. Here, we explored the potential modulatory roles of Rg1 in protection against morphine dependence. METHODS: Conditioned place preference (CPP) was used for establishing a murine model of morphine dependence. 16S rRNA gene sequencing and metabolomics were performed for microbial and metabolite analysis. Molecular analysis was tested for evaluating the host serum and brain responses. RESULTS: Rg1 prevented morphine-induced CPP in mice. The 16S rRNA gene-based microbiota analysis demonstrated that Rg1 ameliorated morphine-induced gut microbiota dysbiosis, specifically for Bacteroidetes. Moreover, Rg1 also inhibited gut microbiota-derived tryptophan metabolism and reduced the serotonin, 5-hydroxytryptamine receptor 1B (5-HTR1B), and 5-hydroxytryptamine receptor 2 A (5-HTR2A) levels. However, the Rg1-induced amelioration of CPP was not observed in mice when their gut microbiome was depleted by non-absorbable antibiotics. Subsequently, gavage with Bacteroides vulgatus increased the abundance of Bacteroidetes. B. vulgatus supplementation synergistically enhanced Rg1-alleviated morphine-induced CPP in mice with microbiome knockdown. Co-treatment with B. vulgatus and Rg1 produced suppressive effects against morphine dependency by inhibiting tryptophan metabolism and reducing the serotonin and 5-HTR1B/5-HTR2A levels. CONCLUSIONS: The gut microbiota-tryptophan metabolism-serotonin plays an important role in gut-brain signaling in morphine disorders, which may represent a novel approach for drug dependence treatment via manipulation of the gut microbial composition and tryptophan metabolite.

Isoalantolactone Induces Cell Cycle Arrest, Apoptosis and Autophagy in Colorectal Cancer Cells.

Colorectal cancer (CRC) is an aggressive cancer. Isoalantolactone (IATL) has been reported to exert cytotoxicity against various cancer cells, but not CRC. In this study, we explored the anti-CRC effects and mechanism of action of IATL in vitro and in vivo. Our results demonstrated that IATL inhibited proliferation by inducing G0/G1 phase cell cycle arrest, apoptosis and autophagy in CRC cells. Repression of autophagy with autophagy inhibitors chloroquine (CQ) and Bafilomycin A1 (Baf-A1) enhanced the anti-CRC effects of IATL, suggesting that IATL induces cytoprotective autophagy in CRC cells. Mechanistic studies revealed that IATL lowered protein levels of phospho-AKT (Ser473), phospho-mTOR (Ser2448), phospho-70S6K (Thr421/Ser424) in CRC cells. Inhibition of AKT and mTOR activities using LY294002 and rapamycin, respectively, potentiated the inductive effects of IATL on autophagy and cell death. In vivo studies showed that IATL suppressed HCT116 tumor growth without affecting the body weight of mice. In consistent with the in vitro results, IATL lowered protein levels of Bcl-2, Bcl-XL, phospho-AKT (Ser473), phospho-mTOR (Ser2448), and phsopho-70S6K (Thr421/Ser424), whereas upregulated protein levels of cleaved-PARP and LC3B-II in HCT116 tumors. Collectively, our results demonstrated that in addition to inhibiting proliferation, inducing G0/G1-phase cell cycle arrest and apoptosis, IATL initiates cytoprotective autophagy in CRC cells by inhibiting the AKT/mTOR signaling pathway. These findings provide an experimental basis for the evaluation of IATL as a novel medication for CRC treatment.

Extracellular Vesicle-Encapsulated miR-183-5p from Rhynchophylline-Treated H9c2 Cells Protect against Methamphetamine-Induced Dependence in Mouse Brain by Targeting NRG1.

Methamphetamine (Meth) is a highly addictive substance and the largest drug threat across the globe. There is evidence to indicate that Meth use has serious damage on central nervous system (CNS) and heart in several animal and human studies. However, the connection in the process of Meth addiction between these two systems has not been determined. Emerging data suggest that extracellular vesicles (EVs) carrying behavior-altering microRNA (miRNAs) play a crucial role in cell communication between CNS and peripheral system. Rhynchophylline (Rhy), an antiaddictive alkaloid, was used to protect the brain and heart from Meth-induced damage, which has caught our attention. Here, we used Meth-dependent conditioned place preference (CPP) animal model and cell model to verify the protective effect of Rhy-treated EVs. Further, small RNA sequencing analysis, qPCR, dual-luciferase reporter assay, and transfection test were used to identify the key EVs-encapsulated miRNAs, isolated from cultured H9c2 cells with different treatments, involved in the therapeutic effect and the underlying mechanisms of Rhy. The results demonstrate that Rhy-treated EVs exert protective effects against Meth dependence through the pathway of miR-183-5p-neuregulin-1 (NRG1). Our collective findings provide novel insights into the roles of EVs miRNAs in Meth addiction and support their potential application in the development of novel therapeutic approaches.

[Study on purification of polysaccharides in root of Salvia miltiorrhiza by macroporous adsorption resin and ion-exchange resin methods].

OBJECTIVE: To study the purification and isolation of polysaccharides from Salvia miltiorrhiza. METHODS: The root was extracted by water purified preliminarily by alcohol precipitation, and then four different types macroporous adsorption resin and one ion-exchange resin were comparatively investigated in the purification and isolation of Salvia polysaccharides. RESULTS: The total polysaccharides of crude extracts was 40.35%, and protein content amounted to 8.96%. Compared with the traditional methods, AB-8, DB-301 type of resin used in purification of polysaccharides could simplify the working process and obtain better effect. Then the obtained crude polysaccharides through AB-8 resin were purified by ion-exchange resin DEAE-52. Three portions of powder were obtained through lyophilization and named as SMP1, SMP2, SMP3. CONCLUSION: Purification of Salvia polysaccharides can be conducted by adoping AB-8, DB-301 type of resin and DEAE-52 ion-exchange resin.