[Analysis of constituents in different parts of Forsythia suspensa by UPLC-Q-TOF-MS and evaluation of their anti-inflammatory activity].

This study aims to characterize and identify the chemical constituents in 11 parts of Forsythia suspensa by using ultra-performance liquid chromatography-quadrupole time of flight-mass spectrometry(UPLC-Q-TOF-MS) combined with a self-established chemical constituent database, including leaves, flowers, fruits, green F. suspensa, old F. suspensa, and seeds. The quality attributes and differences of different parts of F. suspensa were evaluated by principal component analysis, partial least square discriminant analysis, and other stoichiometric methods. A total of 79 compounds were identified, including 13 phenylethanol glycosides, 10 lignans, 12 flavonoids, 10 organic acids, 14 terpenoids, and 20 other types of compounds. Among them, 34 compounds were the main variables of difference between the different parts of F. suspensa, and the content of each component was relatively higher in the leaves and green F. suspensa. The LPS-induced inflammation model of RAW264.7 cells was applied to study the anti-inflammatory activity of the extracts of the different parts of F. suspensa and the main constituents. The results show that the extracts of green F. suspensa, flower, twig, and stem exhibited anti-inflammatory activity, and the constituents such as forsythoside A, phyllyrin, phillygenin, and(+)-pinoresinol-ß-D-glucopyranoside could significantly inhibit anti-inflammatory activity released by NO. The chemical constituent in different parts of F. suspensa is analyzed comprehensively, and the anti-inflammatory activity is evaluated in this study, which provides a reference for the development and comprehensive utilization of F. suspensa resources.

Network pharmacology analysis and experimental verification of the antithrombotic active compounds of trichosanthis pericarpium (Gualoupi) in treating coronary heart disease.

ETHNOPHARMACOLOGICAL RELEVANCE: Trichosanthis pericarpium (TP; Gualoupi, pericarps of Trichosanthes kirilowii Maxim) has been used in traditional Chinese medicine (TCM) to reduce heat, resolve phlegm, promote Qi, and clear chest congestion. It is also an essential herbal ingredient in the "Gualou Xiebai" formula first recorded by Zhang Zhongjing (from the Eastern Han Dynasty) in the famous TCM classic "Jin-Guì-Yào-Lüe" for treating chest impediments. According to its traditional description, Gualou Xiebai is indicated for symptoms of chest impediments, which correspond to coronary heart diseases (CHD). AIM OF THE STUDY: This study aimed to identify the antithrombotic compounds in Gualoupi for the treatment of CHD. MATERIALS AND METHODS: A CHD rat model was established with a combination of high-fat diet and isoproterenol hydrochloride (ISO) administration via subcutaneous multi-point injection in the back of the neck. This model was used to evaluate the antithrombotic effect of two mainstream cultivars of TP ("HaiShi GuaLou" and "WanLou") by analyzing the main components and their effects. Network pharmacology, molecular docking-based studies, and a zebrafish (Danio rerio) thrombosis model induced by phenylhydrazine was used to validate the antithrombosis components of TP. RESULTS: TP significantly reduced the body weight of the CHD rats, improved myocardial ischemia, and reduced collagen deposition and fibrosis around the infarcted tissue. It reduced thrombosis in a dose-dependent manner and significantly reduced inflammation and oxidative stress damage. Cynaroside, isoquercitrin, rutin, citrulline, and arginine were identified as candidate active TP compounds with antithrombotic effects. The key potential targets of TP in thrombosis treatment were initially identified by molecular docking-based analysis, which showed that the candidate active compounds have a strong binding affinity to the potential targets (protein kinase C alpha type [PKCα], protein kinase C beta type [PKCß], von Willebrand factor [vWF], and prostaglandin-endoperoxide synthase 1 [PTGS1], fibrinogen alpha [Fga], fibrinogen beta [Fgb], fibrinogen gamma [Fgg], coagulation factor II [F2], and coagulation factor VII [F7]). In addition, the candidate active compounds reduced thrombosis, improved oxidative stress damage, and down-regulated the expression of thrombosis-related genes (PKCα, PKCß, vWF, PTGS1, Fga, Fgb, Fgg, F2, and F7) in the zebrafish model. CONCLUSION: Cynaroside, isoquercitrin, rutin, citrulline, and arginine were identified as the active antithrombotic compounds of TP used to treat CHD. Mechanistically, the active compounds were found to be involved in oxidative stress injury, platelet activation pathway, and complement and coagulation cascade pathways.

Alginate oligosaccharide assimilation by gut microorganisms and the potential role in gut inflammation alleviation.

Dietary fiber metabolism by gut microorganisms plays important roles in host physiology and health. Alginate, the major dietary fiber of daily diet seaweeds, is drawing more attention because of multiple biological activities. To advance the understanding of alginate assimilation mechanism in the gut, we show the presence of unsaturated alginate oligosaccharides (uAOS)-specific alginate utilization loci (AUL) in human gut microbiome. As a representative example, a working model of the AUL from the gut microorganism Bacteroides clarus was reconstructed from biochemistry and transcriptome data. The fermentation of resulting monosaccharides through Entner-Doudoroff pathway tunes the metabolism of short-chain fatty acids and amino acids. Furthermore, we show that uAOS feeding protects the mice against dextran sulfate sodium-induced acute colitis probably by remodeling gut microbiota and metabolome. IMPORTANCE: Alginate has been included in traditional Chinese medicine and daily diet for centuries. Recently discovered biological activities suggested that alginate-derived alginate oligosaccharides (AOS) might be an active ingredient in traditional Chinese medicine, but how these AOS are metabolized in the gut and how it affects health need more information. The study on the working mechanism of alginate utilization loci (AUL) by the gut microorganism uncovers the role of unsaturated alginate oligosaccharides (uAOS) assimilation in tuning short-chain fatty acids and amino acids metabolism and demonstrates that uAOS metabolism by gut microorganisms results in a variation of cell metabolites, which potentially contributes to the physiology and health of gut.

Stability of tryptophan-containing LOs in flaxseed oil and their response towards γ-tocopherol.

Linusorbs (LOs), significantly influence oil quality and sensory properties of flaxseed oil. Trp-containing LOs exhibit distinct oxidative behavior when γ-tocopherol (γ-T) is present. Polar fractions of crude flaxseed oil were stripped via silica absorption, and reintroduced (LO and γ-T) separately into the oil matrix to investigate their interaction during storage. Compared with crude oil, LOs account for 18.49% reduction of p-anisidine value, while LOs with γ-T contributed to most of the endogenous antioxidant effect in crude oil. γ-T was found to suppress oxidation of Trp-containing LO at early stage (Met form), while facilitate oxidation while at their mid-stage (MetO form, Methionine sulfoxide). In vitro oxidation shows that CLD more likely cleaved into peptide fragments, while few products retain intact ring structures. LC-MS/MS analysis and silicon simulation revealed proximity between MetO and Trp residues, facilitating inter- or intra-molecular reactions and ring structure rupture. Remarkably, the presence of γ-T facilitate these phenomena.

Asparagine-Glucose Amadori Compounds: Formation, Characterization, and Analysis in Dry Jujube Fruit.

Amadori rearrangement products of asparagine with glucose (Asn-Glc-ARP) were first prepared through Maillard model reactions and identified via liquid chromatography-mass spectroscopy. With the study on the effect of the reaction temperature, pH values, and reaction time, the ideal reaction condition for accumulation of Asn-Glc-ARP was determined at 100 °C for 40 min under pH 7. Asparagine (Asn) was prone to degrade from Asn-Glc-ARP in alkaline pH values within a lower temperature range, while in an acidic environment with high temperatures, deamidation of Asn-Glc-ARP to Asp-Glc-ARP (Amadori rearrangement products of aspartic acid with glucose) was displayed as the dominant pathway. The deamidation reaction on the side chain of the amide group took place at Asn-Glc-ARP and transferred it into the hydroxyl group, forming Asp-Glc-ARP at the end. Considering that lyophilization as pretreatment led to limited water activity, a single aspartic acid was not deamidated from Asn directly nor did it degrade from Asp-Glc-ARP even at 120 °C. The degradation of Asn-Glc-ARP through tandem mass spectrometry (MS/MS) analysis showed the obvious fragment ion at m/z 211, indicating that the stable oxonium ion formed during fragmentation. The structure of Asn-Glc-ARP was proposed as 1-deoxy-1-l-asparagino-d-fructose after separation and purification. Also, the content of Asn-Glc-ARP within dry jujube fruit (HeTianYuZao) was quantitated as high as 8.1 ± 0.5 mg/g.

Discovery of sesquiterpene from Youngia japonica with antitumor effect.

Fourteen sesquiterpenes, including one undescribed sesquiterpene lactone, were isolated from Youngia japonica, and their structures were identified by NMR, HRESIMS, ECD and calculated ECD. Cytotoxic activities of all isolates against A549, HeLa, and 4 T1 cell lines were detected by CCK8 assay. Among them, 2 showed obvious cytotoxic activity against A549 cells. Subsequently, the production of ROS, and apoptosis of A549 cells treated with 2 were evaluated. The result showed that 2 distinctly increased the ROS level, and induced the apoptosis of A549 cells. Further anticancer mechanism studies showed that 2 increased the expression of cleaved caspase 3. Taken together, our results demonstrated that 2 might become potential leading compounds for the treatment of lung cancer.

Design, synthesis and biological evaluation of bakuchiol derivatives as multi-target agents for the treatment of Alzheimer's disease.

The concept of multi-target-directed ligands offers fresh perspectives for the creation of brand-new Alzheimer's disease medications. To explore their potential as multi-targeted anti-Alzheimer's drugs, eighteen new bakuchiol derivatives were designed, synthesized, and evaluated. The structures of the new compounds were elucidated by IR, NMR, and HRMS. Eighteen compounds were assayed for acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in vitro using Ellman's method. It was shown that most of the compounds inhibited AChE and BuChE to varying degrees, but the inhibitory effect on AChE was relatively strong, with fourteen compounds showing inhibition of >50% at the concentration of 200 µM. Among them, compound 3g (IC50 = 32.07 ± 2.00 µM) and compound 3n (IC50 = 34.78 ± 0.34 µM) showed potent AChE inhibitory activities. Molecular docking studies and molecular dynamics simulation showed that compound 3g interacts with key amino acids at the catalytically active site (CAS) and peripheral anionic site (PAS) of acetylcholinesterase and binds stably to acetylcholinesterase. On the other hand, compounds 3n and 3q significantly reduced the pro-inflammatory cytokines TNF-α and IL-6 released from LPS-induced RAW 264.7 macrophages. Compound 3n possessed both anti-acetylcholinesterase activity and anti-inflammatory properties. Therefore, an in-depth study of compound 3n is expected to be a multi-targeted anti-AD drug.

Pharmacologically significant constituents collectively responsible for anti-sepsis action of XueBiJing, a Chinese herb-based intravenous formulation.

Sepsis, a life-threatening health issue, lacks effective medicine targeting the septic response. In China, treatment combining the intravenous herbal medicine XueBiJing with conventional procedures reduces the 28-day mortality of critically ill patients by modulating septic response. In this study, we identified the combined active constituents that are responsible for the XueBiJing's anti-sepsis action. Sepsis was induced in rats by cecal ligation and puncture (CLP). The compounds were identified based on their systemic exposure levels and anti-sepsis activities in CLP rats that were given an intravenous bolus dose of XueBiJing. Furthermore, the identified compounds in combination were assessed, by comparing with XueBiJing, for levels of primary therapeutic outcome, pharmacokinetic equivalence, and pharmacokinetic compatibility. We showed that a total of 12 XueBiJing compounds, unchanged or metabolized, circulated with significant systemic exposure in CLP rats that received XueBiJing. Among these compounds, hydroxysafflor yellow A, paeoniflorin, oxypaeoniflorin, albiflorin, senkyunolide I, and tanshinol displayed significant anti-sepsis activities, which involved regulating immune responses, inhibiting excessive inflammation, modulating hemostasis, and improving organ function. A combination of the six compounds, with the same respective doses as in XueBiJing, displayed percentage survival and systemic exposure in CLP rats similar to those by XueBiJing. Both the combination and XueBiJing showed high degrees of pharmacokinetic compatibility regarding interactions among the six active compounds and influences of other circulating XueBiJing compounds. The identification of XueBiJing's pharmacologically significant constituents supports the medicine's anti-sepsis use and provides insights into a polypharmacology-based approach to develop medicines for effective sepsis management.

Soil properties and fungal community jointly explain N2O emissions following N and P enrichment in an alpine meadow.

Nutrient enrichment, such as nitrogen (N) and phosphorus (P), typically affects nitrous oxide (N2O) emissions in terrestrial ecosystems, predominantly via microbial nitrification and denitrification processes in the soil. However, the specific impact of soil property and microbial community alterations under N and P enrichment on grassland N2O emissions remains unclear. To address this, a field experiment was conducted in an alpine meadow of the northeastern Qinghai-Tibetan Plateau. This study aimed to unravel the mechanisms underlying N and P enrichment effects on N2O emissions by monitoring N2O fluxes, along with analyzing associated microbial communities and soil physicochemical properties. We observed that N enrichment individually or in combination with P enrichment, escalated N2O emissions. P enrichment dampened the stimulatory effect of N enrichment on N2O emissions, indicative of an antagonistic effect. Structural equation modeling (SEM) revealed that N enrichment enhanced N2O emissions through alterations in fungal community composition and key soil physicochemical properties such as pH, ammonium nitrogen (NH4+-N), available phosphorus (AP), microbial biomass carbon (MBC), and microbial biomass nitrogen (MBN)). Notably, our findings demonstrated that N2O emissions were significantly more influenced by fungal activities, particularly genera like Fusarium, rather than bacterial processes in response to N enrichment. Overall, the study highlights that N enrichment intensifies the role of fungal attributes and soil properties in driving N2O emissions. In contrast, P enrichment exhibited a non-significant effect on N2O emissions, which highlights the critical role of the fungal community in N2O emissions responses to nutrient enrichments in alpine grassland ecosystems.

AaLaeA targets AaFla1 to mediate the production of antitumor compound in Alternaria alstroemeria.

Endophytic fungi are an important source of novel antitumor substances. Previously, we isolated an endophytic fungus, Alternaria alstroemeria, from the medicinal plant Artemisia artemisia, whose crude extracts strongly inhibited A549 tumor cells. We obtained a transformant, namely AaLaeAOE26 , which completely loses its antitumor activity due to overexpression of the global regulator AaLaeA. Re-sequencing analysis of the genome revealed that the insertion site was in the noncoding region and did not destroy any other genes. Metabolomics analysis revealed that the level of secondary antitumor metabolic substances was significantly lower in AaLaeAOE26 compared with the wild strain, in particular flavonoids were more downregulated according to the metabolomics analysis. A further comparative transcriptome analysis revealed that a gene encoding FAD-binding domain protein (Fla1) was significantly downregulated. On the other hand, overexpression of AaFla1 led to significant enhancement of antitumor activity against A549 with a sevenfold higher inhibition ratio than the wild strain. At the same time, we also found a significant increase in the accumulation of antitumor metabolites including quercetin, gitogenin, rhodioloside, liensinine, ginsenoside Rg2 and cinobufagin. Our data suggest that the global regulator AaLaeA negatively affects the production of antitumor compounds via controlling the transcription of AaFla1 in endophytic A. alstroemeria.

Effect of Repeated Application of Flos Daturae on Sedative and Hypnotic in Mice.

Insomnia is one of the most common sleep-related diseases. In traditional Chinese medicine, Flos daturae has been used as a traditional herbal totreatment of sizens of diseases. The research objective was to investigate the sedative and hypnotic effects of Flos Daturae. Kunming mice were divided into control group, Estazolam (positive drug, 0.0005 g/kg) group and Flos Daturae groups (0.01, 0.02, 0.04g/kg) with random, ig once a day for 7 days. The central sedative effect of flos Daturae on the spontaneous activity of mice was observed using the locomotive activity test, and the hypnotic effect of Flos Daturae was observed in mice using the direct sleep test and the sleep latency with synergistic supra-and sub-threshold doses of pentobarbital sodium. Flos Daturae (0.04g/kg) significantly inhibited mice locomotive activity (P<0.05) and had no direct sleeping effect (P>0.05), increased the number rate of sleep (P<0.05), and significantly shortening sleep latency (P<0.05), enhanced pentobarbital sodium-induced sleep. Flos Daturae possesses have sedative-hypnotic properties.

Efficacy and safety of moxibustion on cancer-related fatigue: a systematic review and meta-analysis of randomized controlled trials.

OBJECTIVE: The goal of this research was to review the literature from randomized controlled trials (RCTs) on the impacts of moxibustion on cancer-related fatigue (CRF) as well as provide credible evidence to guide clinical practice. METHODS: Three English electronic medical databases (PubMed, Embase, and the Cochrane Library) and two Chinese databases (China National Knowledge Infrastructure and Wanfang) were searched. Only randomized controlled trials on the effect of moxibustion on CRF were included in this systematic review. Study selection, data extraction, and validation were all carried out independently by two reviewers. The revised Cochrane Risk of Bias tool was used to assess the quality of the RCTs (RoB 2.0). The Grading of Recommendations Assessment, Development and Evaluation (GRADE) system was applied to assess effect sizes in individual RCTs and pooled effect sizes in meta-analyses. Data were meta-analyzed using Stata (version 14.0). RESULTS: In a random-effects meta-analysis of 24 RCTs with 1894 participants, the aggregated standardized mean difference (SMD) revealed a statistically significant association between moxibustion and alleviation from cancer-related fatigue (SMD = - 1.66, 95% CI = - 2.05, - 1.28, p = 0.000). Pooled results, however, show significant heterogeneity (I2 = 92.5%), and the evidence is insufficient to determine whether this association varies systematically by measuring tools and moxibustion modalities. Furthermore, evidence ranging from very low to low showed that moxibustion had an immediate positive effect on patients with CRF. CONCLUSION: Moxibustion may have a therapeutic effect on cancer-related fatigue. However, further large-scale, multicenter, high-quality RCTs on moxibustion for fatigue relief and safety are still needed because of the handful of studies included and the low methodological quality.

[Effect of electroacupuncture pretreatment on ferroptosis in neurons of rats with cerebral ischemia-reperfusion injury].

OBJECTIVE: To observe the effect of electroacupuncture（EA）preconditioning on ferroptosis in rats with cerebral ischemia-reperfusion injury （CIRI）, so as to explore the neuroprotective mechanism of EA preconditioning. METHODS: Male SD rats were randomly divided into sham operation, model, EA, inhibitor and inducer groups with 20 rats in each group. The CIRI model was established by modified Zea Longa occlusion of the middle cerebral artery. Before modeling, EA treatment （2 Hz/15 Hz, 1-2 mA） was applied to "Baihui"（GV20）, "Fengfu"（GV16） and "Dazhui"（GV14） for rats of the EA group, 20 min a day for 7 consecutive days. Rats of the inhibitor group were intraperitoneally injected with ferristatin-1（25 mg/kg）at a slow and uniform rate. Rats of the inducer group were intraperitoneally injected with Erastin（100 mg/kg） after 7 days of EA preconditioning, once every 2 h for a total of 4 times. The CIRI models were prepared 2 d later after the above interventions finished by thread-occlusion. The degree of neurological impairment was evaluated by modified Zea Longa score. The percentage of infarct size was calculated by TTC staining. The ultrastructure of neurons in hippocampus was observed by transmission electron microscope. The contents of ferrous ion （Fe2+）, malondialdehyde （MDA） and glutathione （GSH） in cerebral tissue and reactive oxygen species （ROS） in serum were determined by biochemical method. The changes of mitochondrial membrane potential in rats brain tissues were detected by flow cytometry. The mRNA and protein expression levels of glutathione peroxidase 4 （GPX4）, acyl-CoA synthetase long-chain family member 4 （ACSL4）, transferrin receptor （TFRC）, 15-lipoxygenase （15-LOX） and cyclooxygenase-2 （COX-2） in the ischemic hippocampal region of CIRI rats were detected by real-time quantitative PCR and Western blot, respectively. RESULTS: Compared with the sham operation group, the neurological impairment score, the percentage of cerebral infarction area, the contents of MDA and Fe2+ in cerebral tissue as well as ROS in serum, the protein and mRNA expression levels of ACSL4, TFRC, 15-LOX, COX-2 in hippocampal tissue were increased （P<0.01）, while the content of GSH in cerebral tissue, the protein and mRNA expression levels of GPX4 in hippocampal tissue were decreased （P<0.01）, and mitochondria in brain tissue were significantly damaged （P<0.01） in the model group. Compared with the model group, the above indexes were all reversed （P<0.05, P<0.01） in the EA group and inhibitor group. Compared with the EA group, the neurological impairment score, the percentage of cerebral infarction area, the contents of MDA and Fe2+ in cerebral tissue as well as ROS in serum, the protein and mRNA expression le-vels of ACSL4, TFRC, 15-LOX, COX-2 in hippocampal tissue were increased （P<0.05, P<0.01）, while the content of GSH in cerebral tissue, the protein and mRNA expression levels of GPX4 in hippocampal tissue were decreased （P<0.01, P<0.05）, and mitochondria in brain tissue were significantly damaged （P<0.05） in the inducer group. CONCLUSION: EA preconditioning has neuroprotective effect on CIRI rats, which may be related to inhibiting ACSL4/TFRC/15-LOX/COX-2 expression and increasing GSH/GPX4 expression.

Protective effect of mussel polysaccharide on cyclophosphamide-induced intestinal oxidative stress injury via Nrf2-Keap1 signaling pathway.

The hard-shelled mussel (Mytilus coruscus) has been used as a traditional Chinese medicine and health food in China for centuries. Polysaccharides from mussel has been reported to have multiple biological functions, however, it remains unclear whether mussel polysaccharide (MP) exerts protective effects in intestinal functions, and the underlying mechanisms of action remain unclear. The aim of this study was to investigate the protective effects and mechanism of MP on intestinal oxidative injury in mice. In this study, 40 male BALB/C mice were used, with 30 utilized to produce an animal model of intestinal oxidative injury with intraperitoneal injection of cyclophosphamide (Cy) for four consecutive days. The protective effects of two different doses of MP (300 and 600 mg/kg) were assessed by investigating the change in body weight, visceral index, and observing colon histomorphology. Moreover, the underlying molecular mechanisms were investigated by measuring the antioxidant enzymes and related signaling molecules through ELISA, real-time PCR, and western blot methods. The results showed that MP pretreatment effectively protected the intestinal from Cy-induced injury: improved the colon tissue morphology and villus structure, increased superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities, and reduced malondialdehyde (MDA) content in serum and colon tissues. Meanwhile, MP also significantly increased the expression levels of SOD, GSH-Px, heme oxygenase-1 (HO-1), and nuclear factor E2-related factor 2 (Nrf2) mRNA in colon tissues. Further, western blot results showed that the expression of Nrf2 protein was significantly upregulated while kelch-like ECH-associated protein 1 (Keap1) was significantly downregulated by MP in the colonic tissues. This study indicates that MP can ameliorate Cy-induced oxidative stress injury in mice, and Nrf2-Keap1 signaling pathway may mediate these protective effects.

[Effect of Tongdu Tiaoshen electroacupuncture pretreatment on PPARγ-mediated pyroptosis of cerebral cortex in rats with cerebral ischemia reperfusion injury].

OBJECTIVE: To observe the effect of Tongdu Tiaoshen (promoting the circulation of the governor vessel and regulating the spirit) electroacupuncture (EA) pretreatment on pyroptosis mediated by peroxisome proliferators-activated receptor γ (PPARγ) of the cerebral cortex in rats with cerebral ischemia reperfusion injury (CIRI) and explore the potential mechanism of EA for the prevention and treatment of CIRI. METHODS: A total of 110 clean-grade male SD rats were randomly divided into a sham-operation group, a model group, an EA group, an EA + inhibitor group and an agonist group, 22 rats in each group. In the EA group, before modeling, EA was applied to "Baihui" (GV 20), "Fengfu" (GV 16) and "Dazhui" (GV 14), with disperse-dense wave, 2 Hz/5 Hz in frequency, 1 to 2 mA in intensity, lasting 20 min; once a day, consecutively for 7 days. On the base of the intervention as the EA group, on the day 7, the intraperitoneal injection with the PPARγ inhibitor, GW9662 (10 mg/kg) was delivered in the EA + inhibitor group. In the agonist group, on the day 7, the PPARγ agonist, pioglitazone hydrochloride (10 mg/kg) was injected intraperitoneally. At the end of intervention, except the sham-operation group, the modified thread embolization method was adopted to establish the right CIRI model in the rats of the other groups. Using the score of the modified neurological severity score (mNSS), the neurological defect condition of rats was evaluated. TTC staining was adopted to detect the relative cerebral infarction volume of rat, TUNEL staining was used to detect apoptosis of cerebral cortical nerve cells and the transmission electron microscope was used to observe pyroptosis of cerebral cortical neural cells. The positive expression of PPARγ and nucleotide-binding to oligomerization domain-like receptor protein 3 (NLRP3) in the cerebral cortex was detected with the immunofluorescence staining. The protein expression of PPARγ, NLRP3, cysteinyl aspartate specific protease-1 (caspase-1), gasdermin D (GSDMD) and GSDMD-N terminal (GSDMD-N) in the cerebral cortex was detected with Western blot. Using the quantitative real-time fluorescence-PCR, the mRNA expression of PPARγ, NLRP3, caspase-1 and GSDMD of the cerebral cortex was detected. The contents of interleukin (IL)-1ß and IL-18 in the cerebral cortex of rats were determined by ELISA. RESULTS: Compared with the sham-operation group, the mNSS, the relative cerebral infarction volume and the TUNEL positive cells rate were increased (P<0.01), pyroptosis was severe, the protein and mRNA expression levels of PPARγ, NLRP3, caspase-1 and GSDMD were elevated (P<0.01); and the protein expression of GSDMD-N and contents of IL-1ß and IL-18 were increased (P<0.01) in the model group. When compared with the model group, the mNSS, the relative cerebral infarction volume and the TUNEL positive cells rate were decreased (P<0.01), pyroptosis was alleviated, the protein and mRNA expression levels of PPARγ were increased (P<0.01), the protein and mRNA expression levels of NLRP3, caspase-1 and GSDMD were decreased (P<0.01), the protein expression of GSDMD-N was reduced (P<0.01); and the contents of IL-1ß and IL-18 were lower (P<0.01) in the EA group and the agonist group; while, in the EA + inhibitor group, the protein expression of PPARγ was increased (P<0.01), the protein and mRNA expression levels of NLRP3 and GSDMD were decreased (P<0.01, P<0.05), the mRNA expression of caspase-1 was reduced (P<0.01); and the contents of IL-1ß and IL-18 were lower (P<0.01). When compared with the EA + inhibitor group, the mNSS, the relative cerebral infarction volume and the TUNEL positive cells rate were decreased (P<0.05, P<0.01), pyroptosis was alleviated, the protein and mRNA expression levels of PPARγ were increased (P<0.01), the protein and mRNA expression levels of NLRP3, caspase-1 and GSDMD were decreased (P<0.01), the protein expression of GSDMD-N was reduced (P<0.01); and the contents of IL-1ß and IL-18 were declined (P<0.01) in the EA group. Compared with the agonist group, in the EA group, the relative cerebral infarction volume and the TUNEL positive cells rate were increased (P<0.05, P<0.01), the mRNA expression of PPARγ was decreased (P<0.01) and the protein expression of GSDMD-N was elevated (P<0.05); and the contents of IL-1ß and IL-18 were higher (P<0.01). CONCLUSION: Tongdu Tiaoshen EA pretreatment can attenuate the neurological impairment in the rats with CIRI, and the underlying mechanism is related to the up-regulation of PPARγ inducing the inhibition of NLRP3 in the cerebral cortex of rats so that pyroptosis is affected.

First Report of 16SrI-B Subgroup Related Phytoplasma Associated with Yellows Symptoms of Rubus cochinchinensis in China.

Rubus cochinchinensis, an important traditional Chinese medicine in China is used to treat rheumatic arthralgia, bruises and lumbocrural pain (He et al.2005). In January 2022, yellow leaves of R. cochinchinensis were found in Tunchang City, Hainan Province, a tropical island in China. Chlorosis spread along the direction of vascular tissue while the leaf veins remain green (Fig. 1). In addition, the leaves were slightly shrunken and the growth vigor is poor (Fig. 1). By survey, we found the incidence of this disease was about 30%. Three etiolated samples and three healthy samples (0.1g each) were used to extract total DNA (TIANGEN plant genomic DNA extraction kit). Using nested PCR method, phytoplasma universal primers P1 / P7 (Schneider et al., 1995) and R16F2n / R16R2 (Lee et al. 1993) were used to amplified phytoplasma 16S rDNA gene. Primers rp F1 / R1 (Lee et al. 1998) and rp F2 / R2 (Martini et al. 2007) were used to amplified rp gene. 16S rDNA gene and rp gene fragments were amplified from three leaf etiolated samples, but not from healthy samples. The amplified fragments were cloned and sequenced, and the sequences were assembled by DNASTAR11. By sequence alignment, we found the obtained 16S rDNA and rp gene sequences of three leaf etiolated samples were same. The length of 16S rDNA fragment was 1237 bp (accession number: ON944105) and the length of rp gene fragment was 1212 bp (accession number: ON960069). The phytoplasma strain was named as 'R. cochinchinensis' yellows leaf phytoplasma (RcT), RcT-HN1 strain. The 16S rDNA gene sequence of RcT-HN1is 99.8% consistent with 16SrI-B subgroup members such as the 'Brassica napus' dwarf phytoplasma strain WH3 (MG599470.1), Chinaberry yellows phytoplasma strain LJM-1(KX683297.1) and Arecanut yellow leaf disease phytoplasma strain B165 (FJ694685.1). The rp gene sequence of RcT-HN1 is 100% consistent with rpI-B subgroup members such as the 'Salix tetradenia' witches'-broom phytoplasma strain YM-1 (KC117314.1) and Chinaberry witches'-broom phytoplasma strain Hainan (EU348781.1). The phylogenetic tree analysis, based on concatenated 16S rDNA-rp gene sequence of same group phytoplasma by MEGA 7.0 employing neighbor-joining (NJ) method with 1000 bootstrap value, were performed (Kumar et al., 2016). The results showed that RcT-HN1 phytoplasma strain formed a subclade in aster yellows group B subgroup (Fig. 2). The virtual RFLP analysis based on the 16S rRNA gene fragment of RcT-HN1 phytoplasma strain was performed by the interactive online phytoplasma classification tool iPhyClassifier (Zhao et al., 2009). The results showed that the phytoplasma strain was same as the reference pattern of the onion yellows phytoplasma of 16SrI-B (GenBank accession: AP006628), and the similarity coefficient was 1.00. This is the first report that 16SrI-B subgroup related phytoplasma infected R. cochinchinensis and caused yellows symptoms in China. The discovery of the disease is helpful to the study of the spread of phytoplasma-related diseases and protect R. cochinchinensis resources.

[Manufacturing classification system for oral solid dosage forms of traditional Chinese medicines(â ): classification of processing routes].

Oral solid dosage(OSD) occupies a key position in the market of Chinese patent medicines and new traditional Chinese medicines. Processing route is the foundation for the research and development of traditional Chinese medicine OSDs. On the basis of prescriptions and preparation methods of 1 308 traditional Chinese medicine OSDs recorded in the Chinese Pharmacopoeia, we summarized the patterns of processing routes of both modern dosage forms(tablets, granules, and capsules) and traditional dosage forms(pills and powder) and constructed a manufacturing classification system(MCS) based on the processing routes. Based on the MCS, statistical analyses were conducted respectively on medicinal materials, pharmaceutical excipients, extraction solvents in the pretreatment process, crushed medicinal materials, methods of concentration and purification, and methods of drying and granulation, aiming to uncover the process features. The results showed that each dosage form can be prepared via different routes with different processing methods of decoction pieces and raw materials for dosage preparation. The raw materials for dosage form preparation of traditional Chinese medicine OSDs included total extract, semi-extract, and total crushed powder, which accounted for different proportions. The raw materials for traditional dosage forms are mainly decoction pieces powder. Semi-extracts are the main raw materials for tablets and capsules, which account for 64.8% and 56.3%, respectively. Total extracts are the main raw materials for granules, with a proportion of 77.8%. Compared with tablets and capsules, traditional Chinese medicine granules with dissolubility requirements had a larger proportion of water extraction process, a higher proportion of refining process(34.7%), and a lower proportion of crushed medicinal mate-rials in semi-extract granules. There are four ways to add volatile oil to the modern dosage forms of traditional Chinese medicine. In addition, some new technologies and processes have been used in concentration, filtration, and granulation processes of traditional Chinese medicine OSDs, and the application of pharmaceutical excipients is diversified. The results of this study are expected to provide reference for the processing route design and upgrading of OSDs for new traditional Chinese medicines.

[Manufacturing classification system for oral solid dosage forms of traditional Chinese medicines (â ¡): classification of tablets disintegration behavior].

In this paper, 50 batches of representative traditional Chinese medicine tablets were selected and the disintegration time was examined with the method in Chinese Pharmacopoeia. The disintegration time and disintegration phenomenon were recorded, and the dissolution behaviors of water-soluble and ultraviolet-absorbent components during the disintegration process of tablets were characterized by self-control method. The results revealed that coating type and raw material type influenced the disintegration time of tablets. It was found that only 4% of traditional Chinese medicine tablets had obvious fragmentation during the disintegration process, while 96% of traditional Chinese medicine tablets showed gradual dissolution or dispersion. Furthermore, according to the disintegration speed, disintegration phenomenon, and whether the cumulative dissolution of measured components was > 90% at complete disintegration, a disintegration behavior classification system(DBCS) was created for the regular-release traditional Chinese medicine tablets. As a result, the disintegration behaviors of 50 batches of traditional Chinese medicine tablets were classified into four categories, i.e. ⅠA_2, ⅠB_1, ⅡB_1, and ⅡB_2. traditional Chinese medicine tablets(Class I) with disintegration time ≤ 30 min were defined to be rapid in disintegration, which can be the objective of optimization or improvement of Chinese herbal extract(semi extract) tablets. Different drug release models were used to fit the dissolution curve of traditional Chinese medicine tablets with gradual dissolution or dispersion phenomenon(i.e. Type B tablets). The results showed that the dissolution curves of water-soluble components in the disintegration process conformed to the zero order kinetics and the Ritger-Peppas model. It could be inferred that the disintegration mechanisms of type B tablets were a combination of dissolution controlled and swelling controlled mechanisms. This study contributes to understanding the disintegration behavior of traditional Chinese medicine tablets, and provides a reference for the design and improvement of disintegration performance of traditional Chinese medicine tablets.

[Modification of C20 oxidase in tanshinone biosynthesis pathway].

Tanshinones are one of the main effective components of Salvia miltiorrhiza, which play important roles in the treatment of cardiovascular diseases. Microbial heterogony production of tanshinones can provide a large number of raw materials for the production of traditional Chinese medicine(TCM) preparations containing S. miltiorrhiza, reduce the extraction cost, and relieve the pressure of clinical medication. The biosynthetic pathway of tanshinones contains multiple P450 enzymes, and the catalytic element with high efficiency is the basis of microbial production of tanshinones. In this study, the protein modification of CYP76AK1, a key P450-C20 hydroxylase in tanshinone pathway, was researched. The protein modeling methods SWISS-MODEL, Robetta, and AlphaFold2 were used, and the protein model was analyzed to obtain the reliable protein structure. The semi-rational design of mutant protein was carried out by molecular docking and homologous alignment. The key amino acid sites affecting the oxidation activity of CYP76AK1 were identified by molecular docking. The function of the obtained mutations was studied with yeast expression system, and the CYP76AK1 mutations with continuous oxidation function to 11-hydroxysugiol were obtained. Four key amino acid sites that affected the oxidation acti-vity were analyzed, and the reliability of three protein modeling methods was analyzed according to the mutation results. The effective protein modification sites of CYP76AK1 were reported for the first time in this study, which provides a catalytic element for different oxidation activities at C20 site for the study of the synthetic biology of tanshinones and lays a foundation for the analysis of the conti-nuous oxidation mechanism of P450-C20 modification.

Clinical efficacy of selenium supplementation in patients with Hashimoto thyroiditis: A systematic review and meta-analysis.

BACKGROUND: Evidence suggests that selenium supplementation could be useful in the treatment of Hashimoto thyroiditis (HT), but the available trials are heterogeneous. This study investigates clinically relevant effects of selenium supplementation in patients with HT. METHODS: A systematic search was performed in PubMed, Web of Science, EMBASE, Scopus, and the Cochrane Library. The latest update was performed on December 3, 2022. We investigated the changes in thyroid peroxidase antibodies (TPOAb) and thyroglobulin antibodies (TgAb) after selenium supplementation. The effect sizes were expressed as weighted mean difference (WMD) with 95% confidence intervals (CIs). RESULTS: After screening and full-text assessment, 7 controlled trials comprising 342 patients were included in the systematic review. The results showed that there was no significant change in TPOAb levels (WMD = -124.28 [95% CI: -631.08 to 382.52], P = .631, I2 = 94.5%) after 3 months of treatment. But there was a significant decrease in TPOAb levels (WMD = -284.00 [95% CI: -553.41 to -14.60], P < .05, I2 = 93.9%) and TgAb levels (WMD = -159.86 [95% CI: -293.48 to -26.24], P < .05, I2 = 85.3%) after 6 months of treatment. CONCLUSIONS: Selenium supplementation reduces serum TPOAb and TgAb levels after 6 months of treatment in patients with HT, but future studies are warranted to evaluate health-related quality or disease progression.