Cationic Nanoemulsion-Encapsulated Retinoic Acid as an Adjuvant to Promote OVA-Specific Systemic and Mucosal Responses.

Cationic nanostructures have emerged as an adjuvant and antigen delivery system that enhances dendritic cell maturation, ROS generation, and antigen uptake and then promotes antigen-specific immune responses. In recent years, retinoic acid (RA) has received increasing attention due to its effect in activating the mucosal immune response; however, in order to use RA as a mucosal adjuvant, it is necessary to solve the problem of its dissolution, loading, and delivery. Here, we describe a cationic nanoemulsion-encapsulated retinoic acid (CNE-RA) delivery system composed of the cationic lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOTAP), retinoic acid, squalene as the oil phase, polysorbate 80 as surfactant, and sorbitan trioleate 85 as co-surfactant. Its physical and chemical properties were characterized using dynamic light scattering and a spectrophotometer. Immunization of mice with the mixture of antigen (ovalbumin, OVA) and CNE-RA significantly elevated the levels of anti-OVA secretory immunoglobulin A (sIgA) in vaginal lavage fluid and the small intestinal lavage fluid of mice compared with OVA alone. This protocol describes a detailed method for the preparation, characterization, and evaluation of the adjuvant effect of CNE-RA.

Eremophilane and cadinane sesquiterpenoids from the fruits of Alpinia oxyphylla and their anti-inflammatory activities.

The fruits of Alpinia oxyphylla have been used for centuries in China as both edible resources and traditional Chinese medicine. In order to identify structurally interesting and bioactive constituents from the fruits of A. oxyphylla, bioassay-guided fractionation and purification of the crude extracts were performed, which led to the isolation of 38 sesquiterpenoids, including six previously undescribed eremophilane sesquiterpenoids (1-6), six new cadinane sesquiterpenoids (23-24, 26-29), and 26 known analogues (7-22, 25 and 31-38). The structures of these compounds were elucidated by comprehensive spectroscopic data analysis, single crystal X-ray diffraction, quantum chemistry calculations (13C-NMR and ECD), and Mo2(OAc)4 reaction. Several of the isolated compounds (8, 13, 17, 18, 30, 31 and 35) showed moderate to strong inhibition of the secretion of cytokines (NO, TNF-α and IL-6) in LPS-stimulated BV-2 cells. Furthermore, western blot, immunofluorescence, and real-time PCR assays indicated that 18 could down-regulate the mRNA levels of TNF-α, IL-6, COX-2, and iNOS and the protein expression of COX-2 and iNOS. Meanwhile, 18 was able to partially inhibit the phosphorylation of ERK1/2, JNK, and p38. Thus, the discovery of structurally diverse anti-inflammatory sesquiterpenoids from the fruits of A. oxyphylla in this study could benefit the further development and utilization of this plant.

Synthetic Self-Adjuvanted Lipopeptide Vaccines Conferred Protection against Helicobacter pylori Infection.

Helicobacter pylori (H. pylori) colonizes the stomach epithelium of half the world's population and is responsible for various digestive diseases and even stomach cancer. Vaccine-mediated protection against H. pylori infection depends primarily on the specific mucosal and T-cell responses. In this study, the synthetic lipopeptide vaccines, Hp4 (Pam2 Cys modified UreB T-cell epitope) and Hp10 (Pam2 Cys modified CagA T/B cell combined epitope), not only induce the bone marrow derived dendritic cells (BMDCs) maturation by activating a variety of pattern-recognition receptors (PRRs) such as Toll-like receptor (TLR), Nod-like receptor (NLR), and retinoic acid-inducing gene (RIG) I-like receptor (RLR), and but also stimulate BMDCs to secret cytokines that have the potential to modulate T-cell activation and differentiation. Although intranasal immunization with Hp4 or Hp10 elicits robust epitope-specific T-cell responses in mice, only Hp10 confers protection against H. pylori infection, possibly due to the fact that Hp10 also induces substantial specific sIgA response at mucosal sites. Interestingly, Hp4 elevates the protective response against H. pylori infection of Hp10 when administrated in combination, characterized by better protective effect and enhanced specific T-cell and mucosal antibody responses. The results suggest that synthetic lipopeptide vaccines based on the epitopes derived from the protective antigens are promising candidates for protection against H. pylori infection.

[Observation of effect of Wu Yun Liu Qi on reproductive hormones in varicocele patients].

Objective： To observe roproductive hormone levels in varicocele patients during a cycle (6 years) of Wu Yun Liu Qi, and explore whether the cycle had effect on the roproductive hormone levels. Methods： Data of roproductive hormone levels in varicocele patients from 2015 to 2020 were analyzed retrospectively. FSH、LH、T、PRL、E2 levels and T/E2 ratio were compared among the six years. According to Chinese sexagenary cycle heavenly stems and earthly branches of each year from 2015 to 2020 its yunqi characteristics were determined. Results: Totally data of 848 cases of varicocele patients were collected from 2015 to 2020. Among which, in 2015 (Yiwei year) there were 57 cases, in 2016 (Bingshen year) 83 cases, in 2017 (Dingyou year ) 133 cases, in 2018(Wuxu year) 156 cases, in 2019(Sihai year) 274 cases, and in 2020(Gengzi year) 145 cases. The levels of FSH、LH、PRL、T were not diferrent statistically from the six years except individual year. However, the level of E2 in 2016 when the Yunqi was Shao Yang Xiang Huo Si Tian and Jue Yin Feng Mu Zai Quan obviously higher than other years excpet 2018( All P< 0.05). And T/E2 ratio was lower in 2016 than other years except 2018 and 2020( All P< 0.05). Conclusions: Shi Xiang factors of Wu Yun Liu Qi had effect on roproductive hormone levels in varicocele patients, showing by higher E2 level in Yinshen year when the Shi Xiang factors may have bad effect on human fertility.

Successful treatment of stage IIIB intrahepatic cholangiocarcinoma using neoadjuvant therapy with the PD-1 inhibitor camrelizumab: A case report.

BACKGROUND: The prognosis of intrahepatic cholangiocarcinoma (ICC) with lymph node metastasis is poor. The feasibility of surgery is not certain, which is a contraindication according to the National Comprehensive Cancer Network guidelines. The role of immunotherapy as a neoadjuvant therapy for ICC is not clear. We herein describe a case of ICC with lymph node metastasis that was successfully treated with neoadjuvant therapy. CASE SUMMARY: A 60-year-old man with a liver tumor was admitted to our hospital. Enhanced computed tomography and magnetic resonance imaging revealed a space-occupying lesion in the right lobe of the liver. Multiple subfoci were found around the tumor, and the right posterior branch of the portal vein was invaded. Liver biopsy indicated poorly differentiated cholangiocytes. According to the American Joint Committee on Cancer disease stage classification, ICC with hilar lymph node metastasis (stage IIIB) and para-aortic lymph node metastasis was suspected. A report showed that two patients with stage IIIB ICC achieved a complete response (CR) 13 mo and 16 mo after chemotherapy with a PD-1 monoclonal antibody. After multidisciplinary consultation, the patient was given neoadjuvant therapy, surgical resection and lymph node dissection, and postoperative adjuvant therapy. After three rounds of PD-1 immunotherapy (camrelizumab) and two rounds of gemcitabine combined with cisplatin regimen chemotherapy, the tumor size was reduced. Therefore, a partial response was achieved. Exploratory laparotomy found that the lymph nodes of Group 16 were negative, and the tumor could be surgically removed. Therefore, the patient underwent right hemihepatectomy plus lymph node dissection. The patient received six rounds of chemotherapy and five rounds of PD-1 treatment postoperatively. After 8 mo of follow-up, no recurrence was found, and a CR was achieved. CONCLUSION: Neoadjuvant therapy combined with surgical resection is useful for advanced-stage ICC. This is the first report of successful treatment of stage IIIB ICC using neoadjuvant therapy with a PD-1 inhibitor.

Eight new phenolic acids from the leaves of Illicium dunnianum and their osteoprotective activities.

Eight previously unreported phenolic acids (1-8), including three new phenylpropenoid glycosides (1-3), and five undescribed shikimic acid derivatives (4-8), together with six known analogues (9-14), were obtained from the dried leaves of Illicium dunnianum. The structures of these new compounds were elucidated by extensive spectroscopic analyses (1D, 2D-NMR, HRESIMS, IR, UV) and chemical methods. Compounds 1, 2, 4, and 5 were tested for their promotion effect on osteoblastogenesis of pre-osteoblastic MC3T3-E1 cells and inhibitory effect on osteoclastogenesis of RANKL-induced RAW264.7 cells. As a result, 1 and 4 exerted a promotion effect on osteoblastogenesis, but without activity on osteoclastogenesis. Our studies not only enrich the structural diversity of phenolic acids in nature, but also discover new lead compounds from folk plants with activities on osteoblastogenesis or osteoclastogenesis.

Two new iridoid glycosides from the fruit of Gardenia jasminoides.

Two new iridoid glycosides, 2'-O-cis-coumaroylgardoside (1), and 6'-O-caffeoylioxide (2), were isolated from the fruit of Gardenia jasminoides. The structures of these compounds were elucidated based on spectroscopic analysis (HR-ESI-MS, NMR) and chemical methods. The anti-inflammatory activities of the isolates were evaluated by measuring their inhibitory effects on PGE2 production in LPS stimulated RAW 264.7 macrophages, compounds 1 and 2 could reduce PGE2 levels in LPS-activated RAW 264.7 macrophages with IC50 values of 121.4 and 83.38 µM, respectively.

Arteannoides U-Z: Six undescribed sesquiterpenoids with anti-inflammatory activities from the aerial parts of Artemisia annua (Qinghao).

Four previously unreported sesquiterpenoid diasteromers, arteannoides U-X (1-4), together with one new norsesquiterpenoid 5 (arteannoide Y) and one undescribed rearranged cadinene sesquiterpenoid 6 (arteannoide Z) were obtained from the dried aerial parts of Artemisia annua (Qinghao). Notably, arteannoides U-X (1-4) are four stereoisomers that possess the same molecules and the same planar connectivity, but differ from each other in configuration at a certain stereocenter. Their accurate structures were unambiguously identified and distinguished by extensive spectroscopic analyses, NMR calculations with DP4+ analysis, electronic circular dichroism (ECD) calculations and X-ray diffraction analyses. Compounds 1, 3, and 4 showed inhibitory activities against the production of inflammatory cytokines (PGE2, NO, IL-6 and TNF-α) in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages.

Reference intervals for preterm thyroid function during the fifth to seventh day of life.

BACKGROUND: Due to the lack of reference intervals for serum free triiodothyronine (FT3), free thyroxine (FT4) and thyroid stimulating hormone (TSH) in preterm neonates during the 5th to 7th day of life, we performed a retrospective study using the chemiluminescence immunoassay system. METHODS: A total of 2040 preterm neonates with a gestational age (GA) of 26-35 weeks in the neonatal intensive care unit from 2014 to 2019 were included. Their serum FT3, FT4 and TSH values were calculated and analyzed to establish reference intervals for preterm neonates stratified by GA. The comparisons of FT3, FT4 and TSH were made by sex (males and females) and gestational age (26-28 weeks; 29-32 weeks; 33-35 weeks). RESULTS: The reference intervals for FT3, FT4 and TSH in preterm neonates with a GA of 26-35 weeks were (1.65~5.21) pmol/L, (8.64~25.41) pmol/L, and (0.406~12.468) mlU/L, respectively. There were significant differences between serum FT3 and FT4 values and GA, while TSH levels were not significantly different (P < 0.01). The serum FT3 values of males were lower than those of females, especially in the 29-32 weeks group. No significant differences in serum values between sexes were found in FT4 or TSH (P > 0.05). CONCLUSION: Reference intervals of thyroid function tests were established to determine the early diagnostic criteria of thyroid diseases for neonates with a GA of 26-35 weeks and to avoid unnecessary retesting and interventions. The reference intervals of FT4 can be used as an indicator to regulate the doses of thyroid hormone supplement in the treatments of congenital hypothyroidism.

Systematically identifying the anti-inflammatory constituents of Cimicifuga dahurica by UPLC-Q/TOF-MS combined with network pharmacology analysis.

Cimicifuga dahurica (Turcz.) Maxim, which is also regarded as the main origin of "Shengma" in the Chinese Pharmacopoeia, has been used as a cooling and detoxification agent for thousands of years. Our previous phytochemical investigations of C. dahurica extracts (CDEs) led to the isolation of a series of 9,19-cycloalkane triterpenoids and phenolic acids showing a potential anti-inflammatory activity. However, the chemical profiling of CDEs and the material basis of its anti-inflammatory effect in vivo has not been clarified. In the present study, the CDE chemical profile and prototype components in rat plasma were identified via ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. As a result, a total of 106 components were identified or tentatively characterized in CDEs, including 54 triterpenoids, 35 phenolic acids, eight amides and nine other type constituents (39 compounds were confirmed with the reference standards). In addition, 20 prototype components (15 triterpenoids and five phenolic acids) were identified in rat plasma, which potentially related to the anti-inflammatory effects of CDEs. Moreover, the anti-inflammatory activities of the main prototype components were further evaluated by their inhibitory effects on the production of NO, as well as the expressions of iNOS and COX-2 in lipopolysaccharide-stimulated RAW264.7 cells, which indicated that 9,19-cycloalkane triterpenoids may play an anti-inflammatory role by down-regulating the expression of iNOS.

[Research on chemical constituents from Artemisia annua â ].

Chemical constituents were isolated and purified from the water extract of Artemisia annua by column chromatography of HP-20 macroporous resin, silica gel, ODS, Sephadex LH-20, HW-40, and semi-preparative RP-HPLC. Their structures were elucidated by physicochemical properties and spectral analyses. As a result, Fifteen compounds were isolated and identified as vitexnegheteroin M(1), sibricose A5(2), securoside A(3), citrusin D(4), annphenone(5), E-melilotoside(6), esculetin(7), scopoletin-7-O-ß-D-glucoside(8), eleutheroside B_1(9), chrysosplenol D(10), patuletin-3-O-ß-D-glucopyranoside(11), quercetin-7-O-ß-D-glucoside(12), rutin(13), apigenin 6,8-di-C-ß-D-glucopyranoside(14), isoschaftoside(15), among them, compounds 1-4 were identified from Artemisia for the first time. Additionally, the isolates were evaluated for their inhibitory effects on the production of PGE_2 in LPS-simulated RAW264.7 macrophages. The results showed that compounds 1, 2, 8, and 10-15 could reduce PGE_2 levels, to a certain extent.

Metabolic profiles of Jin-hong tablets in rats by ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry.

Jin-hong tablets (JHTs), a well-known traditional Chinese patent medicine (TCPM), have been effectively used for the treatment of chronic superficial gastritis (CSG). The metabolic profile of TCPMs is performed to determine their bioactive components. In this study, a five-step strategy based on ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry and metabolynx™ software combined with mass defect filter technique was developed to delineate the metabolic profile of JHT in vivo. As a result, a total of 163 JHT-related xenobiotics (38 prototypes and 125 metabolites) were identified or tentatively characterized in rat biological samples, and the phase I and II metabolism processes mainly included demethylation, hydroxylation, sulfation, and glucuronidation. In addition, after oral administration of JHT, a large amount of alkaloid-related ingredients was detected in rat plasma samples, indicating that alkaloids may play an important role in the treatment of CSG with JHT. This study is beneficial for understanding the JHT's in vivo metabolic profiles and characteristics, which helps to reveal its in vivo effective components and provides a solid basis for further studies on its functional mechanism.

[Transcriptome analysis of rhizome of Polygonatum cyrtonema and identification of candidate genes involved in biosynthetic pathway of steroidal saponin].

To enrich the transcriptome data in rhizome of Polygonatum cyrtonema seedlings, identify candidate functional genes involved in steroidal saponin biosynthesis and provide genetic resources for the research on anabolism pathway and regulatory mechanism of active components in P. cyrtonema, Illumina platform was applied to perform transcriptomic sequencing of rhizome of P. cyrtonema, followed by a series of bioinformatics analysis on RNA-seq data, including de novo assembly, annotation, classification and metabolic pathway analysis of the assembled unigene. Meanwhile, a deep analysis on the steroidal saponin biosynthesis in secondary metabolism pathway was performed. The results showed a total of 126 546 unigene were obtained by de novo transcriptome assembly, of which 47 226 were annotated. Of these, 16 499 unigene were mapped to 132 specific pathways, of which 2 768 were identified to be involved in 22 secondary metabolic pathways. One hundred and thirteen unigene were identified from the transcriptome database, which encoded 27 metabolic enzymes associated with steroidal saponin biosynthesis and shared similarity with 45 functional genes in Arabidopsis thaliana. In conclusion, a series of candidate functional genes, which might be involved in steroidal saponin biosynthesis, were selected from the transcriptome database of P. cyrtonema rhizome. Further investigation of these candidate genes will provide insight into their actual functions in the steroidal saponin biosynthetic pathway in P. cyrtonema. In addition, this study also provide abunant reference data for transcriptome characterization of P. cyrtonema and has important significance for functional genomics of P. cyrtonema.

[Selection and validation of internal reference genes for qPCR in Polygonatum cyrtonema tubers at different development stages and in response to abiotic stress].

In order to analyze the expression of genes involved in steroidal saponin biosynthesis pathway in Polygonatum cyrtonema tubers, it is very important to select internal reference genes that are stably expressed at different development stages and in response to abiotic stress. According to the previously established P. cyrtonema transcriptome database and reported internal reference genes in plant, this study systematically analyzed eight candidate internal reference genes including histone H2 A, glyceraldehyde-3-phosphate dehydrogenase, ACTIN, ß-tubulin, ubiquitin-conjugating enzyme-E2-10, elongation factor 1-alpha isoform, 18 S rRNA and α-tubulin 4 for expression stability in P. cyrtonema tubers at different development stages and in response to methyl jasmonate(MeJA) stress by using Real time fluorescence quantitative PCR(qPCR). Based on the statistical analysis of qPCR results by using GeNorm, NormFinder and BestKeeper softwares, the expression of ubiquitin-conjugating enzyme-E2-10 and elongation factor 1-alpha isoform are the most stable in P. cyrtonema tubes at different development stages and in response to MeJA stress. The two internal reference genes were further validated by analyzing the expression of 4 genes involved in steroidal saponin biosynthesis pathways. In conclusion, ubiquitin-conjugating enzyme-E2-10 and elongation factor 1-alpha isoform can be used as the most appropriate internal reference genes for qPCR analysis in P. cyrtonema. This study also provide a foundation for future investigate the molecular mechanism of steroidal saponin biosynthesis pathways in P. cyrtonema.

Monoterpene glycosides with anti-inflammatory activity from Paeoniae Radix.

Six new monoterpene glycosides, named 6'-O-nicotinoylalbiflorin (1), 4'-O-vanillylalbiflorin (2), paeonidanin L (3), paeoniflorigenin-1-O-ß-d-xyloside (4), 6'-(2-hydroxypropanoyl)-paeoniflorin (5), oxylactiflorin (6), together with 16known ones (7-22) were isolated from the 70% ethanol extract of Paeoniae Radix. Their structures were elucidated based on spectroscopic analysis (1D and 2D NMR, HRESIMS, IR and UV), chemical evidences and comparison with literatures. The inhibitory effects of all the isolates were evaluated against lipopolysaccharide (LPS) stimulated PGE2 production in RAW 264.7 macrophages.

Phenolic acids and their glycosides from the rhizomes of Cimicifuga dahurica.

Phytochemical study on rhizomes of Cimicifuga dahurica resulted in the isolation of nine new neolignan and phenylpropanoid glycosides, cimicifugasides A-E (1, 2, 7-9), cimicifugamides B-D (3-5), shomaside G (6) along with four known compounds (10-13). Their structures were identified by extensive spectroscopic analyses (1D-, 2D-NMR, MS, CD, IR, UV) and chemical methods. Their anti-inflammatory potentials were evaluated by measuring their effects on PGE2 production of LPS-stimulated RAW264.7 cells, and compounds 12 and 13 showed moderate anti-inflammatory activities.

Two new labdane diterpenoids from aerial parts of Leonurus japonicus and their anti-inflammatory activity.

Two new labdane diterpenoids, Leojaponin E (1) and F (2), together with three known compounds were isolated from the dried herb of Leonurus japonicus Houtt., Lamiaceae. Their structures were determined based on extensive spectroscopic analyses. The absolute configurations of 1 and 2 were elucidated on the basis of experimental and calculated electronic circular dichroism spectra. In addition, compounds 1 and 2 exerted inhibition of LPS-induced PGE2 production in a dose-dependent manner at concentrations ranging from 5 to 20 µM.

[Simultaneous determination of thirteen components in Tong'an injection by LC-MS/MS].

The aim of this study was to develop a simple, sensitive ultra performance liquid chromatography mass spectrometry (UPLC-MS/MS) method for the determination of syringaresinol, N-trans-feruloyltyramine, chelerythrine chloride, sinomenine, coptisine chloride, sanguinarine, chelidonine, magnolflorine, allocryptopine, protopine, farrerol, stylopine and dihydrosanguin-arine in Tong'an injection (TAI), which could be used for the quality control of TAI. The UPLC analysis was performed on Agilent Zorbax SB-Aq column (2.1 mm×150 mm,3.5 µm), with 0.1% formic acid solution (A) -acetonitrile (B) as the mobile phase for gradient elution (0.01-2 min, 5%B; 2-8 min, 5%-30%B; 8-11 min, 30%-95%B; 11-13 min, 95%B; 13-13.1 min, 95%-5%B; 13.1-14 min, 5%B). The flow rate was 0.5 mL•min⁻¹, and the column temperature was 25 ℃; multiple reaction monitoring (MRM) was performed in electrospray ion source positive ion mode for quantitative determination. The calibration curves for the above thirteen compounds showed good linear relationship in corresponding mass concentration range (r>0.999 0). The average recovery rate of the compounds ranged from 95.70% to 104.8%, with RSD of less than 1.9%. The contents of thirteen active components in 10 batches of TAI were 0.021 2-0.029 0, 0.001 7-0.002 3, 0.000 9-0.001 3, 5.952-6.205 2, 0.195 4-0.240 5, 0.002 0-0.002 9, 0.693-0.798 2, 0.069 3-0.078 2, 0.089 29-0.102 9, 0.386 5-0.420 1, 0.014 3-0.015 9, 0.755 3-0.842 1, and 0.008 2-0.011 2 g•L⁻¹ respectively. Methodology validation proved that this method was simple, rapid, sensitive and accurate, which can be used to provide reference for the comprehensive evaluation of TAI quality. The determination results of 10 batches of TAI showed the content of each batch had no significant difference. The results may provide a basis for the quality control of TAI.

A new hetero dimeric terpenoid derivative, japonicaside C, from the flower buds of Lonicera japonica.

A rare hetero dimeric terpenoid derivative, named japonicaside C, together with five known secoiridoid gloucosides were isolated from the flower buds of Lonicera japonica. The structures of these compounds were established on the basis of spectroscopic analyses. Japonicaside C is the first representative of a novel type of hetero dimeric terpenoid, biogenetically originated from a guaiane-type sesquiterpenoid and a secoiridoid glucoside. Anti-virus activity of the isolated compounds was evaluated in vitro.

[Effect of rat intestinal flora on in vitro metabolic transformation of pumiloside].

To study the metabolic transformation of pumiloside by rat intestinal flora in vitro and identify its metabolites. Pumiloside was incubated in the rat intestinal flora in vitro. HPLC was used to monitor the metabolic process, and HPLC-Q-TOF-MS was used to identify the structures of biotransformation products. In vitro, pumiloside was easily metabolized by rat intestinal flora, and with the prolongation of metabolic time, pumiloside was transformed into several metabolites. Three metabolites were initially identified in this experiment. The study indicated that pumiloside could be extensively metabolized in the rat intestinal flora in vitro.