[Spectral characteristics of sesquiterpene pyridine alkaloids from Tripterygium plants].

Sesquiterpene pyridine alkaloids are important components in Tripterygium plants, possessing a wide range of pharmacological activities, such as anti-inflammation immunosuppression, anti-tumor, anti-virus, and deinsectization, and are of great research value. They are composed of highly oxidized dihydro-ß-furansquiterpene and pyridine dicarboxylic acid through ester bonds. According to the structural characteristics of pyridine dicarboxylic acid fragments, they can be divided into various structural subtypes. Up to now, more than 110 sesquiterpene pyridine alkaloids have been isolated and identified from Tripterygium plants. This study reviewed the structural features and spectral(i.e., UV, IR, MS, and NMR) characteristics of sesquiterpene pyridine alkaloids and summarized the structural elucidation process in detail to provide references for their further research and development.

[Study on potential hepatotoxicity of rhein in Rhei Radix et Rhizoma based on liver metabolism].

The bilirubin metabolism mediated by the phase Ⅱ metabolizing enzyme UGT1A1 in the liver was evaluated to study the potential hepatotoxicity risk based on investigation on the inhibitory effect of rhein and its metabolites on the UGT1A1 enzyme in Rhei Radix et Rhizoma. Firstly, in vitro liver microsomes incubation was used to initiate the phase Ⅱ metabolic reaction to investigate the inhibitory effect of rheinon UGT1A1 enzyme. Secondly, the phase Ⅰ and phase Ⅱ metabolic reactions were initiated to investigate the hepatotoxicity risk of rhein metabolites. It was found that the rhein and its phase Ⅱ metabolites had no significant inhibitory effect on UGT1A1 enzyme, but its phase Ⅰ metabolites significantly reduced UGT1A1 enzyme activity. Based on the metabolites analysis, it is speculated that the rhein phase Ⅰ metabolite rheinhydroxylate and its tautomers have certain hepatotoxicity risks, while the toxicity risk induced by the prototype and phase Ⅱ metabolites of rheinglucoside, rheinglucuronic acid and rhein sulfate is small.

[Prediction of potential drug interactions of apigenin based on molecular docking and in vitro inhibition experiments].

The purpose of this study was to investigate the effect of apigenin on UGT1 A1 enzyme activity and to predict the potential drug-drug interaction of apigenin in clinical use. First,on the basis of previous experiments,the binding targets and binding strength of apigenin to UGT1 A1 enzyme were predicted by computer molecular docking method. Then the inhibitory effect of apigenin on UGT1 A1 enzyme was evaluated by in vitro human liver microsomal incubation system. Molecular docking results showed that apigenin was docked into the active region of UGT1 A1 enzyme protein F,consistent with the active region of bilirubin docking,with moderate affinity. Apigenin flavone mother nucleus mainly interacted with amino acid residues ILE343 and VAL345 to form hydrophobic binding Pi-Alkyl. At the same time,the hydroxyl group on the mother nucleus and the amino acid residue LYS346 formed an additional hydrogen bond,which increased the binding of the molecule to the protein. These results suggested that the flavonoid mother nucleus structure had a special structure binding to the enzyme protein UGT1 A1,and the introduction of hydroxyl groups into the mother nucleus can increase the binding ability. In vitro inhibition experiments showed that apigenin had a moderate inhibitory effect on UGT1 A1 enzyme in a way of competitive inhibition,which was consistent with the results of molecular docking. The results of two experiments showed that apigenin was the substrate of UGT1 A1 enzyme,which could inhibit the activity of UGT1 A1 enzyme competitively,and there was a risk of drug interaction between apigenin and UGT1 A1 enzyme substrate in clinical use.

[Research progress on chemical constituents and quality control of Tripterygium wilfordii preparations].

Tripterygium wilfordii preparations,with various biological activities such as immunosuppressive,anti-inflammatory and anti-cancer effects,are widely used in the treatment of autoimmune diseases such as rheumatoid arthritis,lupus erythematosus,and nephrotic syndrome. They have definite therapeutic effect,but often cause serious adverse reactions and result in damages to liver,kidney,blood,reproduction,and other systems due to their complex compositions,great toxicity,and narrow margin between the toxic and therapeutic dosages. At present,T. wilfordii preparations produced by different manufacturers exhibit large variations in clinical efficacy and side effects in account of their different chemical compositions and quality fluctuation due to differences in raw materials and production process. However,the existing quality standards are controversial in terms of index components and content limit,which cannot be effectively used for the overall quality control of the preparations. In this paper,the research progress on chemical constituents,quality standard and quality control methods of four T. wilfordii preparations including Tripterygium Tablets,Tripterygium Zongtie Tablets,Tripterygium Shuangceng Tablets and Tripterygium Glycosides Tablets was reviewed,in order to provide ideas and reference for the quality improvement of this type of preparations.

[Study on hepatotoxicity of physcion based on liver metabolism in vitro].

To evaluate the hepatotoxicity risks of physcion on the basis of the bilirubin metabolism mediated by glucuronidation of UDP-glucuronosyltransferases 1A1(UGT1A1 enzyme). The monomers were added into the rat liver microsomes to test the hepatotoxicity by using bilirubin as UGT1A1 enzyme substrate, with apparent inhibition constant K_i as the evaluation index. Liver microsome incubation in vitro was adopted to initiate phase Ⅱ metabolic reaction and investigate the inhibitory effect of physcion. Then the phase Ⅰ and Ⅱ metabolic reactions were initiated to investigate the comprehensive inhibition of metabolites and prototype components. The results showed that when only the phase Ⅱ reaction was initiated, physcion directly acted on the UGT1A1 enzyme in a prototype form, exhibited weak inhibition and the inhibition type was mixed inhibition; When the phase Ⅰ and Ⅱ reactions were initiated simultaneously, the inhibitory effects of physcion on UGT1A1 enzyme became strong and the inhibition type was mixed inhibition, suggesting that physcion had phase Ⅰ and Ⅱ metabolic processes, and the metabolites had strong inhibitory effect on UGT1A1 enzyme. This experiment preliminarily proved that the metabolites of physcion may be the main components to induce hepatotoxicity.

[Quality control of Guci tablets using UPLC-ELSD fingerprint analysis coupled with chemometrics].

Ultra-performance liquid chromatography-evaporative light scattering detection (UPLC-ELSD) fingerprint analysis method was established for quality control of Guci tablets. Chromatographic separation was performed on Waters Acquity UPLC BEH C18 column (2.1 mm×100 mm, 1.7 µm) at 30 °C of column temperature. Acetonitrile-0.1% formic acid solution was adopted as mobile phase for gradient elution. The flow rate was set at 0.3 mL·min⁻¹, and the injection volume was 3 µL. Detection was carried out on an ELSD with a nitrogen pressure of 0.28 MPa, drift tube temperature of 60 °C, and gain of 400. A total of 39 batches of samples produced by six manufacturers were measured by using the above method and the data were analyzed by ChemPattern software. The peak present in more than 75% of the samples was defined as a common peak, and 30 common peaks were determined. Among them, 19 peaks were identified by rapid resolution liquid chromatography/tandem mass spectrometry (RRLC-MS/MS) method, 16 of which were confirmed by reference substances. The similarity of the tested samples was 0.47-0.98, suggesting that the quality of the samples from different manufacturers varied greatly. Furthermore, principal component analysis (PCA) and hierarchical analysis (HCA) were performed to clarify the main different components in samples. The results indicated that there might be some feeding problems about Paeoniae Radix Alba, Notoginseng Radix et Rhizoma, and Clematidis Radix et Rhizoma in a few manufacturers. This study provided some evidences for the overall quality control of Guci tablets, as well as its quality standard improvements.

A comparative study on bioactive constituents in different parts of Lonicera japonica determined by HPLC-ESI-MS(n).

Lonicera japonica Thunb. is a well-known traditional herbal medicine in most East Asian countries. In China, the flower bud and stem of this plant are used for various clinical therapies, while the leaf is not officially recognized as an active part. Due to the similarities in their chemical constituents but great differences in their commercial values, the flower bud has been found to be adulterated with leaf and/or stem during the production of formulations by some drug manufactures. In order to identify adulteration in products and enable quality control, it is necessary to chemically discriminate these three parts of L. japonica. In the current study, an HPLC-ESI-MS(n) method was developed and validated for the quantitative analysis of 18 bioactive compounds: 7 organic acids, 6 iridoids, and 5 flavonoids, in batches of flower bud, stem, and leaf samples. Subsequently, chemometric analyses, such as one-way analysis of variance, principal component analysis, and hierarchical clustering analysis, were performed based on the quantitative data. The results indicated that there were remarkable differences in the distribution of the investigated compounds among the three parts of L. japonica, and that they could be straightforwardly and reliably distinguished according to their chemical profiles. Twelve compounds were selected as potential differential metabolites, which would be useful for quality control of L. japonica. As the content of caffeic acid was low in the flower bud but much higher in the stem and leaf, it could be used as a chemical marker to identify adulteration.

[Simultaneous determination of ten bioactive constituents in Lonicerae Flos by HPLC-MS-MS].

In this study, an HPLC-MS/MS method was developed and validated for simultaneous determination of six iridoids and four flavonoids in batches of Lonicerae Flos samples. Chromatographic separation was performed on a Shiseido Capcell Pak-C18 column (4.6 mm×250 mm, 5 µm). 0.1% Aqueous formic and acid (A) and acetonitrile (B) were adopted as mobile phase. Detection was carried out on a triple quadrupole mass spectrometer in the negative ion mode using an electrospray source. Multiple reaction monitoring (MRM) mode was employed. The developed method showed good linearity (R² ≥0.999 0) for all the analytes within the test ranges and the limits of quantification (LOQs) ranged from 7.4 to 31.0 µg•L⁻¹. The recoveries varied between 94.16% and 105.3%. The quantitative data indicated that the total content of iridoids (0.338%-1.440%) was much higher than that of flavonoids (0.015 4%-0.057 5%) in all samples. Moreover, it was found that there were significant differences in the content of six compounds among the samples from three different original plants, which might provide scientific evidences for the origin identification and quality control of Lonicerae Flos.

Five new compounds from Rhododendron mariae Hance.

Five new compounds (-)-(7R,8S,7'R,8'S)-4,9,4',9'-tetrahydroxy-3,3'-dimethoxy-7,7'-epoxylignan 9-O-ß-d-xylopyranoside (1), (-)-(7'R,8'S)-5'-methoxyl(dimeric coniferyl acetate) (2), (+)-(1R,2S)-1,2-bis(4-hydroxy-3-methoxyphenyl)-3-acetyl-1,3-propanediol (3), (-)-3-((2R,3R)-2-ethoxy-3-(hydroxymethyl)-7-methoxy-2,3-dihydrobenzofuran-5-yl)propan-1-ol (4), and (+)-3-((2S,3S)-2-ethoxy-3-(hydroxymethyl)-7-methoxy-2,3-dihydrobenzofuran-5-yl)propan-1-ol (5), together with 12 known compounds, were isolated from an ethanol extract of the dried stems of Rhododendron mariae Hance. Their structures were elucidated by NMR, HR-MS, CD, ORD experiments and chemical methods. Compounds 2, 3, 6, and 17 exhibited significant inhibitory effects on the nitric oxide production in lipopolysaccharide activated C57BL6/J mouse macrophages.

[Chemical constituents from roots of Illicium majus].

Ten compounds, including seven sesquiterpenes, two phenols and one phenylpropanoid, were isolated from the roots of Illicium majus by means of silica gel, ODS, Sephadex LH-20, and preparative HPLC. On analysis of MS and NMR spectroscopic data , their structures were established as cycloparviflorolide (1), cycloparvifloralone (2), tashironin (3), tashironin A (4), anislactone A(5), anislactone B (6), pseudomajucin (7), syringaldehyde (8), methyl-4-hydroxy-3, 5-dimethoxybenzoate (9), and (E)-3-methoxy-4,5-methylenedioxycinnamic alchol (10), respectively. Compounds 1-4 and 8-10 were first isolated from this plant. In the in vitro assays, at a concentration of 1.0 x 10(-5) mol x L(-1), compounds 5 and 6 were active against LPS induced NO production in microglia with a inhibition rate of 75.31% and 53.7%, respectively.

Gene silencing of the BDNF/TrkB axis in multiple myeloma blocks bone destruction and tumor burden in vitro and in vivo.

Osteolytic bone diseases are a prominent feature of multiple myeloma (MM), resulting from aberrant osteoclastic bone resorption that is uncoupled from osteoblastic bone formation. Myeloma stimulates osteoclastogenesis, which is largely dependent on an increase in receptor activator of NF-κB ligand (RANKL) and a decrease in osteoprotegerin (OPG) within the bone marrow milieu. Recently, brain-derived neurotrophic factor (BDNF) was identified as a MM-derived factor that correlates with increased RANKL levels and contributes to osteolytic bone destruction in myeloma patients. Because tyrosine receptor kinase B (TrkB), the receptor of BDNF, is abundantly expressed in osteoblasts, we sought to evaluate the role of BDNF/TrkB in myeloma-osteoblast interactions and the effect of this pathway on the RANKL/OPG ratio and osteoclastogenesis. Coculture systems constructed with noncontact transwells revealed that, in vitro, MM-derived BDNF increased RANKL and decreased OPG production in osteoblasts in a time- and dose-dependent manner. These effects were completely abolished by a specific small interfering RNA for TrkB. BDNF regulates RANKL/OPG expression in osteoblasts through the TrkB/ERK pathway. To investigate the biological effects of BDNF on myeloma in vivo, a SCID-RPMI8226 mice model was constructed using lentiviral short hairpin RNA-transfected RPMI8226 cells. In this system, stable knockdown of BDNF in MM cells significantly restored the RANKL/OPG homostasis, inhibited osteolytic bone destruction and reduced angiogenesis and tumor burden. Our studies provide further support for the potential osteoclastogenic effects of BDNF, which mediates stroma-myeloma interactions to disrupt the balance of RANKL/OPG expression, ultimately increasing osteoclastogenesis in MM.

Diterpenoids and sesquiterpenoids from the roots of Illicium majus.

Five new diterpenoids (1-5), five new sesquiterpenoids (6-10), and three known compounds (11-13) were isolated from the roots of Illicium majus. Their structures were elucidated by extensive spectroscopic analysis. The absolute configuration of 1 was assigned by X-ray crystallography, whereas those of the 1,2-diol moieties in 3 and 4 were determined using Snatzke's method. The abietane acids 1, 2, 11, 12, and 13 displayed antiviral activity against the Coxsackie B3 virus, with IC50 values of 3.3-51.7 µM/mL.

Identification and Functional Analysis of ToBPI1/LBP and ToBPI2/LBP in Anti-Bacterial Infection of Trachinotus ovatus.

Bactericidal/permeability-increasing protein (BPI) and lipopolysaccharide-binding protein (LBP) are a group of antibacterial proteins that play an important role in the host's innate immune defense against pathogen infection. In this study, two BPI/LBPs, named ToBPI1/LBP (1434 bp in length, 478 amino acids) and ToBPI2/LBP (1422 bp in length, 474 amino acids), were identified from the golden pompano. ToBPI1/LBP and ToBPI2/LBP were significantly expressed in immune-related tissues after being challenged with Streptococcus agalactiae and Vibrio alginolyticus. The two BPI/LBPs showed significant antibacterial activity against Gram-negative Escherichia coli and Gram-positive S. agalactiae and Streptococcus iniae. In contrast, the antibacterial activity against Staphylococcus aureus, Corynebacterium glutamicum, Vibrio parahaemolyticus, V. alginolyticus and Vibrio harveyi was low and decreased with time. The membrane permeability of bacteria treated with recombinant ToBPI1/LBP and ToBPI2/LBP was significantly enhanced. These results suggest that ToBPI1/LBP and ToBPI2/LBP may play important immunological roles in the immune response of the golden pompano to bacteria. This study will provide basic information and new insights into the immune response mechanism of the golden pompano to bacteria and the function of BPI/LBP.

Comparative analyses of complete chloroplast genomes reveal interspecific difference and intraspecific variation of Tripterygium genus.

The genus Tripterygium was of great medicinal value and attracted much attention on the taxonomic study using morphological and molecular methods. In this study, we assembled 12 chloroplast genomes of Tripterygium to reveal interspecific difference and intraspecific variation. The sequence length (156,692-157,061 bp) and structure of Tripterygium were conserved. Comparative analyses presented abundant variable regions for further study. Meanwhile, we determined the ndhB gene under positive selection through adaptive evolution analysis. And the phylogenetic analyses based on 15 chloroplast genomes supported the monophyly of Tripterygium hypoglaucum and the potential sister relationship between Tripterygium wilfordii and Tripterygium regelii. Molecular dating analysis indicated that the divergence time within Tripterygium was approximately 5.99 Ma (95% HPD = 3.11-8.68 Ma). The results in our study provided new insights into the taxonomy, evolution process, and phylogenetic construction of Tripterygium using complete plastid genomes.

Development and validation of an online calculator to predict the pathological nature of colorectal tumors.

BACKGROUND: No single endoscopic feature can reliably predict the pathological nature of colorectal tumors (CRTs). AIM: To establish and validate a simple online calculator to predict the pathological nature of CRTs based on white-light endoscopy. METHODS: This was a single-center study. During the identification stage, 530 consecutive patients with CRTs were enrolled from January 2015 to December 2021 as the derivation group. Logistic regression analysis was performed. A novel online calculator to predict the pathological nature of CRTs based on white-light images was established and verified internally. During the validation stage, two series of 110 images obtained using white-light endoscopy were distributed to 10 endoscopists [five highly experienced endoscopists and five less experienced endoscopists (LEEs)] for external validation before and after systematic training. RESULTS: A total of 750 patients were included, with an average age of 63.6 ± 10.4 years. Early colorectal cancer (ECRC) was detected in 351 (46.8%) patients. Tumor size, left semicolon site, rectal site, acanthosis, depression and an uneven surface were independent risk factors for ECRC. The C-index of the ECRC calculator prediction model was 0.906 (P = 0.225, Hosmer-Lemeshow test). For the LEEs, significant improvement was made in the sensitivity, specificity and accuracy (57.6% vs 75.5%; 72.3% vs 82.4%; 64.2% vs 80.2%; P < 0.05), respectively, after training with the ECRC online calculator prediction model. CONCLUSION: A novel online calculator including tumor size, location, acanthosis, depression, and uneven surface can accurately predict the pathological nature of ECRC.

A general procedure for establishing composite quality evaluation indices based on key quality attributes of traditional Chinese medicine.

Licorice, a medicinal herb and food flavor ingredient, has been widely used in traditional Chinese medicine (TCM) for the past 4000 years. In this study, we propose a new quality evaluation approach for licorice quality control based on the key quality attributes commonly used in TCM. The high quality of TCM formulations is ensured by verifying the genuine origin and implementing good agricultural and collection practices for each medicinal herb. In our study, the genuine production area, the harvest season, and the number of growth years were considered the key quality attributes of TCM. To ensure the representativeness of our analysis, we obtained a total of 158 licorice sample batches that differed in the number of growth years, the location of the production areas, and the season for harvesting. Initially, the 158 sample batches were subjected to ultra-high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS/MS). A preliminary screen identified 11 licorice compounds related to the three key quality attributes of TCM . An analysis by ultra-high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UHPLC-TQ-MS/MS) verified the presence of 34 compounds in all licorice samples. These 34 compounds included the 11 compounds related to the three key quality attributes of the samples, along with other bioactive components identified in previous studies. After using UHPLC-TQ-MS/MS to assess the signal peak intensities of the 34 compounds, we selected 17 licorice compounds to establish sample content evaluation indices, which were determined by high-performance liquid chromatography at four different wavelengths in all 158 licorice sample batches. Finally, the screen identified nine compounds that were closely associated with the quality attributes of licorice based on principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). Our results suggested that liquiritin and eight other compounds could be used as quality control indicators of licorice, which provided a foundation to establish the TCM quality composite evaluation index (TCM QCEI). In summary, this research concept can serve as a reference for research on quality markers and the evaluation of TCM.

Diagnostic performance of narrow-band imaging international colorectal endoscopic and Japanese narrow-band imaging expert team classification systems for colorectal cancer and precancerous lesions.

BACKGROUND: In recent years, two new narrow-band imaging (NBI) classifications have been proposed: The NBI international colorectal endoscopic (NICE) classification and Japanese NBI expert team (JNET) classification. Most validation studies of the two new NBI classifications were conducted in classification setting units by experienced endoscopists, and the application of use in different centers among endoscopists with different endoscopy skills remains unknown. AIM: To evaluate clinical application and possible problems of NICE and JNET classification for the differential diagnosis of colorectal cancer and precancerous lesions. METHODS: Six endoscopists with varying levels of experience participated in this study. Eighty-seven consecutive patients with a total of 125 lesions were photographed during non-magnifying conventional white-light colonoscopy, non-magnifying NBI, and magnifying NBI. The three groups of endoscopic pictures of each lesion were evaluated by the six endoscopists in randomized order using the NICE and JENT classifications separately. Then we calculated the six endoscopists' sensitivity, specificity, accuracy, positive predictive value, and negative predictive value for each category of the two classifications. RESULTS: The sensitivity, specificity, and accuracy of JNET classification type 1 and 3 were similar to NICE classification type 1 and 3 in both the highly experienced endoscopist (HEE) and less-experienced endoscopist (LEE) groups. The specificity of JNET classification type 1 and 3 and NICE classification type 3 in both the HEE and LEE groups was > 95%, and the overall interobserver agreement was good in both groups. The sensitivity of NICE classification type 3 lesions for diagnosis of SM-d carcinoma in the HEE group was significantly superior to that in the LEE group (91.7% vs 83.3%; P = 0.042). The sensitivity of JNET classification type 2B lesions for the diagnosis of high-grade dysplasia or superficial submucosal invasive carcinoma in the HEE and LEE groups was 53.8% and 51.3%, respectively. Compared with other types of JNET classification, the diagnostic ability of type 2B was the weakest. CONCLUSION: The treatment strategy of the two classification type 1 and 3 lesions can be based on the results of endoscopic examination. JNET type 2B lesions need further examination.

Diagnostic value of novel retroflexion colonoscopy in the right colon: A randomized controlled trial.

BACKGROUND: Colonoscopy is the accepted gold standard for the detection of colorectal cancer. However, colonoscopy is less effective in preventing colon cancer in the right side compared with the left side. AIM: To investigate the feasibility of a novel type of retroflexion colonoscope, EC-3490Ti colonoscope, for detection of proximal colon lesions. METHODS: In this prospective trial, we recruited patients who underwent colonoscopy for screening or surveillance. When the endoscopists could not grasp the whole observation of the right-side colon mucosa in the forward view (FV), insertion and withdrawal were repeatedly performed in the FV group with the EC38-i10F colonoscope while retroflexion was performed in the retroflexed view (RV) group with the EC-3490Ti colonoscope. Adenoma detection rate, the total number of adenomas per positive participant, the success rate of retroflexion, and endoscope withdrawal time were recorded and compared. RESULTS: The total adenoma detection rate (39.3% vs 37.7%, P = 0.646) did not show any significant difference between the two groups. However, the polyp detection rate (59.6% vs 51.0%, P = 0.002), adenoma detection rate in the right colon (21.6% vs 14.4%, P = 0.012), and the total number of adenomas per positive participant (2.1 vs 1.7, P = 0.011) reached statistical significance. Retroflexion was achieved in 91.7% of our cohort. Compared with the FV group, the withdrawal time was significantly prolonged in the RV group (586.1 ± 124.4 s vs 508.8 ± 129.6 s, P < 0.001). In contrast, the proportion of additional ancillary pressure decreased (27.4% vs 45.7%, P < 0.001), and the visual analog scale pain scores did not increase (2.7 ± 1.4 vs 2.8 ± 1.4, P = 0.377). CONCLUSION: Retroflexion in the proximal colon could be performed successfully and safely with the EC-3490Ti colonoscope. This maneuver could detect more adenomas effectively.

Accumulated Clinical Experiences from Successful Treatment of 1377 Severe and Critically Ill COVID-19 Cases.

In late December 2019, COVID-19 was firstly recognized in Wuhan, China and spread rapidly to all of the provinces of China. The West Campus of Wuhan Union Hospital, the designated hospital to admit and treat the severe and critically ill COVID-19 cases, has treated a large number of such patients with great success and obtained lots of valuable experiences based on the Chinese guideline (V7.0). To standardize and share the treatment procedures of severe and critically ill cases, Wuhan Union Hospital has established a working group and formulated an operational recommendation, including the monitoring, early warning indicators, and several treatment principles for severe and critically ill cases. The treatment experiences may provide some constructive suggestions for treating the severe and critically ill COVID-19 cases all over the world.

[Effect of RNA interference targeting brain-derived neurotrophic factor gene on HeLa cell proliferation and apoptosis].

OBJECTIVE: To screen effective sequences of short hairpin RNA on brain-derived neurotrophic factor (BDNF) gene and the effect of RNA interference on the proliferation and apoptosis of HeLa cells, a cervix carcinoma cell line with high expression of BDNF. METHODS: Two recombinant eukaryotic human-BDNF siRNA expression vectors were designed and constructed. Sequences were confirmed by restrictive endonuclease digestion and DNA sequencing. The empty vector pGenesil-1 and two recombinant plasmids, pGenesil-shRNA-BDNF1 and pGenesil-shRNA-BDNF2, were transfected into HeLa cells using Lipofectamine 2000 (groups: P(0), P(1) and P(2), respectively). The mRNA and protein levels of BDNF in HeLa cells were detected by RT-PCR and Western blot, respectively. The cellular proliferation rates were determined by MTT assay and the apoptotic rates were measured by flow cytometry and Hoechest 33258. RESULTS: The recombinant eukaryotic BDNF siRNA expression vectors were successfully constructed. The expression of mRNA and protein of BDNF in P(1) group were significantly decreased, comparing with non-transfected group, P(0) and P(2) groups (F = 48.19, P < 0.01). P(2) group failed to meet the expected results (P > 0.05). In addition, the proliferation activity was reduced in P(1) group and the peak point of proliferation curve was prolonged. Moreover, the early cell apoptotic rates were statistically increased in P(1)[(53.4 +/- 4.2)%] VS. non-transfected [(0.8 +/- 0.4)%], P(0) [(5.1 +/- 1.8)%] and P(2)[(7.9 +/- 2.4)%] groups (F = 269.77, P < 0.01). CONCLUSION: HeLa cells express a high level of BDNF. BDNF gene silencing by RNA interference increases the apoptosis of HeLa cells and inhibits cell proliferation, offering a possible target for efficient tumor therapy.