AIM To study the chemical constituents from the petroleum ether fraction of the roots of Gypsophila licentiana Hand.-Mazz.METHODS Silica gel,Sephadex LH-20 and semi-preparative HPLC were used for isolation and purification,then the structures of obtained compounds were identified by physicochemical properties and spectral data.RESULTS Eighteen compounds were isolated and identified as dibutyl phthalate(1),glyceryl arachidate(2),bis(2-ethylhexyl)terephthalate(3),9,12-octadecadienoic acid(Z,Z)-methyl ester(4),(3'S,4'S)-3'-acetoxy-4'-angeloyloxy-3',4'-dihydroseselin(5),3-(4-hydroxy-3-methoxyphenyl)-propanoic acid(6),bis(2-ethylhexyl)phthalate(7),2,2'-oxybis(1,4)-di-tert-butylbenzene(8),gypsogenin(9),3-keto,16α-hydroxy,24-noroleanolic acid(10),3-oxo-olean-12-en-28-oic acid(11),10-eicosenoic acid(12),hexacosanic acid(13),enniatin B(14),(R,Z)-21-methyl-8-pentatriacontene(15),ethyl gallate(16),stellarine A(17),pentacosane(18).CONCLUSION All compounds are isolated from this plant for the first time.

Subunit 5B of constitutively photomorphogenic 9 signalosome(CSN5B)is an inhibitory factor for the biosynthesis of vitamin C in the L-galactose synthesis pathway in plants.To create mutants with richer vitamin C in tomato fruits,a dual-target vector of pKSE402-SlCSN5Bwas constructed and trans-formed into the breeding parent of 1912.Based on the molecular biological assay and DNA sequencing,17 transgenic positive lines were determined,and 6 lines of them were genetically mutated at the target site of SlCSN5B,with an editing efficiency of 35.3％.Among the mutants,2 lines were homozygous mu-tants,csn5b-6 with 180 bp deletion and csn5b-8 with 3 bp deletion.The biological traits of two types of deficiency homozygotes without exogenous T-DNA insertion derived from the T1 generation were observed,and there were no significant differences in phenotypes of the plant and fruit.Through physiological tes-ting of red-ripe fruits from T,generation lines of csn5b-6,the GMPase activity and the vitamin C content significantly increased by 43％and 37.8％,respectively,the content of hydrogen peroxide significantly decreased by 25.9％,and the content of soluble solids did not obviously change,compared with the wild type.There were no significant differences in plant traits and physiological characteristics between T1 gen-eration lines of csn5b-8 and the wild type.Based on gene expression analysis and protein structure predic-tion,the results showed that the gene of SlCSN5B normally transcribed,and the loss of large peptide seg-ments of CSN5B encoded by SlCSN5B caused changes in its structure to affect functioning,which led to an increase of vitamin C content in the line of csn5b-6-1 1.The findings suggest that the vitamin C content of tomato fruit can be improved by CRISPR/Cas9-mediated SlCSN5B gene editing,which provides the valuable resources for high-quality breeding in tomato.

Subunit 5B of constitutively photomorphogenic 9 signalosome(CSN5B)is an inhibitory factor for the biosynthesis of vitamin C in the L-galactose synthesis pathway in plants.To create mutants with richer vitamin C in tomato fruits,a dual-target vector of pKSE402-SlCSN5Bwas constructed and trans-formed into the breeding parent of 1912.Based on the molecular biological assay and DNA sequencing,17 transgenic positive lines were determined,and 6 lines of them were genetically mutated at the target site of SlCSN5B,with an editing efficiency of 35.3％.Among the mutants,2 lines were homozygous mu-tants,csn5b-6 with 180 bp deletion and csn5b-8 with 3 bp deletion.The biological traits of two types of deficiency homozygotes without exogenous T-DNA insertion derived from the T1 generation were observed,and there were no significant differences in phenotypes of the plant and fruit.Through physiological tes-ting of red-ripe fruits from T,generation lines of csn5b-6,the GMPase activity and the vitamin C content significantly increased by 43％and 37.8％,respectively,the content of hydrogen peroxide significantly decreased by 25.9％,and the content of soluble solids did not obviously change,compared with the wild type.There were no significant differences in plant traits and physiological characteristics between T1 gen-eration lines of csn5b-8 and the wild type.Based on gene expression analysis and protein structure predic-tion,the results showed that the gene of SlCSN5B normally transcribed,and the loss of large peptide seg-ments of CSN5B encoded by SlCSN5B caused changes in its structure to affect functioning,which led to an increase of vitamin C content in the line of csn5b-6-1 1.The findings suggest that the vitamin C content of tomato fruit can be improved by CRISPR/Cas9-mediated SlCSN5B gene editing,which provides the valuable resources for high-quality breeding in tomato.

AIM To study the chemical constituents from the petroleum ether fraction of the roots of Gypsophila licentiana Hand.-Mazz.METHODS Silica gel,Sephadex LH-20 and semi-preparative HPLC were used for isolation and purification,then the structures of obtained compounds were identified by physicochemical properties and spectral data.RESULTS Eighteen compounds were isolated and identified as dibutyl phthalate(1),glyceryl arachidate(2),bis(2-ethylhexyl)terephthalate(3),9,12-octadecadienoic acid(Z,Z)-methyl ester(4),(3'S,4'S)-3'-acetoxy-4'-angeloyloxy-3',4'-dihydroseselin(5),3-(4-hydroxy-3-methoxyphenyl)-propanoic acid(6),bis(2-ethylhexyl)phthalate(7),2,2'-oxybis(1,4)-di-tert-butylbenzene(8),gypsogenin(9),3-keto,16α-hydroxy,24-noroleanolic acid(10),3-oxo-olean-12-en-28-oic acid(11),10-eicosenoic acid(12),hexacosanic acid(13),enniatin B(14),(R,Z)-21-methyl-8-pentatriacontene(15),ethyl gallate(16),stellarine A(17),pentacosane(18).CONCLUSION All compounds are isolated from this plant for the first time.

Objective To explore the regulatory role of transient receptor potential channel 6(TRPC6)on podocyte autophagy under the influence of homocysteine(Hcy)in mouse kidney.Methods Mouse renal podocytes were divided into control group and Hcy groups(stimulated by Hcy at 40,60,80 and 100 μmol/L for 48 h).The level of TRPC6 mRNA was assessed using quantitative reverse transcription polymerase chain reaction(qRT-PCR)to identify the optimal Hcy concentration for subsequent experiments.Western blotting was employed to evaluate the expression levels of autophagy-related proteins LC3 Ⅱ and p62,as well as the expression levels of podocyte structural proteins Nephrin and Podocin.The expression levels of TRPC6 mRNA and protein in both groups were determined using qRT-PCR,Western blotting and immunofluorescence.Transfections of cells with TRPC6 overexpression or interference were set as follows:(1)control group(untreated),negative control group of TRPC6 overexpression,and TRPC6 overexpression group;(2)control group(untreated),negative control group of TRPC6 interference,and TRPC6 interference group(si-1,si-2,si-3).The expression level of TRPC6 was detected using qRT-PCR.The cells after overexpressing or interfering of TRPC6 were further set as follows:(1)control group(untreated),Hcy group(80 μmol/L Hcy added),TRPC6 overexpression control+Hcy group,TRPC6 overexpression+Hcy group;(2)control group(untreated),Hcy group,TRPC6 interference control+Hcy group,and TRPC6 interference+Hcy group.The expression levels of p62,LC3 Ⅱ,and TRPC6 proteins were detected using Western blotting.Results qRT-PCR detection results showed that compared with control group,the expression level of TRPC6 mRNA in Hcy group increased with the increase of Hcy concentration,with the highest expression level observed at 80 μmol/L Hcy.Therefore,80 μmol/L Hcy was selected as the optimal concentration for intervention.At this time,the expression level of autophagy-related protein LC3 Ⅱ increased,and the expression level of p62 decreased(P<0.05).Western blotting results showed that compared with control group,the expression levels of podocyte-related proteins Nephrin and Podocin in Hcy group were significantly decreased(P<0.05).qRT-PCR results showed that compared with control group,the expression level of TRPC6 mRNA in Hcy group was significantly increased(P<0.05).Compared with negative control group for TRPC6 overexpression,both mRNA and protein expression levels of TRPC6 in TRPC6 overexpression group were significantly higher(P<0.05).Compared with negative control group for TRPC6 interference,both mRNA and protein expression levels of TRPC6 in TRPC6 interference group were significantly decreased(P<0.05).Western blotting results showed that compared with negative control group for TRPC6 overexpression,the expression level of autophagy-related protein LC3 Ⅱ in TRPC6 overexpression+Hcy group was significantly increased,and the expression level of p62 was significantly decreased(P<0.05).Compared with TRPC6 negative control+Hcy group for TRPC6 interference+Hcy,the expression level of autophagy-related protein LC3 Ⅱ in TRPC6 interference+Hcy group was significantly decreased,and the expression level of p62 was significantly increased(P<0.05).Conclusion Hcy can induce autophagy of renal podocytes.Inhibiting the expression of TRPC6 can significantly reduce the autophagy damage to podocytes.

This study aimed to investigate the therapeutic effect of Yunkang Oral Solution on the improvement of spleen deficiency and pregnancy outcomes in pregnant mice with spleen deficiency syndrome induced by irregular diet and over consumption of cold and bitter foods. To simulate human irregular diet and over consumption of cold and bitter foods leading to spleen deficiency, the pregnant mice with spleen deficiency syndrome were prepared using an alternate-day fasting and high-fat diet combined with oral administration of Sennae Folium. During the experiment, spleen deficiency-related indicators and diarrhea-related parameters were measured. Gastric and intestinal motility(gastric emptying rate and intestinal propulsion rate) were evaluated. The levels of serum ghrelin, growth hormone(GH), gastrin(Gas), total cholesterol(TC), low-density lipoprotein cholesterol(LDL-c), chorionic gonadotropin β(β-CG), progesterone(P), and estradiol(E_2) were measured. Intestinal barrier function in pregnant mice with spleen deficiency syndrome was assessed. Conception rate, ovarian coefficient, litter-bearing uterine coefficient, number of live fetuses, average fetal weight, and fetal length were calculated. The results showed that Yunkang Oral Solution significantly improved spleen deficiency-related indicators and diarrhea in pregnant mice with spleen deficiency syndrome, increased gastric emptying rate and intestinal propulsion rate, elevated the levels of gastrointestinal hormones(ghrelin, GH, and Gas) in the serum, and reduced lipid levels(TC and LDL-c), thereby improving lipid metabolism disorders. It also improved colonic tissue morphology, increased the number of goblet cells, and promoted the mRNA and protein expression of occludin and claudin-1 in colonic tissues, thereby alleviating intestinal barrier damage. Yunkang Oral Solution also regulated the levels of pregnancy hormones(β-CG, P, and E_2) in the serum of pregnant mice with spleen deficiency syndrome. Moreover, it increased the conception rate, ovarian coefficient, litter-bearing uterine coefficient, number of live fetuses, average fetal weight, and fetal length. These findings suggest that Yunkang Oral Solution can improve spleen deficiency-related symptoms in pregnant mice before and during pregnancy, regulate pregnancy-related hormones, and improve pregnancy outcomes.
Pregnancy ; Female ; Mice ; Humans ; Animals ; Spleen ; Ghrelin ; Fetal Weight ; Cholesterol, LDL ; Diarrhea

Inductively coupled plasma mass spectrometry (ICP-MS) was applied to determine the concentrations of lead (Pb), cadmium (Cd) and arsenic (As) in Lindera aggregata (Sims) Kosterm. The physiologically based extraction test (PBET) digestion in vitro/Caco-2 cell model was established to investigate the bioaccessible contents of Pb, Cd and As in decoction of Lindera aggregata (Sims) Kosterm. The target-organ toxicity dose modification of HI method (TTD) was used to evaluate the cumulative risk caused by the combined exposure of the total levels of Pb, Cd and As in Lindera aggregata (Sims) Kosterm. and the bioaccessible contents in the decoction. The results showed that the total contents of Pb, Cd and As in 4 batches of samples were in the range of 2.901-3.872, 1.299-1.800 and 0.062-0.216 mg·kg^-1, respectively. After transportation by Cacco-2 cells, the bioaccessible contents of Pb, Cd, and As in the decoction were in the range of 0.045-0.080, 0.070-0.112 and 0.004-0.018 mg·kg^-1. The results of risk assessment showed that calculated by the total amounts of heavy metals in the Lindera aggregata (Sims) Kosterm., for the end points of nervous system, the cumulative risks of co-exposure of heavy metals in 3 batches of samples were of concern. After decoction and transportation by Caco-2 cells, for the end points of cardiovascular system, blood, nervous system, kidney and testis, the TTD modification of HI values of all batches of samples were less than 1, and the health risks were acceptable. The study provided methodology basis for a more objective assessment of the health risks of heavy metals and harmful elements in traditional Chinese medicine and for a more scientific limit standard of heavy metals and harmful elements.

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.
Oxidoreductases ; Biosynthetic Pathways ; Molecular Docking Simulation ; Reproducibility of Results ; Salvia miltiorrhiza/chemistry* ; Amino Acids/metabolism* ; Plant Roots/genetics*

Objective: This article investigated the clinical characteristics and distribution of drug resistance mutation sites in HBV RT region of hepatitis B infected patients. Methods: Retrospective analysis was made on 1 948 patients with HBV infection, who had been tested for NAs resistance mutation and had a medical history of NAs in the Laboratory Department of the Fifth Medical Center of the PLA General Hospital from January 2020 to December 2021. Basic clinical information and drug resistance related mutation information were recorded. Meanwhile, the serological index data of hepatitis B were collected. Drug resistance gene mutant group and non-mutated group were grouped according to whether the drug resistance genes had a mutation in HBV RT region, and the clinical characteristics and genotype distribution of the two groups were statistically analyzed. The pattern of drug resistance gene mutation, number of mutation sites, drug resistance type and mutation of NAs resistance-related sites were analyzed in 917 patients with drug resistance gene mutation in HBV RT region. χ² Inspection was used for counting data. Meanwhile, two independent samples t-test and Wilcoxon rank sum test were used for measurement data. Results: Among the 1 948 patients with chronic HBV infection, 917 patients had drug resistance gene mutation in RT region (47.07%). The proportion of patients with acute hepatitis B and CHB in HBV RT resistance gene mutant group was lower than that in the non-mutated group, while the proportion of patients with HBV-related cirrhosis was higher than that in the non-mutated group, these differences were statistically significant. Compared with the non-mutated group in HBV RT region, the age, the positive rates of HBeAg and HBV DNA, and HBV DNA load of these patients were increased in drug resistance gene mutant group, these differences were statistically significant. Genotypes of patients in both groups were dominated by C, followed by B and D. The proportion of patients with genotype C in HBV RT drug resistance gene mutant group was higher than that of non-mutated group, the difference was statistically significant. There were 53 gene mutation patterns in 917 patients with drug resistance gene mutation in HBV RT region, and the main pattern was rtL180M+rtM204V+rtS202G (9.70%). The mutation sites were dominated by 3 (20.74%). There were 5 types of drug resistance, LAM+Ldt (21.25%) was the most. Among the 18 sites that were clearly associated with LAM, ADV, ETV and Ldt resistance in the HBV RT region, 14 sites were mutated, and the most common mutation sites were rtL180M, rtM204V, rtM204 and rtS202G. what's more, the proportion of patients with NAs drug resistance was LAM>Ldt>ETV>ADV. Conclusion: In order to prevent adverse consequences of this study such as disease recurrence or disease progression caused by HBV drug resistance, HBV infected patients, who have long-term use of NAs antiviral therapy, should monitor the level of HBV DNA and drug resistance genes in HBV RT region in order to optimize the treatment plan in time or guide individualized treatment.
Humans ; Hepatitis B virus/genetics* ; Hepatitis B, Chronic ; Antiviral Agents/therapeutic use* ; DNA, Viral/therapeutic use* ; Retrospective Studies ; Mutation ; Drug Resistance, Viral/genetics* ; Lamivudine/therapeutic use*

Humans ; Consensus ; Hypersensitivity ; Desensitization, Immunologic/adverse effects* ; Immunotherapy/adverse effects* ; Injections, Subcutaneous ; Allergens