Natural products (NPs) have long held a significant position in various fields such as medicine, food, agriculture, and materials. The chemical space covered by NPs is extensive but often underexplored. Therefore, high-throughput and efficient methodologies for the annotation and discovery of NPs are desired to address the complexity and diversity of NP-based systems. Mass spectrometry (MS) has emerged as a powerful platform for the annotation and discovery of NPs. MS databases provide vital support for the structural characterization of NPs by integrating extensive mass spectral data and sample information. Additionally, the released annotation methodologies, based on a variety of informatics tools, continuously improve the ability to annotate the structure and properties of compounds. This review examines the current mainstream databases and annotation methodologies, focusing on their advantages and limitations. Prospects for future technological advancements are then discussed in terms of novel applications and research objectives. Through a systematic overview, this review aims to provide valuable insights and a reference for MS-based NPs annotation, thereby promoting the discovery of novel natural entities.
Biological Products/chemistry* ; Mass Spectrometry/methods* ; Databases, Factual ; Drug Discovery/methods* ; Humans

BACKGROUND:Previous studies found that the proliferative scar-specific long non-coding RNA lncRNA HSFAS is a novel biomarker that can be used in the diagnosis of hypertrophic scar,but how it functions in hypertrophic scar is not clear. OBJECTIVE:To investigate the role and mechanism of lncRNA HSFAS in hypertrophic scar.METHODS:Fresh scar tissue and surrounding normal skin tissue samples from three patients with hypertrophic scar were collected,and tissue immunofluorescence was used to detect the expression of lncRNA HSFAS in frozen sections of two skin tissues. Primary fibroblasts were isolated from proliferative scarred skin tissue and normal skin tissue and cultured by enzyme digestion method. Quantitative real-time PCR was used to detect the mRNA expression of lncRNA HSFAS in cells. The proteins bound to lncRNA HSFAS were detected by RNA pull-down combined mass spectrometry. GO and KEGG were used to analyze the main functions and pathways of lncRNA HSFAS involved in hypertrophic scar progression. The targeted binding of lncRNA HSFAS to proteins was determined by catRAPID and RPISeq website analysis. RESULTS AND CONCLUSION:Compared with normal skin tissue and fibroblasts from normal skin tissue,the expression of lncRNA HSFAS in human hypertrophic scar tissue and primary fibroblasts from hypertrophic scar tissue was significantly increased (P<0.05). There were 510 proteins clearly bound to lncRNA HSFAS by RNA pull-down combined mass spectrometry. The results of GO and KEGG analyses showed that these proteins were mainly involved in RNA splicing and processing,chromosome synthesis and separation,and cell cycle. Among them,the proteins involved in RNA splicing and processing included scaffold attachment factor B2 and DICER1,and the binding fraction with lncRNA HSFAS was higher. The results of bioinformatics analysis showed that lncRNA HSFAS was bound to scaffold attachment factor B2 and DICER1 proteins. To conclude,lncRNA HSFAS may affect gene expression by interacting with scaffold attachment factor B2 and DICER1 proteins to regulate RNA splicing and processing modification,thus promoting the occurrence and development of hypertrophic scar.

Objective To investigate the role of Toll-like receptor 4(TLR4)in the regulation of homocys-teine(Hcy)-induced ferroptosis in macrophages.Methods Mouse macrophage cells RAW264.7 were cultured and divided into control group,Hcy intervention group(Hcy group),and Hcy plus ferroptosis inhibitor group(Hcy+Fer-1 group).After transfection with interference fragments,macrophages were treated with Hcy,and then divided into control group,Hcy intervention group(Hcy group),TLR4 interference negative control plus Hcy intervention group(si-NC+Hcy group),and TLR4 interference plus Hcy intervention group(si-TLR4+Hcy group).Macrophages were transfected with overexpression lentivirus and treated with Hcy,then were divided into control group,Hcy intervention group(Hcy group),a TLR4 overexpression negative control plus Hcy intervention group(OE-NC+Hcy group),and a TLR4 overexpression plus Hcy intervention group(OE-TLR4+Hcy group).After 48 hours of intervention,real-time fluorescent quantitative PCR and western blot were used to detect the expression levels of TLR4 in macrophages treated with Hcy;western blot was used to detect the expression levels of ferroptosis-related proteins ACSL4,GPX4,and FTH1 in macrophages,and ferrous ion assay kit to detect the concentration of Fe2+in macrophages;reactive oxygen species(ROS)assay kit and laser confocal microscopy were used to detect the content of intracellular reactive oxygen species.Results Compared with those in the control group,the expression level of the pro-ferroptosis protein ACSL4 was increased in the Hcy group(P<0.05),while the expression levels of anti-ferroptosis proteins GPX4 and FTH1 were decreased(P<0.05);the concentration of Fe2+was increased(P<0.05),and the content of ROS was increased.Meanwhile,the protein and mRNA expres-sion levels of TLR4 were both increased in the Hcy group(P<0.05).After macrophages were transfected with TLR4 interference fragments,compared with those in the si-NC+Hcy group,the expression levels of GPX4 and FTH1 were increased(P<0.05);the expression level of ACSL4 was decreased(P<0.05);the concentration of Fe2+was decreased(P<0.05),and the content of ROS was reduced in the si-TLR4+Hcy group.After macro-phages were transfected with TLR4 overexpression lentivirus,compared with those in the OE-NC+Hcy group,the expression levels of GPX4 and FTH1 were decreased(P<0.05),and the expression level of ACSL4 was increased(P<0.05)in the OE-TLR4+Hcy group.Conclusion Hcy induces the occurrence of ferroptosis in macrophages,and Toll-like receptor 4 has a positive feedback regulatory effect on ferroptosis in macrophages.

Objective To screen for differentially expressed apoptosis-related circular RNAs(circRNAs)in osteoblasts from steroid-induced osteonecrosis of the femoral head(SONFH)and to investigate their roles and mechanisms in osteoblast apoptosis.Methods MC3T3-E1 cells were cultured and divided into two groups:Control and DEX-treated.Western blot analysis was employed to evaluate the expression levels of BCL2-Associated X protein(Bax)and B-cell lymphoma 2(Bcl-2).Cell apoptosis was assessed using TUNEL staining and flow cytometry.RNA was extracted from both normal and DEX-treated MC3T3-E1 cells,followed by RNA-seq to identify differen-tially expressed circular RNAs(circRNAs).The functions and pathways of these circRNAs were analyzed using Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG).The differentially expressed mmu_circ_0000818 was selected for further verification at the cellular level.Its chromosomal location and conservation were examined using the UCSC Genome Browser Gateway and circBase.Overexpression plasmids and small interfering RNAs(siRNAs)targeting mmu_circ_0000818 were constructed and transfected into the cells.Subsequently,apop-tosis in MC3T3-E1 cells from each group was evaluated by flow cytometry.Results Compared with the control group,the apoptosis rate of MC3T3-E1 cells was significantly increased in the DEX group(P<0.01).Differen-tially expressed circRNAs were identified based on log2foldchange(≥2)and P value(P<0.05).Relative to the control group,there were 234 differentially expressed circRNAs in the DEX group,including 138 up-regulated and 96 down-regulated circRNAs.GO and KEGG enrichment analyses of the target genes of these differentially expressed circRNAs revealed significant associations with apoptosis and the PI3K-Akt signaling pathway.qRT-PCR results demonstrated that the expression level of mmu_circ_0000818 was markedly higher in the DEX group com-pared to the control group(P<0.01).Analysis using the UCSC Genome Browser and CircBase indicated that mmu_circ_0000818,located at chromosome 17:78712463-78715086,is formed by the cyclization of exons 6-7 of the Crim1 gene and exhibits high conservation across species.Flow cytometry results indicated that knockdown of mmu_circ_0000818 attenuated DEX-induced apoptosis in MC3T3-E1 cells,while overexpression of mmu_circ_0000818 exacerbated apoptosis.Conclusions CircRNA mmu_circ_0000818 was significantly upregulated in DEX-treated MC3T3-E1 cells,and its downregulation mitigated DEX-induced apoptosis.Consequently,mmu_circ_0000818 may represent a promising therapeutic target for the prevention and treatment of SONFH.

Objective To investigate the role of Toll-like receptor 4(TLR4)in the regulation of homocys-teine(Hcy)-induced ferroptosis in macrophages.Methods Mouse macrophage cells RAW264.7 were cultured and divided into control group,Hcy intervention group(Hcy group),and Hcy plus ferroptosis inhibitor group(Hcy+Fer-1 group).After transfection with interference fragments,macrophages were treated with Hcy,and then divided into control group,Hcy intervention group(Hcy group),TLR4 interference negative control plus Hcy intervention group(si-NC+Hcy group),and TLR4 interference plus Hcy intervention group(si-TLR4+Hcy group).Macrophages were transfected with overexpression lentivirus and treated with Hcy,then were divided into control group,Hcy intervention group(Hcy group),a TLR4 overexpression negative control plus Hcy intervention group(OE-NC+Hcy group),and a TLR4 overexpression plus Hcy intervention group(OE-TLR4+Hcy group).After 48 hours of intervention,real-time fluorescent quantitative PCR and western blot were used to detect the expression levels of TLR4 in macrophages treated with Hcy;western blot was used to detect the expression levels of ferroptosis-related proteins ACSL4,GPX4,and FTH1 in macrophages,and ferrous ion assay kit to detect the concentration of Fe2+in macrophages;reactive oxygen species(ROS)assay kit and laser confocal microscopy were used to detect the content of intracellular reactive oxygen species.Results Compared with those in the control group,the expression level of the pro-ferroptosis protein ACSL4 was increased in the Hcy group(P<0.05),while the expression levels of anti-ferroptosis proteins GPX4 and FTH1 were decreased(P<0.05);the concentration of Fe2+was increased(P<0.05),and the content of ROS was increased.Meanwhile,the protein and mRNA expres-sion levels of TLR4 were both increased in the Hcy group(P<0.05).After macrophages were transfected with TLR4 interference fragments,compared with those in the si-NC+Hcy group,the expression levels of GPX4 and FTH1 were increased(P<0.05);the expression level of ACSL4 was decreased(P<0.05);the concentration of Fe2+was decreased(P<0.05),and the content of ROS was reduced in the si-TLR4+Hcy group.After macro-phages were transfected with TLR4 overexpression lentivirus,compared with those in the OE-NC+Hcy group,the expression levels of GPX4 and FTH1 were decreased(P<0.05),and the expression level of ACSL4 was increased(P<0.05)in the OE-TLR4+Hcy group.Conclusion Hcy induces the occurrence of ferroptosis in macrophages,and Toll-like receptor 4 has a positive feedback regulatory effect on ferroptosis in macrophages.

BACKGROUND:Previous studies found that the proliferative scar-specific long non-coding RNA lncRNA HSFAS is a novel biomarker that can be used in the diagnosis of hypertrophic scar,but how it functions in hypertrophic scar is not clear. OBJECTIVE:To investigate the role and mechanism of lncRNA HSFAS in hypertrophic scar.METHODS:Fresh scar tissue and surrounding normal skin tissue samples from three patients with hypertrophic scar were collected,and tissue immunofluorescence was used to detect the expression of lncRNA HSFAS in frozen sections of two skin tissues. Primary fibroblasts were isolated from proliferative scarred skin tissue and normal skin tissue and cultured by enzyme digestion method. Quantitative real-time PCR was used to detect the mRNA expression of lncRNA HSFAS in cells. The proteins bound to lncRNA HSFAS were detected by RNA pull-down combined mass spectrometry. GO and KEGG were used to analyze the main functions and pathways of lncRNA HSFAS involved in hypertrophic scar progression. The targeted binding of lncRNA HSFAS to proteins was determined by catRAPID and RPISeq website analysis. RESULTS AND CONCLUSION:Compared with normal skin tissue and fibroblasts from normal skin tissue,the expression of lncRNA HSFAS in human hypertrophic scar tissue and primary fibroblasts from hypertrophic scar tissue was significantly increased (P<0.05). There were 510 proteins clearly bound to lncRNA HSFAS by RNA pull-down combined mass spectrometry. The results of GO and KEGG analyses showed that these proteins were mainly involved in RNA splicing and processing,chromosome synthesis and separation,and cell cycle. Among them,the proteins involved in RNA splicing and processing included scaffold attachment factor B2 and DICER1,and the binding fraction with lncRNA HSFAS was higher. The results of bioinformatics analysis showed that lncRNA HSFAS was bound to scaffold attachment factor B2 and DICER1 proteins. To conclude,lncRNA HSFAS may affect gene expression by interacting with scaffold attachment factor B2 and DICER1 proteins to regulate RNA splicing and processing modification,thus promoting the occurrence and development of hypertrophic scar.

Objective To screen for differentially expressed apoptosis-related circular RNAs(circRNAs)in osteoblasts from steroid-induced osteonecrosis of the femoral head(SONFH)and to investigate their roles and mechanisms in osteoblast apoptosis.Methods MC3T3-E1 cells were cultured and divided into two groups:Control and DEX-treated.Western blot analysis was employed to evaluate the expression levels of BCL2-Associated X protein(Bax)and B-cell lymphoma 2(Bcl-2).Cell apoptosis was assessed using TUNEL staining and flow cytometry.RNA was extracted from both normal and DEX-treated MC3T3-E1 cells,followed by RNA-seq to identify differen-tially expressed circular RNAs(circRNAs).The functions and pathways of these circRNAs were analyzed using Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG).The differentially expressed mmu_circ_0000818 was selected for further verification at the cellular level.Its chromosomal location and conservation were examined using the UCSC Genome Browser Gateway and circBase.Overexpression plasmids and small interfering RNAs(siRNAs)targeting mmu_circ_0000818 were constructed and transfected into the cells.Subsequently,apop-tosis in MC3T3-E1 cells from each group was evaluated by flow cytometry.Results Compared with the control group,the apoptosis rate of MC3T3-E1 cells was significantly increased in the DEX group(P<0.01).Differen-tially expressed circRNAs were identified based on log2foldchange(≥2)and P value(P<0.05).Relative to the control group,there were 234 differentially expressed circRNAs in the DEX group,including 138 up-regulated and 96 down-regulated circRNAs.GO and KEGG enrichment analyses of the target genes of these differentially expressed circRNAs revealed significant associations with apoptosis and the PI3K-Akt signaling pathway.qRT-PCR results demonstrated that the expression level of mmu_circ_0000818 was markedly higher in the DEX group com-pared to the control group(P<0.01).Analysis using the UCSC Genome Browser and CircBase indicated that mmu_circ_0000818,located at chromosome 17:78712463-78715086,is formed by the cyclization of exons 6-7 of the Crim1 gene and exhibits high conservation across species.Flow cytometry results indicated that knockdown of mmu_circ_0000818 attenuated DEX-induced apoptosis in MC3T3-E1 cells,while overexpression of mmu_circ_0000818 exacerbated apoptosis.Conclusions CircRNA mmu_circ_0000818 was significantly upregulated in DEX-treated MC3T3-E1 cells,and its downregulation mitigated DEX-induced apoptosis.Consequently,mmu_circ_0000818 may represent a promising therapeutic target for the prevention and treatment of SONFH.

Rosuvastatin(RVS)is an excellent drug with anti-inflammatory and lipid-lowering properties in the aca-demic and medical fields.However,this drug faces a series of challenges when used to treat atherosclerosis caused by hyperhomocysteinemia(HHcy),including high oral dosage,poor targeting,and long-term toxic side effects.In this study,we applied nanotechnology to construct a biomimetic nano-delivery system,macrophage membrane(M?m)-coated RVS-loaded Prussian blue(PB)nanoparticles(MPR NPs),for improving the bioavailability and targeting capacity of RVS,specifically to the plaque lesions associated with HHcy-induced atherosclerosis.In vitro assays demonstrated that MPR NPs effectively inhibited the Toll-like receptor 4(TLR4)/hypoxia-inducible factor-1α(HIF-1α)/nucleotide-binding and oligomerization domain(NOD)-like receptor thermal protein domain associated protein 3(NLRP3)signaling pathways,reducing pyroptosis and inflammatory response in macrophages.Additionally,MPR NPs reversed the abnormal distribution of adenosine triphosphate(ATP)-binding cassette transporter A1(ABCA1)/ATP binding cassette transporter G1(ABCA1)/ATP binding cassette transporter G1(ABCG1)caused by HIF-1α,promoting cholesterol efflux and reducing lipid deposition.In vivo studies using apolipoprotein E knockout(ApoE-/-)mice confirmed the strong efficacy of MPR NPs in treating atherosclerosis with favorable bio-security,and the mechanism behind this efficacy is believed to involve the regulation of serum metabolism and the remodeling of gut microbes.These findings suggest that the synthesis of MPR NPs provides a promising nanosystem for the targeted therapy of HHcy-induced atherosclerosis.

OBJECTIVE：To study th e improvement effect and mechanism of Dajianzhong decoction on irritable bowel syndrome（IBS）visceral pain model rats. METHODS ：Totally 48 male non-weaning rats were randomly divided into normal control group ，model group ，pinaverium bromide group （positive control ，45 mg/kg）and Dajianzhong decoction high-dose ， medium-dose and low-dose groups （2.16，1.08，0.54 g/kg，by crude drug ），with 8 rats in each group. Except for normal control group，IBS visceral pain model was established by mother and child separation ，acetic acid enema ，ovalbumin intraperitoneal Remote limb precondition - ing protects against ischemia induced neuronal death through ameliorating neuronal oxidative DNA damage injection in other groups for 57 d. On the 58th day ，the rats in administration groups were given the corresponding drugs intragastrically，and model group and normal control group were given constant volume of purified water ，once a day ，for consecutive 14 d. The general condition of rats was observed ；abdominal wall withdrawal reaction （AWR）was adopted to evaluate the visceral sensitivity of rats in each group under 20，40，60，and 80 mmHg （1 mmHg＝0.133 kPa）pressure；HE staining method was used to observe the colon pathological features of rats in each group. Western blotting assay was used to detect the protein expression of intestinal glial cells markers fibrillary acidic protein （GFAP），nerve growth factor （NGF）and its receptor TrkA in colon tissue. RESULTS ：The mucosal layer of colon tissue in rats of model group was discontinuous ，gland edema was observed and lymphocytes ，neutrophils and eosinophils were scattered in the lamina propria. In Dajianzhong decoction low-does group，the mucosal layer of colon tissue was incomplete ，some glands were slightly edematous ，and a few lymphocytes ， neutrophils in the lamina propria. The colonic mucosa epithelial structure was intact ，glands arranged regularly ，and no degenerative necrosis and inflammatory cells were observed in Dajianzhong decoction medium- and high-dose groups and pinaverium bromide group. Compared with normal control group ，AWR scores under 20，40，and 60 mmHg pressure ，relative protein expression of GFAR ，NGF and TrkA were all increased significantly in model group and Dajianzhong decoction low-does groups（P＜0.05 or P＜0.01）. Compared with model group ，AWR scores under 20，and 40 mmHg pressure in Dajianzhong decoction medium- and high-dose groups and pinaverium bromide group ，AWR scores under 60 mmHg pressure in Dajianzhong decoction high-dose group and pinaverium bromide group ，relative protein expression of GFAP ，NGF，TrkA in colon tissue in Dajianzhong decoction medium- and high-dose groups and pinaverium bromide group were all decreased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS ：Dajianzhong decoction could improve the visceral pain of IBS model rats by inhibiting the activation of intestinal glial cells and reducing the expression of NGF and TrkA.

Objective Compare the efficacy of superselective arterial embolization with observation in the treatment of HFP caused by trauma.Methods From Jan,2013 to Dec,2017,6 traumatic HFP patients were involved into the study,the age range from 18 to 44 years old,with an average age of 26.2 years.The fistula was measured by doppler ultrasonography before the treatment with its longest diameter,and which ranges from 0.8 to 1.4 cm,the average diameter is 1.14 cm.All patients are divided into two groups according to their treatment.Operation group:4 patients underwent the pudendal artery superselective gelatin sponge vascular embolization.Control group:2 cases were treated with local cold compressing under watchful waiting.In control group,patients are treated by local cold compress with ice bag for 3 weeks:15 mins × 10 times every day.The erectile hardness of the patients was observed and the fistula was evaluated by doppler ultrasonography 3 weeks later,and all the patients were reexamined by doppler ultrasonography 1 year later.The erectile hardness was divided into four grades according to the patient~ subjective feelings:grade Ⅰ,the penis was enlarged but not rigid;grade Ⅱ,the penis had hardness,but not enough to insert into the vagina;grade Ⅲ,the penis could be inserted into the vagina,but not to achieve complete erection;grade Ⅳ,the penis fully erect and firm.The erectile function was evaluated with the International Index of Erectile Function-5(IIEF-5) after 1-year's follow-up.Results 4 patients in the operation group were operated successfully without obvious complications after operation.Median follow-up time of all the patients was 21 months (5-50).In group operation,4 cases of surgical patients did not have the recurrence of priapism.There was no obvious abnormality in the color doppler ultrasound of penis.The score of IIEF-5 was 25,24,24 and 23,respectively.Postoperative erectile function made a complete recovery.In group observation,2 patients under watchful waiting still have grade Ⅱ-Ⅲ sustained priapism,IIEF-5 score is 21 and 19 respectively,the penis color doppler ultrasound scan still showed the change of arteriovenous fistula.Conclusion Superselective arterial embolization is effective in the treatment of HFP,and the recovery of sexual function is better after operation compared with watchful waiting.