Saussurea involucrata (SI) has long been used in traditional medicine to treat RA, but its underlying mechanism remains unclear. This study utilized network pharmacology and molecular docking to explore the potential pharmacological effects of bioactive compounds in SI on RA. A total of 27 active compounds were identified, along with 665 corresponding targets. Additionally, 593 disease-related targets were obtained from multiple databases, with 119 common targets shared with SI. The high-ranking targets mainly belong to the MAPK family and NF-κB pathway, including MAPK14, MAPK1, RELA, TNF, and MAPK8, all of which are associated with inflammation and joint destruction in RA. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed significant pathways related to IL-17 signaling, Th17 cell differentiation, and osteoclast differentiation. Molecular docking and dynamic simulations demonstrated strong interactions between several flavonoids and RA-related targets. Xuelianlactone, Involucratin, and Flazin exhibit outstanding binding efficacy with targets such as MAPK1, MAPK8, and TNF. These findings provide valuable insights into the therapeutic potential of SI for RA and offer directions for further drug development.Rheumatoid arthritis (RA) is a chronic autoimmune disease with a global prevalence of approximately 0.46%, causing significant impairments in patients’ quality of life and an economic burden.  Rheumatoid arthritis (RA), a chronic autoimmune disease, exhibited a global prevalence of 0.46% between 1980 and 2018, with significant regional and country-specific variations , highligThere is no cure for RA at present, but there are some pharmacological and non-pharmacological treatments that can relieve symptoms, delay disease progression, and improve quality of life . The maiSaussurea, a medicinal plant, was first documented in the ancient Tibetan book Sman-dpyad Zla-ba’i-rgyal-po [Saussurea involucrata (SI), grown in Xinjiang, China, is particularly noteworthy and widely used in Uyghur Medicine, known as “Tage leylishi” [rgyal-po  in Chinaeylishi” . Accordieylishi” . Accordieylishi” ,20. Ongoeylishi” ,22.Saussurea has found extensive clinical use in the treatment of RA, available in various dosage forms such as injections, capsules, and more [In China, and more . Howevertcmspw.com/tcmsp.php, accessed on 27 February 2023) [organchem.csdb.cn/scdb/default.htm, accessed on 5 April 2023) [www.symmap.org, accessed on 12 April 2023) [Saussurea involucrata” was employed for these searches, yielding relevant results. Furthermore, additional active ingredients were supplemented by searching the literature in the China National Knowledge Infrastructure(CNKI) [To ensure a comprehensive collection of the bioactive components of SI, this study conducted searches across multiple databases. The databases used included the Traditional Chinese Medicine Systems Pharmacology Database  , the Chiil 2023) , and Symil 2023) . The keyre(CNKI) ,27,28.pubchem.ncbi.nlm.nih.gov, accessed on 12 April 2023) using the pubchempy Python toolkit, acquiring the Simplified Molecular Input Line Entry System (SMILES) notations. These notations were then imported into the SwissADME database  and subjected to screening based on criteria such as a gastrointestinal absorption (GI) rating of “High” and drug-likeness having two or more items marked as “Yes” [The obtained components were screened based on parameters such as oral bioavailability and drug-likeness to ensure their potential for metabolism within the human body. For the query results from the SymMap database, components with an oral bioavailability (OB) threshold of ≥30% were selected . These sas “Yes” . Subsequas “Yes” . Finallywww.uniprot.org, accessed on 27 February 2023). For other components, their SMILES were input into SwissTargetPrediction  with the species attribute set to “Homo sapiens” for target prediction, and targets with a probability greater than 0 were collected [For the components queried from the TCMSP, their targets were also obtained from this database, and their gene names were queried from the Uniprot database  was queried using the keyword “rheumatoid arthritis”, and disease-related target genes were obtained by applying a screening criterion of Score ≥ 0.3 [www.genecards.org, accessed on 27 February 2023) was queried, and data with a Relevance score > 10 were selected [www.omim.org/search/advanced/geneMap, accessed on 13 April 2023) and the TTD database  were employed to query targets associated with RA [Disease targets were collected through searches across multiple databases. The DisGeNet database  to generate a Venn diagram [Components exert their effects on the disease by targeting specific genes. The target genes for both the components and the disease were inputted into the Bioinformatics website  with a confidence level set at >0.9, and the species selected as H. sapiens (human) [In order to elucidate the relationships among target proteins and further refine key targets, the intersecting target genes were inputted into the STRING database ( (human) . Based o (human) . The medp.adjust < 0.05 [p.adjust, and GeneRatio values. As for the GO analysis results, they were sorted and visualized separately for the cellular component (CC), molecular function (MF), and biological process (BP) categories.The key targets were uploaded to the Bioinformatics website for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The results were subjected to screening based on the criterion of t < 0.05 , and subInformation such as component–target, disease–target, drug–component, pathway–target, function–target, etc., were summarized and imported into Cytoscape for network visualization, to construct their relationship network. Then, network analysis was performed using the network analysis function , and accwww.rcsb.org, accessed on 18 May 2023) to retrieve structures with species as H. sapiens and a resolution of less than or equal to 2.5 Å. The structures were selected based on parameters such as Gene Name, Resolution (Å), ligand, R-Value, pH, Experimental Method, Publication Year, etc. [The core targets were searched in the RCSB PDB database  [To determine the position and size of the Grid Box, priority was given to the original ligand positions within the structures, taking into account the target position descriptions from the Uniprot database. In the case of structures without their own ligands, the Deepsite tool (ay 2023)  on the Pay 2023)  was emplhttp://sobereva.com/soft/Sobtop, accessed on 4 September 2023) was used to generate topology files with the GAFF force field. The receptor–ligand complex was subjected to solvation, ion balance and energy minimization. In order to simulate the human body environment, the ion concentration was set at 0.145 M when adding ions, and the MD simulation of the system was carried out at a temperature of 310 K and a pressure of 1 bar for 50 ns. The cut-off value of the non-bonded interaction was set to 1.2 nm, and the long-distance electrostatic interaction was calculated [The well-combined target protein–ligand complex was selected from the molecular docking results, and MD simulations were performed using Gromacs 2023.2. For protein molecules, Charmm36 force field and TIP3P water model were chosen for processing . Ligand lculated  and gmx_After the above component-mining, a total of 27 components of SI were obtained , correspp.adjust < 0.05 were obtained. Among these pathways, the top 20 pathways with the smallest p.adjust values were selected and presented in The key targets were input into the Bioinformatics website and, after screening, a total of 131 pathways with p.adjust < 0.05. The top 10 results with the smallest p.adjust values were selected for plotting. As for the CC analysis, only four results met the requirements, and all of them were included in the plot. The plot exhibits a color gradient shifting from blue to red, indicating an increasing level of significance. Additionally, the length of each bar corresponds to the number of genes enriched within the specific category, with longer bars signifying a higher gene enrichment count.Based on the output results from the Bioinformatics website, the findings were categorized into three groups, as shown in Information such as bioactive components, key targets, related pathways, and the functions obtained above was formatted in a network diagram using Cytoscape software . In the p < 0.05). However, the binding effect of IL6, ILB, CHUK, and IKBKB to each ligand is not outstanding.According to the network analysis results of Cytoscape, 10 compounds with the highest degree values, Quercetin, Luteolin, Acacetin, Xuelianlactone, Moslosooflavone, Hispidulin, Mosloflavone, Involucratin, 5,7-Dihydroxy-6,8-Dimethoxyflavone and Flazin were selected as molecular docking ligands (core components). Ten targets with the highest degree values, MAPK14, MAPK1, RELA, TNF, MAPK8, IL6, IL1B, CHUK, IKBKB and NFKBIA, were selected as the core targets for molecular docking. Nine protein structures were selected as the receptors, with CHUK and IKBKB targets located in different segments of the same protein structure . The resFor each target protein, the ligand with the lowest affinity was selected from the molecular docking results. Visualization was achieved using Biovia Discovery Studio 2021 software, and the docking results are presented in Based on the degree values obtained in network analysis and molecular docking results, the following study primarily focuses on the MAPK1 and TNF. For both targets, the top four compounds with the best docking results, as well as the original ligands (38Z and VGY) of the target proteins, were selected for MD simulations. In addition, for MAPK8, two compounds and the original ligand (38Z) were chosen for simulations. Considering the certainty of the target position, the simulation was conducted with NFKIBA as the representative target for the NF-κB pathway, focusing on the top two best-performing compounds based on the docking results. In MD simulations, RMSD measures the average deviation or fluctuations in atomic positions in a molecular system from their initial positions, providing insight into the stability of a target protein–ligand complex. Generally, it is considered that once the RMSD value stabilizes and the fluctuation range is less than 0.2 nm, the system can be deemed stable . It can 2. Throughout the entire simulation process, the SASA values of these compounds show small fluctuations, indicating their stable interaction with the protein complexes (Solvent-Accessible Surface Area (SASA) quantifies the surface area of a molecule that is accessible to solvent molecules and can indicate changes in a molecule’s conformation or exposure during simulation, aiding in the assessment of binding interactions in a target protein–ligand complex. The average SASA values for complexes XT (black), AT (red), IM (blue), LM (green), and FM (purple) during MD simulation are as follows: 202.49 ± 3.72, 201.65 ± 3.17, 179.03 ± 3.25, 175.97 ± 2.59, and 179.9 ± 2.9 nmomplexes .total) between the ligand, receptor and their respective complexes in the stable phase of MD simulation was calculated, as shown in total curves of each compound have small fluctuations, and the average is below −10 kcal/mol. Furthermore, by utilizing the gmx_mmpbsa.bsh script, the entropy change in each complex is calculated at a time interval of 1000 ps, allowing for the calculation of the total binding energy (ΔG). total computed using gmx_mmpbsa.py. The results from both methods exhibit a close resemblance, which enhances the credibility of the computed free energy values. It can be observed from the figure and the table that ΔG and ΔH for XT are significantly lower than that of the other ligands, consistent with the results observed in molecular docking.Molecular Mechanics–Poisson Boltzmann Surface Area (MMPBSA) is a method used to estimate the binding energy of a target protein–ligand complex by post-processing the MD trajectory. MMPBSA can characterize the strength and stability of the protein–ligand interaction by calculating the difference between the free energy of the bound state and the free energy of the unbound state. For the five complexes mentioned above, we selected segments with stable RMSD values and extracted 10 ns trajectories for MMPBSA energy analysis. The total binding energy (ΔG) is the sum of the change in enthalpy (ΔH) and the change in entropy (–TΔS) . Using gThis study investigated the binding free energy and residue contributions of active components of SI to the RA target using methods such as MD simulations and MMPBSA energy calculations. As shown in The analysis of residue interactions with different ligands targeting the same receptor reveals crucial regions that facilitate complex binding. In future drug design efforts, it is essential to pay attention to the contributions of these residues. This can be achieved by introducing functional groups or optimizing their molecular spatial structures, aiming to retain residues with strong binding forces and reduce the impact of residues with significant resistance .In this study, we employed network pharmacology to analyze SI, resulting in the identification of 27 bioactive components and 119 targets that intersect with RA. Among these targets, 33 were identified as key targets. Additionally, we conducted KEGG and GO analyses to investigate the pathways and biological processes involved. The findings suggest a potential correlation between the components present in SI and the treatment of RA.Among the core targets, ERK (MAPK1), JNK (MAPK8), and p38 MAPK (MAPK14) are well-defined families of MAPKs, activated by phosphorylation, and play significant roles in the inflammatory and destructive mechanisms observed in rheumatoid arthritis, including regulating pro-inflammatory cytokine production and mediating downstream signaling from IL-1, IL-17, and TNF-α receptors. These properties make them attractive therapeutic targets for rheumatic diseases ,52. RELAFrom a pathway perspective, the pathways exhibiting high gene enrichment and confidence levels include IL-17, TNF, TLR (Toll-like receptor), NLR (NOD-like receptor), and CLR (C-type lectin receptor) signaling pathways, as well as Th17, Th1, and Th2 cell differentiation pathways, among which the IL-17 and Th17 pathways are the most confident. Interactions between RA, Fibroblast-Like Synoviocytes (FLSs), and Th17 cells further contribute to tumorous FLS growth and joint pannus formation . PreviouThe GO analysis primarily centers on responses to biotic stimuli, specifically lipopolysaccharides and other molecules of bacterial origin, known to trigger immune and inflammatory responses. Previous research has indicated that bacterial infection plays a significant role in contributing to RA pathogenesis . The patBased on the above analysis results, components Luteolin, Involucratin, Xuelianlactone, Flazin, Quercetin, and Acacetin, all belonging to the flavonoid class, exhibit favorable binding effects with the targets. Quercetin exerted a bone-protective effect by reducing MMPs, RANKL production, and osteoclast formation via the MAPKs and NF-κB pathways . CurrentIn summary, the network pharmacology analysis suggests that the mechanism of SI in treating RA mainly involves its flavonoids, which target key proteins through MAPK and NF-κB-related pathways. By doing so, they effectively inhibit inflammation and mitigate bone destruction, leading to therapeutic effects. The binding efficiency of complexes like XT, AT, and FM has been confirmed through MD simulations. This suggests that the action of SI in treating RA is achieved through multiple components acting on various targets. However, it is essential to note that network pharmacology is primarily a data-driven and network-based research method. Therefore, these findings should be further validated and supported by experimental studies to establish their clinical relevance and potential as RA treatment options.


Background: Acute gouty arthritis (AGA) significantly impairs patients’ quality of life. Currently, existing therapeutic agents exhibit definite efficacy but also lead to serious adverse reactions. Therefore, it is essential to develop highly efficient therapeutic agents with minimal adverse reactions, especially within traditional Chinese medicine (TCM). Additionally, food polyphenols have shown potential in treating various inflammatory diseases. The Qingre-Huazhuo-Jiangsuan-Recipe (QHJR), a modification of Si-Miao-San (SMS), has emerged as a TCM remedy for AGA with no reported side effects. Recent research has also highlighted a strong genetic link to gout.Methods: The TCM System Pharmacology (TCMSP) database was used to collect the main chemical components of QHJR and AGA-related targets for predicting the metabolites in QHJR. HPLC-Q-Orbitrap-MS was employed to identify the ingredients of QHJR. The collected metabolites were then used to construct a Drugs-Targets Network in Cytoscape software, ranked based on their “Degree” of significance. Differentially expressed genes (DEGs) were screened in the Gene Expression Omnibus (GEO) database using GEO2R online analysis. Subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed. The DEGs were utilized to construct a Protein-Protein Interaction (PPI) Network via the STRING database. In vivo experimental validation was conducted using colchicine, QHJR, rapamycin (RAPA), and 3-methyladenine (3-MA) as controls to observe QHJR’s efficacy in AGA. Synovial tissues from rats were collected, and qRT-PCR and Western blot assays were employed to investigate Ampk-related factors , autophagy-related factors , and inflammatory-related factors (NLRP3). ELISA assays were performed to measure inflammatory-related factor levels , and H&E staining was used to examine tissue histology.Results: Network analysis screened out a total of 94 metabolites in QHJR for AGA. HPLC-Q-Orbitrap-MS analysis identified 27 of these metabolites. Notably, five metabolites  were not associated with any individual herbal component of QHJR in TCMSP database, while six metabolites  overlapped with the predicted results from the previous network analysis. Further network analysis highlighted key components, such as Caffeic acid, cis-resveratrol, Apigenin, and Isoliquiritigenin. Other studies have found that their treatment of AGA is achieved through reducing inflammation, consistent with this study, laying the foundation for the mechanism study of QHJR against AGA. PPI analysis identified TNF, IL-6, and IL-1β as hub genes. GO and KEGG analyses indicated that anti-inflammation was a key mechanism in AGA treatment. All methods demonstrated that inflammatory expression increased in the Model group but was reversed by QHJR. Additionally, autophagy-related expression increased following QHJR treatment. The study suggested that AMPKα and p-AMPKα1 proteins were insensitive to 3 MA and RAPA, implying that AMPK may not activate autophagy directly but through ULK1 and mTOR.Conclusion: In conclusion, this study confirms the effectiveness of QHJR, a modified formulation of SMS , against AGA. QHJR, as a TCM formula, offers advantages such as minimal safety concerns and potential long-term use. The study suggests that the mechanism by which QHJR treats AGA may involve the activation of the AMPK/mTOR/ULK1 pathway, thereby regulating autophagy levels, reducing inflammation, and alleviating AGA. These findings provide new therapeutic approaches and ideas for the clinical treatment of AGA. Acute gouty arthritis (AGA) represents a major and increasingly serious public health issue, whose incidence is increasing year by year and seriously impacting people’s health and lives . AGA is Traditional Chinese Medicine (TCM) has been used to treat gouty arthritis (GA) for centuries. The most widely used TCM treatments for GA are acupuncture and the Chinese herbal medicine Si-Miao-San . AcupuncThe molecules identified in gout are involved in a multitude of critical processes, including the degradation of the extracellular matrix in cartilage cells, transduction of inflammatory signals, and regulation of fibroblast-like synoviocyte invasion pathways . In addiPP121 can alleviate MSU crystal-induced acute gouty arthritis via inhibition of the NLRP3 inflammasome , the highly conserved serine/threonine-protein kinase, serves as an intracellular energy sensor with significant effects on catabolism and anabolism regulation . Referre+-K+-ATPase signaling impairment and cell injury in renal tubules  (https://www.tcmsp-e.com/load_intro.php?id=29) (We employed the TCMSP database (msp.php)  to explomsp.php) . The crip?id=29) .https://www.uniprot.org/) and Search Tool for the Retrieval of Interacting Genes database (https://string-db.org) were utilized to convert protein gene names and obtain drug component targets. For comprehensive information on human genes, encompassing genome, proteome, transcription, heredity, and function, we turned to the GeneCards Database (https://www.genecards.org/) (The UniProt database (ds.org/) . AGA-relds.org/) , a “Botads.org/) .Homo sapiens” in the search field of the GEO database (https://www.ncbi.nlm.nih.gov/geo/). These keywords were selected to retrieve relevant data. Through this search, we identified gene chip data with the accession number GSE160170. The GSE160170 dataset was based on the GPL21827 platform, specifically the Agilent-079487 Arraystar Human LncRNA microarray V4 (Probe Name version). This dataset comprised a total of 6 specimens from individuals with gout and 6 specimens from individuals serving as normal controls.The initial step of our study involved performing a keyword search using “acute gouty arthritis” and “https://string-db.org/) (To construct a protein-protein interaction (PPI) network, we employed the STRING database (db.org/) . Subsequdb.org/)  was utilhttps://david.ncifcrf.gov/). To identify significantly enriched genes, we set the critical value at p < 0.05 and |log2(FC)| > 1. In this study, the “clusterprofiler” package in the R software was employed to analyze the primary functions of the DEGs.To investigate the functional annotations of DEGs, we performed GO analysis and KEGG 11 botanical drugs of QHJR  were purFirst, fry the Atractylodis rhizoma and Coicis semen until they turn slightly yellow, and prepare the Plantaginis semen by wrapping it with gauze. Soak all the botanical drugs (except Plantaginis semen) in purified water for 2 h, then remove them and prepare for decoction. Next, place all the botanical drugs  in a pot filled with 2 L of water and boil for 0.5 h. Concentrate the decoction to concentrations of 5.15 g/mL , 2.56 g/mL , and 1.3 g/mL  respectively according to the concentration requirements of the QHJR-High, QHJR-Medium, and QHJR-Low groups. The “g/mL” means “drug—solvent ratio”, which is all the botanical drugs of QHJR to pure water. Filter the botanical drugs using gauze to collect the herbal liquid. The decoction was then entrusted to a temperature-controlled refuge within a refrigerator, maintained at a frosty 4 °C.Prepare the drug solution (QHJR)  with a chttps://pubchem.ncbi.nlm.nih.gov/) to obtain their structural information. The obtained structures were then imported into Swiss Target Prediction (http://www.swisstargetprediction.ch/) to predict their target proteins. Disease-related target proteins were retrieved from the GeneCards database (https://www.genecards.org/) (The key metabolites predicted and identified were searched in the PubChem database (ds.org/) and filteds.org/) 3.9.1 forA total of 88 male Sprague-Dawley rats with SPF-grade, weighing 180 ± 20 g, were sourced from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd (License number: Beijing Baishan SCXK 2016–0006). The rats were categorized into 8 groups (n = 11) for further experimentation, including the Normal group, Model group, QHJR-H group, QHJR-M group, QHJR-L group, Colchicine group, RAPA g, group, and 3-MA group. The rate in the Normal group injected normal saline (NS) was used as the control. The others were injected with normal MSU to the sestablishAGA model  The knee joints of rats were dissected to obtain synovial tissues, which were subsequently fixed in 4% paraformaldehyde for a duration exceeding 24 h. The fixed tissues were then embedded in paraffin. Additional synovial tissues were preserved in liquid nitrogen at −80 °C for subsequent Quantitative Real-Time PCR and Western blot assays. Following this, 4-μm thick sections of paraffin-embedded tissue were deparaffinized, stained with hematoxylin and eosin (HE), subjected to dehydration using fractional xylene and ethanol, and ultimately sealed with neutral gums.Blood samples were collected from the abdominal aorta of rats, and the freshly obtained blood was allowed to stand before being centrifuged at 3500 rpm for a duration of 10 min. The serum was extracted at −80 °C for preservation. The levels of TNF-α, IL-1β, and IL-6 were determined using an ELISA kit, following the manufacturer’s instructions.® Green Master Mix , DEPC water (9.5 μL) and cDNA (1 μL). The reaction procedure was presented as follows activation under 95°C; 20-s amplification under 95°C; 30-s un3058°C and 20-s under 72°C for a total of 40 cycles. The 2−ΔΔCT method was performed to quantify PCR analysis. Ex Taq-enzyme Kit (Solarbio PC1100) was employed to extract total synovial RNA, while PrimeScript RTregent Kit  was used to prepare cDNA through reverse transcription. qRT-PCR machine CFX96  was used for qRT-PCR, and the reaction system included respective primers (1 μL each), Power SYBRThe mixture containing RIPA lysates and protease/phosphatase inhibitors was adopted for extracting total synovial proteins. Protein content was quantified by a BCA kit (Solarbio PC0020). Protein aliquots were separated through SDS-PAGE, followed by transfer onto PVDF membranes (Millipore). Then, membranes were blocked using 5% defatted milk for 2 h, followed by overnight primary antibody incubation under 4°C Dc., 189 from Plantaginis Semen, 67 from Radix Clematidis, 315 from Smilacis Glabrae Rhixoma, 21 from Poria Cocos (Schw.) Wolf., 38 from Curcumaelongae Rhizoma, 145 from Perilla Frutescens, and 44 from Coicis Semen. Screening based on OB ≥ 30% and DL ≥ 0.18 yielded 117 metabolites, with 10 from Polygoni Cuspidati Rhizoma Et Radix, 24 from Spatholobus Suberectus Dunn, 2 from Dioscoreae Septemlo Bae Rhizoma, 9 from Atractylodes Lancea (Thunb.)Dc., 9 from Plantaginis Semen, 7 from Radix Clematidis, 15 from Smilacis Glabrae Rhixoma, 15 from Poria Cocos (Schw.) Wolf., 3 from Curcumaelongae Rhizoma, 14 from Perilla Frutescens, and 9 from Coicis Semen. After removing duplicates, 94 metabolites were identified. Protein gene names were converted using the STRING and UniProt databases, and AGA targets were subsequently identified in GeneCards. The intersection of these targets was then employed to construct the “Botanical drug-Metabolite-Target” network, as depicted in The main components in extracts of QHJR were analyzed qualitatively based on HPLC-Q-Orbitrap-MS. The analysis results are shown in p < 0.05 and |log2(FC)| > 1, and it was shown as the volcano . Then, the top 10 hub genes were identified based on their node degree and included TNF, IL6, IL1B, IL10, IFNG, CD80, CXCL8, CCL3, STAT1, and IL1A .etc., based on GO analysis  analysis, indicate that QHJR may reduce inflammation through the involvement of TNF, IL6, IL1B, and other factors. Therefore, we further investigated and evaluated the role of QHJR in the treatment of AGA by establishing a rat model of AGA Firstly, We established the AGA model. Rats in the Normal group were injected with NS, and in other groups were injected with MSU. The previous studies  have demAs depicted in The results of the Biological function analysis suggested that AGA was closely related to inflammation, such as leukocyte chemotaxis, leukocyte migration, NOD-like receptor signaling pathway, and TNF signaling pathway. etc. Especially in the predicted hub genes of AGA, TNF, IL-6, and IL-1β were identified as joint targets of QHJR, suggesting that the primary mechanism underlying the treatment of QHJR for anti-AGA may reduce inflammation. To investigate this further, we analyzed the effects of QHJR on the level of inflammation markers TNF-α, IL-6, and IL-1β using the ELISA method. Our results demonstrate that QHJR treatment significantly inhibited the level of TNF-α, IL-6, and IL-1β induced by MSU . In addiAutophagy has been reported to lower the inflammation. To investigate how autophagy influenced the treatment of AGA, autophagy level was first measured in inflammation. We added 3-MA which is an autophagy inhibitor, and RAPA which is an autophagy promoter as the controls. Many ATG proteins have vital effects on autophagy. Atg5 plays an essential part in canonical autophagy and autophagy-related processes . CompareNext, we examined the expression of autophagy in inflammatory-related factors. H&E assay displayed that the RAPA group had declined inflammatory cell infiltration relative to the Model group . In addiMoreover, the expressions of Atg5 and Atg7 , Beclin-Considering that AMPKα is an energy sensor exerting a positive regulatory effect on autophagy by inhibiting mTOR to remove the phosphorylation inhibition of ULK1 at Ser757 and induce the binding of ULK1 to AMPK , we aimeAMPK phosphorylation at Thr172 exerts a vital role in activating AMPK, which usually acts as the activity indicator . To veriAcute gouty arthritis, is a common ailment, particularly among the middle-aged and elderly demographics . The patCurrently, due to the increasing incidence rates and limitations of Western medicine (such as toxic side effects) , the devin vitro and animal models of many diseases, including rheumatoid arthritis and gouty arthritis  formulations . OB and rthritis . TCMSP irthritis . Howeverrthritis . Thus, tPharmMapper and SwissTarget, along with other target prediction databases, are essential tools for unraveling molecular interactions governing drug targets. These resources enable researchers to explore complex mechanisms and elucidate drug efficacy. With advanced algorithms and data integration, these platforms empower comprehensive investigation of the intricate drug-target relationship. Leveraging these databases, researchers gain insights into candidate drug therapeutics, driving novel pharmaceutical discovery. SwissTarget Prediction utilizes reverse screening to predict possible protein targets for drug candidates . We condThe publicly available GEO platform was utilized to identify DEGs between healthy individuals and patients. The DEGs present promising therapeutic targets for combating the disease. Leveraging the GEO platform, researchers can gain critical insights into the molecular changes underlying the pathological condition, facilitating the development of targeted therapeutic interventions . Thus, tetc. Moreover, Beclin 1 is required for apoptotic cell engulfment and to coordinate actin dynamics and membrane phospholipid synthesis to promote efficient apoptotic cell engulfment (pP121 via inhibition of the NLRP3 inflammasome (KEGG analysis indicates that apoptosis may be a key mechanism. Previous studies have shown that anti-inflammatory effects mediated by cell apoptosis are closely related to autophagy , and a sgulfment . Autophagulfment . Previougulfment . Alteredgulfment . Howevergulfment . MSU cryammasome . Autophaammasome . Uric acammasome . Howeverammasome . Autophaammasome . In our AMPK could regulate autophagy through phosphorylating autophagy-related factors, coordinately regulating ULK1 to induce autophagy with mTORC1 . The preTo sum up, all the findings confirmed that QHJR played an important role in AGA, and anti-inflammation is the main mechanism of QHJR for AGA. In our study, it was confirmed the high expression of the predicted key genes in AGA, and their expression was reduced after QHJR treatment, laying the foundation for the mechanism study of QHJR in the treatment of AGA. Following identification, we found the presence of 27 metabolites in QHJR, and the TCMSP database indicates that five of these metabolites do not belong to any single herbal component of QHJR. It is hypothesized that these metabolites may arise from interactions among the individual herbal components of QHJR, suggesting that QHJR cannot be substituted by any single herbal constituent. We found that AMPK may not directly activate autophagy. Furthermore, QHJR regulates autophagy by activating the AMPKα/mTOR/ULK1 pathway, thus reducing MSU-induced inflammation and contributing to the treatment of AGA. This provides novel insights and approaches for the clinical treatment of AGA.However, this study still has limitations. For example, we found that there is no direct relationship between Ampk and autophagy, but further verification is needed. Furthermore, due to the limitations of network analysis, the predicted key potential metabolites still need further experimental validation for the accuracy of the research.


Scleritis is a serious inflammatory eye disease that can lead to blindness. The etiology and pathogenesis of scleritis remain unclear, and increasing evidence indicates that some specific genes and proteins are involved. This study aimed to identify pivotal genes and drug targets for scleritis, thus providing new directions for the treatment of this disease.We screened candidate genes and proteins associated with scleritis by text-mining the PubMed database using Python, and assessed their functions by using the DAVID database. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used to identify the functional enrichment of these genes and proteins. Then, the hub genes were identified with CytoHubba and assessed by protein-protein interaction (PPI) network analysis. And the serum from patients with active scleritis and healthy subjects were used for the validation of hub genes. Finally, the DGIdb database was used to predict targeted drugs for the hub genes for treating scleritis.A total of 56 genes and proteins were found to be linked to scleritis, and 65 significantly altered pathways were identified in the KEGG analysis (FDR < 0.05). Most of the top five pathways involved the categories “Rheumatoid arthritis,” “Inflammatory bowel disease”, “Type I diabetes mellitus,” and “Graft-versus-host disease”. TNF and IL6 were considered to be the top 2 hub genes through CytoHubba. Based on our serum samples, hub genes are expressed at high levels in active scleritis. Five scleritis-targeting drugs were found among 88 identified drugs.This study provides key genes and drug targets related to scleritis through bioinformatics analysis. TNF and IL6 are considered key mediators and possible drug targets of scleritis. Five drug candidates may play an important role in the diagnosis and treatment of scleritis in the future, which is worthy of the further experimental and clinical study. Scleritis is an inflammatory condition causing sharp pain in the sclera, which in severe cases may spread to the cornea and uvea . AccordiThe treatment of scleritis mainly involves suppressing inflammation and reducing tissue damage. Currently, the main clinical treatment for infectious scleritis is based on the primary cause of infection. For noninfectious scleritis, the major therapeutic interventions are systemic glucocorticoids, nonsteroidal anti-inflammatory drugs (NSAIDs) and immunosuppressants . HoweverAlthough the lack of specific and reproducible animal models of scleritis has hindered further research into the pathogenesis and treatment of this disease, accumulating evidence has proven that some specific genes and proteins are involved in the occurrence and development of scleritis. In this study, we identified genes and proteins associated with scleritis from the literature, analyzed them through a bioinformatics approach, explored the pathogenesis of scleritis and crucial drug targets for this disease, and provided a new direction for treatment.We comprehensively reviewed the literature related to the keyword “scleritis” in any language published in the PubMed database from 1910 to February 2022, using the computer programming language Python. Then, we performed text mining of common words from the abstract. These publications were summarized in a word cloud, with noninformative common English terms curated and removed manually .According to similar methods previously used by others , 17, we https://david.ncifcrf.gov) , version 2021 (crf.gov) . Accordicrf.gov) . CC indiEnriched candidate genes were assessed with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis on the DAVID platform, and pathways including at least three genes with an FDR<0.05 were selected. KEGG is an online database that systematically analyzes gene functions from the perspective of gene and molecular networks. The KEGG database contains maps of the most known metabolic pathways and regulatory pathways. Graphical information rather than complex text is used in KEGG to introduce each metabolic pathway and its relationship to others .https://cn.string-db.org/). STRING is a biological database of known and predicted PPIs in molecular biology , version 11.5 . Five patients with active scleritis and five healthy control adults were recruited from the Eye Center, Renmin Hospital of Wuhan University (2021–2023), and their serum samples were collected for enzyme-linked immunosorbent assay (ELISA) validation. The inclusion criteria for patients were as follow: (i) the presence of swelling and edema of the scleral tissue and congestion of the deeper episcleral vessels, and diagnosed by an experienced ophthalmologist as scleritis  noP<0.05 was considered significant.Protein expression in serum samples was determined by ELISA. The serum levels of TNF, IL6, IFNγ, IL1β, ICAM1, IL17A, MMP9, IL4, IL12, TGFβ , and HMGB1  were determined by ELISA following the manufacturer instructions. The statistical analyses were carried out using GraphPad Prism 8.0. All experiments were performed by a two-tailed Student’s t-test. http://www.dgidb.org/) provides an interface to search for drug-gene interactions and drug-ready genomes by leveraging existing resources. Potential drugs that interact with the top two genes above were found using DGIdb  are shown in To evaluate the biological characteristics of the identified genes and proteins, GO enrichment analysis was conducted using DAVID online tools. A total of 46 functions were included in the CC analysis, of which 24 were significantly enriched (FDR < 0.05). For the CC analysis, most of the genes and proteins identified were enriched in “extracellular space”, “external side of plasma membrane”, “MHC class II protein complex”, “integral component of lumenal side of endoplasmic reticulum membrane” and “cell surface”. In the MF analysis, 29 functions were involved, of which 17 were significantly enriched (FDR< 0.05). These genes were highly associated with “serine-type endopeptidase activity”, “endopeptidase activity”, “metalloendopeptidase activity”, “zinc ion binding” and “metallopeptidase activity”. The BP analysis identified 143 functions, 99 of which were significantly enriched (FDR < 0.05). The top 5 terms were “immune response”, “collagen catabolic process”, “extracellular matrix organization”, “antigen processing and presentation of peptide or polysaccharide antigen KEGG enrichment pathways were analyzed using DAVID online tools to show the enriched pathways associated with the identified candidate genes and proteins. As a result of KEGG analysis, 65 pathways were significantly altered (FDR<0.05) (The top five pathways were “Graft-versus-host disease (GVHD)”, “Type I diabetes mellitus”, “RA”, “Allograft rejection” and “Inflammatory bowel disease (IBD)” Figure 4Using the STRING database, the PPI network was constructed and uploaded to Cytoscape for the purpose of searching key modules of genes and proteins. All 52 nodes and 896 edges of the PPI network are depicted in To identify the most critical nodes in the PPI network, all nodes were ranked using five algorithms in CytoHubba: MNC, MCC, degree, EPC, and closeness. As shown in P < 0.05). The expressions of ICAM1 and MMP9 were not significantly different between healthy controls and patients, but showed an increasing trend in patients. Additionally, we also investigated the expression of other important Inflammation-related cytokines such as IL4, IL12, TGFβ and HMGB1 (To verify the protein expression levels of TNF, IL6, IFNγ, IL1β, ICAM1, IL17A and MMP9, serum samples from 5 healthy controls and 5 patients with active scleritis were collected to perform ELISA Figure 7As key genes, TNF and IL6 were considered first-grade hub genes. We entered TNF and IL6 into the DGIdb to search for potential drugs. This analysis revealed 88 drugs, and Since scleritis can be divided into different subtypes according to etiology or site of onset, clinical management strategies for them are different. Therefore, we further refined the collected candidate genes and proteins associated with scleritis (In the current study, we summarized and refined the genes and proteins associated with scleritis from the published literature. Through these candidate genes and proteins, we performed a systematic analysis to identify essential pathways and search for potential drugs. Two key modules and eight key genes were finally identified. Eighty-eight potential drugs were considered as candidates for the key genes TNF and IL6, and DALIMUMAB, ETANERCEPT, INFLIXIMAB, PF-04236921, and INSULIN were found to act against both TNF and IL6 and were identified as potentially contributing to the treatment of scleritis.The results of GO concentration analysis showed that cytokines and MHC class II protein complexes have important roles in the development of inflammation in scleritis disease, as suggested in several reports –29. The in vitro experiment, scleral cells stimulated with IL1β showed a significant increase in the expression level of IL6 compared to control cells (In our study, two key genes, TNF and IL6, were eventually identified by CytoHubba and Venn analysis. TNF encodes a multifunctional proinflammatory cytokine belonging to the tumor necrosis factor superfamily. It can bind to and act through its receptors TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR . TNF-α iol cells , indicatol cells . These rWe made network diagrams by drug-gene interactions to find targeted drugs that can interact with TNF and IL6. From the results obtained with DGIdb, most of the 88 targeted drugs were inhibitors, monoclonal antibodies, etc. According to the results of the analysis, a total of five drugs were specific for both TNF and IL6, namely, INFLIXIMAB, ADALIMUMAB, ETANERCEPT, PF-04236921, and INSULIN, among which PF-04236921 has not been currently recognized by the FDA , 53, 54.Since scleritis can be divided into different subtypes according to the etiology or location of disease, the clinical treatment strategies are also different, we further refine the analysis results to identify the potential hub genes and targeted drugs of different subtypes of scleritis. The results confirmed that our prediction of scleritis hub genes and targeted drugs is more suitable for non-infectious scleritis and anterior scleritis, but for infectious scleritis and posterior scleritis, the current analysis methods cannot accurately locate the target drug. This may be due to the small number of cases and the unclear description of the subtype of scleritis in the reports, which requires further study.Some limitations exist in the present study. First, from the data sources, the data analyzed in our study are from the published literature, which has restricted the total amount and scope of data. New studies may have an unpredictable impact on the current results. Second, we could not perform bioinformatic network analysis or gene expression prediction due to a lack of epigenetic data. Third, some bioactive lipids may also play an important role in the pathogenesis of scleritis , but dueThe original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Research Ethics Committee of the Renmin Hospital of Wuhan University. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.FY: Conceptualization, Methodology, Formal Analysis, Writing - Original Draft; YL: Data Curation, Writing - Original Draft; TZ: Validation, Review & Editing; YS: Conceptualization, Funding Acquisition, Resources, Supervision, Writing - Review & Editing. All authors contributed to the article and approved the submitted version.


Neuropathic pain (NP) is the most prevalent form of chronic pain resulting from nerve damage or injury. Despite the widespread use of Duhuo Jisheng decoction (DHJSD) in traditional Chinese medicine (TCM) to treat chronic pain, the mechanism underlying its analgesic action remains unclear.Using network pharmacology, we obtained DHJSD and NP-related target information from public databases to construct protein–protein interactions (PPI) and compound-target networks based on common target genes. These networks were further analyzed using gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG). The interaction between molecules was verified through molecular docking using AutoDock Tools software. Additionally, we treated a chronic constriction injury (CCI) rat model with DHJSD and determined the mechanical withdrawal threshold (MWT). We used an enzyme-linked immunosorbent assay kit to determine the levels of inflammatory cytokines. Furthermore, qRT-PCR was employed to analyze ACHE, NOS2, MAPK3, PTGS2, AKT1, and PPARG mRNA expression, and immunofluorescence was used to evaluate changes in microglia.M1 polarization to improve neuroinflammation, PPARG may have been involved in the process.Our screening of compounds and targets identified 252 potential targets of DHJSD associated with NP. PPI analysis, along with GO and KEGG analyses, revealed that the potential mechanism of DHJSD in NP treatment may be related to inflammatory reactions, the IL-17 signaling pathway, MAP kinase activity, and endocrine activity. Based on molecular docking, the core target showed significant affinity for DHJSD's active components. Moreover, DHJSD treatment repaired the CCI-induced inflammatory reaction in the spinal cord while regulating the expression of ACHE, NOS2, MAPK3, PTGS2, AKT1, and PPARG mRNA. Immunofluorescence results indicated that the active components of DHJSD may regulate microglial The multi-component, multi-target, and multi-pathway actions of DHJSD provide new insights into its therapeutic mechanism in NP.The online version contains supplementary material available at 10.1186/s13018-023-04121-9. Chronic pain is a common health condition that affects 6.2% of the general population worldwide. Neuropathic pain (NP) is the most common form of chronic pain caused by nerve damage or injury . VariousTraditional Chinese Medicine (TCM) monomers and compounds have been shown to be effective in treating NP . For insAngelicae Pubescentis Radix (APR), known as Duhuo Jisheng Decoction (DHJSD), is a medicinal herb widely used to treat sciatica, rheumatoid arthritis, and osteoporosis. APR Extracts and their constituent compounds have potent analgesic and anti-inflammatory effects . For insNetwork pharmacology uses biological networks as targets to analyze the links among drugs, targets, and diseases. A comprehensive and systematic study using network pharmacology is consistent with its holistic nature; thus, it is an important tool for screening active components and elucidating their underlying mechanisms of action. Molecular docking was originally used to detect recognition mechanisms between small and large molecules. In recent years, analytical docking has been applied to drug discovery , where iIn this study, we aimed to identify and analyze the active components, target genes, and related signaling pathways of DHJSD and validate the results in a CCI rat model to elucidate the mechanism of action of DHJSD on NP Fig. . Our dathttp://www.genecards.org/) and DisGeNET (https://www.disgenet.org/home/). Target protein names and their corresponding genes were identified and corrected using UniProt (http://www.uniprot.org). Common target genes from the two databases were analyzed using STRING 11 (https://string-db.org) to determine potential protein interactions with protein–protein interaction (PPI) scores > 0.4, indicating significance. Cytoscape 3.8.2 (http://www.cytoscape.org) was used to visualize the PPI network and Wiki pathways. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted to evaluate target genes at the biological functional level, and key signaling pathways were selected based on enrichment scores. Active compounds, target genes, and prominent signaling pathways were used to construct a compound-target pathway network using Cytoscape 3.8.2. Visualization was performed using https://www.bioinformatics.com.cn and https://www.chiplot.online/By accessing the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), the chemical components of DHJSD were identified, and their possible targets were predicted. The criteria for identifying active components were oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18 in the following processes: absorption, metabolism, distribution, and excretion. The potential target genes were identified using GeneCards (https://pubchem.ncbi.nlm.nih.gov/) and the Protein Data Bank . Receptor proteins were retrieved from the PDB. By utilizing PyMOL 2.3.4 (https://pymol.org/2/) and AutoDock Tools (http://autodock.scripps.edu/resources/adt), receptors were modified by running these processes: dehydration, de-liganding, hydrogenation, and equilibrium charge. AutoDock Vina 1.1.2 (http://vina.scripps.edu) was used to dock the receptors to small-molecule ligands.The affinity of the active components for the crystal structures of the target proteins was verified using PubChem , ground into a powder, and mixed in relative proportions, as described in the Chinese Pharmacopoeia  were procured from Jinan Ponyue Experimental Animal Breeding  and housed under a 12-h diurnal cycle. The rats were provided with water and food ad libitum. The CCI rat models were designed using previously described procedures . Briefly.The rats were then transferred to a vitreous chamber with a wired-mesh floor and allowed to acclimate for 30 min. Mechanical allodynia was assessed using the mechanical withdrawal threshold (MWT) at 0, 1, 4, and 7-day postoperation between 8:00 and 17:00. Measurement of 50% was done with von Frey filaments   using an up-down approach .n = 3/group) were subjected to anesthetize intraperitoneally by administration of 1% sodium pentobarbital (40 mg/kg body weight); the ipsilateral L4-6 spinal cord tissue was rapidly excised, succeeded by using cold phosphate-buffered saline (PBS) for wash and allowed to snap-freeze in liquid nitrogen and − 80 °C storage to be used in further analyses. RNA from the ipsilateral spinal cord was extracted using TRIzol reagent  following the manufacturer’s protocols, and its quantity and purity were assessed at 260/280 nm using a NanoDrop 2000 . The reverse transcription kit  was used for synthesizing cDNA, conducting RT-qPCR on a Bio-Rad iQ5 real-time system  using SYBR mixture  following the protocols, setting the thermal conditions to 25 °C, 55 °C, and 85 °C for 10, 30, and 5 min, respectively, using GAPDH as the reference gene. Primers  were used.The anesthetized rats (described above) were perfused with 200 mL of cold PBS. The ipsilateral L4-6 spinal cord tissue was rapidly excised, fixed in 4% PFA, embedded in paraffin, and sectioned. Paraffin sections were then sequentially dewaxed in xylene, anhydrous ethanol, 85% alcohol, and 75% alcohol, and placed in antigen retrieval buffer . PBS (pH 7.4) was then used to wash the sections three times (5 min each), followed by 30 min of permeabilization with 0.3% Triton X-100 and a second wash with PBS, after which the sections were blocked for 1 h using 3% BSA. For co-staining, PPARG , Iba-1  and CD86  were co-incubated at 4 °C overnight, followed by 3 times of washing (5 min each) and 2 h of incubation in the dark at 20 °C in the presence of Alexa Fluor-488 or -594 goat anti-rabbit , and counterstained with DAPI at 20 °C for 10 min in the dark. The analysis was performed using a 3DHISTECH slide scan system .p ≤ 0.05 indicated statistical significance.GraphPad Prism 9.0 was utilized for conducting the statistical analysis, reporting the data as mean ± SD. One-way ANOVA followed by the Bonferroni post hoc test was used for comparing means among groups. We identified 180 active components  , 96 molecular functions (MF), and 55 cell components (CC) . In contrast, the CCI + DHJSD group had significantly higher MWT values on days 4, 7, 10 and 14 postoperation than the CCI group (p < 0.05)  and significantly decreased in the CCI + DHJSD group compared to the CCI group (p < 0.05). Nevertheless, PPARG had a more significant elevation in the CCI + DHJSD group than in the CCI group (p < 0.05)  Fig. .Fig. 9Cop < 0.05) and significantly decreased in the CCI + DHJSD group compared to the CCI group (p < 0.05) . However, CD86 upregulation was reversed in the CCI + DHJSD group  refers to pain directly caused by an injury or disease involving the somatosensory system ; its treTCM is a holistic and systemic approach to treating complex health conditions . DHJSD iNetwork pharmacology is a useful tool for revealing the mechanisms of action of TCMs in disease treatment. In our study, 83 target genes identified through multiple database searches were subjected to GO and KEGG analyses, revealing that DHJSD might affect NP by governing numerous BP, CC, and MF besides signaling pathways, and wiki pathways, suggesting the importance of neuroinflammation. Furthermore, DHJSD may synergistically regulate key target genes in the interleukin (IL)-17, tumor necrosis factor (TNF), cAMP, T-cell receptor, Toll-like receptor, HIF-1, and MAPK signaling pathways, also involving response to lipopolysaccharide, cellular response to chemical stress, catecholamine binding, and MAP kinase activity. Previous studies found that IL-17A leads to NF-κB, MAPK, and C/EBP cascades activation, inducing IL-6, IL-1β, and TNFα proinflammatory effects following binding to the corresponding target cell receptors  –26. MoreThe PPI and compound-target pathway networks allowed us to identify the top 10 active components and 15 target genes subjected to molecular docking. The results showed that five ingredients, stigmasterol, beta-sitosterol, formononetin, naringenin, and quercetin, were predicted to be functional ingredients of DHJSD in the treatment of NP with ACHE, NOS2, AKT1, MAPK3, PTGS2, and PPARG.M1 polarization in microglia that is upregulated in injured neural macrophages and Schwann cells along with other pain mediators  [M1-type activation in BV-2 microglia, exerting a protective effect against NP [M1/M2 macrophage polarization and enhances functional recovery in spinal cord injury rats [M1 polarization may mediate the anti-inflammatory effects of DHJSD.NOS2 is one of the three nitric oxide synthase (NOS) isoforms, and its end product modulates NP . Both 5-., COX2) , 34. PPA., COX2) . PPARG i., COX2) . PPARG h., COX2) . In addiainst NP . Moreoveury rats . In concm1) and anti-inflammatory (m2) [m1-like microglia secrete TNF, IL-1, and IL-6 as pro-inflammatory substances, which damage neural networks [M1 polarization in microglia attenuates NP [Microglia are the immune cells of the CNS. Neuroinflammation caused by microglia is also involved in NP development . When exory (m2) . In the networks –44. Notauates NP , 45.M1 polarization in the L4–6 spinal cord and the expression of inflammatory factors.In our study, ACHE, MAPK3, and PTGS2 were significantly downregulated, whereas PPARG was upregulated in the CCI + DHJSD group compared to the CCI group. Moreover, DHJSD significantly suppressed NOS2 expression in the spinal cord and decreased CD86/Iba-1 positive cell number in the dorsal horn. These results showed that DHJSD markedly improved neuroinflammation, partly by reducing microglial M1-type microglial polarization can similarly affect astrocytes. Reactive astrocytes exhibit heterogeneous and multifaceted functions in SCI, such as providing trophic and metabolic support to neurons, inhibiting axonal growth, and modulating inflammation [M1 microglia may induce astrocyte necrosis by activating TLR4/MyD88 signaling, thereby favoring neuronal cell survival [M1 phenotype through PPAR-r/CD36-dependent mechanisms, inhibiting astrocyte proliferation and neuroinflammation amelioration [M1-type microglial polarization, thereby reducing astrocyte proliferation and treating neuropathic pain.But there are limitations to our experiments, we focused only on changes in microglia. Recent studies have found that astrocyte-microglia interactions are particularly important for the treatment of neuropathic pain, and bidirectional communication between astrocytes and microglia can modulate central nervous system responses through the secretion of multiple cytokines and inflammatory mediators . Astrocyammation , 49. Necsurvival . MpHE caioration . Thus, wM1 polarization and inflammatory factor expression in the microglia. In future studies, we will focus on exploring the specific therapeutic mechanisms of DHJSD in NP, thereby contributing to developing new analgesic drugs for the effective treatment of NP with mild or no side effects.In conclusion, using network pharmacology, we systematically analyzed the analgesic mechanism of DHJSD and found that stigmasterol, beta-sitosterol, formononetin, naringenin, and quercetin were the active chemical components responsible for its action, while ACHE, NOS2, AKT1, MAPK3, PTGS2, and PPARG were the core targets. The results of the in vivo experiments also suggested that DHJSD significantly ameliorated neuroinflammation following NP treatment, in part by decreasing Network pharmacology, molecular docking, and animal experiments were combined to study the analgesic mechanisms of DHJSD. Based on our data, DHJSD may regulate microglial polarization in the spinal cord to alleviate neuroinflammation; however, further research is required to validate our results. Overall, exploring the active components of DHJSD and the regulatory mechanisms underlying microglial cell activation in the rat spinal cord may help develop new analgesic drugs to effectively treat NP.Additional file 1. Table S1: Chemical compounds of 15 herbs in DHJSD. Table S2: Targets of DHJSD and NPs. Table S3: PPI analysis. Table S4. GO enrichment analysis data. Table S5. KEGG pathway enrichment analysis data. Table S6. C-T-P analysis data. Table S7: Molecular docking data.Additional file 2. Fig S1: M2 polarisation.


To assess the effect of sex and laterality on clinical features of primary rhegmatogenous retinal detachment (RRD).This study is a retrospective analysis of data prospectively collected. We extracted data from two online datasets over a 7-year period of patients older than 16 years who had undergone surgery for primary RRD. Data on baseline characteristics were analyzed to compare males versus females, and right versus left eyes.Of 8133 eyes analyzed, 4342 (53.4%) were right. The overall male predominance (63.7%) was more marked in the age range 50–69 years. Men were more commonly pseudophakic and presented more frequently with baseline posterior vitreous detachment (PVD). Female sex was significantly associated with baseline myopia, retinal holes as causative retinal break, and isolated inferior RD. Men had more frequent foveal involvement, greater RRD extent, greater numbers and larger sized retinal tears including dialysis and giant retinal tears. Regarding laterality, foveal involvement, larger retinal breaks, isolated temporal RD and temporal retinal breaks were more common in right eyes, whereas left eyes were more myopic at baseline and presented more frequently with isolated nasal RD and nasal retinal breaks.This study confirmed the predominance of male sex and right laterality in RRD. Sex and laterality were associated with multiple presenting features of RRD including extent, break distribution, number, size and type, as well as RD distribution. Rhegmatogenous retinal detachment (RRD) has an annual incidence of 1–2:10,000 in the general population, with recent evidence suggesting it is increasing . It has Knowing that accurate characterization of RRD might provide insight into pathogenesis, previous studies have analyzed the effect of several features including lens status on the features and outcomes of RRD , 9. The Indeed, it has been widely recorded that there is a higher incidence of RRD in males –13. AlthSimilarly, an increased incidence of RRD in right eyes has been repeatedly reported in the literature , 13, 17,In this large prospectively collected database analysis, we aimed to investigate the potential effect of sex and eye laterality on the clinical features of primary RRD.https://www.rcophth.ac.uk/standardspublications-research/audit-and-data/clinical-data-sets/retinal-detachment-dataset/) and use the same web-based data collection tool. Only primary RRDs are included in the database, whereas secondary RDs of any type  are excluded. We therefore also excluded inappropriately entered cases where these had been mentioned as comorbidity and patients under 16 years old.We extracted the data from the EURETINA RRD database and the Britain & Eire Association of Vitreoretinal Surgeons (BEAVRS) VR database in March 2020. All primary RRDs that had undergone surgical repair of any type  from May 2012 to May 2019 were extracted. We excluded cases that had incomplete data for age, sex, laterality, and lens status and in whom a retinal drawing hadn’t been completed. The two databases conform to the UK national RD dataset , central vision loss duration, presence and grade of vitreous haemorrhage (VH) , RRD anaTo analyze differences in the distribution of the RRD we derived a number of groups based on their location including those localized to one quadrant only, as well as those with any involvement of the superior, inferior, temporal and nasal retina. Similarly, the position of the lowest break was categorized as being superior (10–2 o’clock), inferior (4–8 o’clock), and either nasal (1–5 o’clock in right eyes and 7–11 o’clock in left eyes) or temporal (7–11 o’clock in right eyes and 1–5 o’clock in left eyes).This study conformed to the UK’s Data Protection Act and the principles of the declaration of Helsinki. No patient could be identified with any of the data contained in the database and a unique random alphanumeric code is used for internal identification. As the dataset is considered a service evaluation, no IRB approval and/or informed consent were needed according to UK guidelines.https://www.r-project.org/). All VA values were converted to logMAR values for analysis, considering count fingers (CF), hand movements (HM), perception of light (PL) and no perception of light (NPL) equivalent to 1.98, 2.28, 2.70 and 3.00, respectively [Descriptive and statistical analysis was performed using R version 3.4.1  or inappropriate entry (28). A total of 84 surgeons entered data with a median of 47 cases per surgeon (interquartile range 10–115).The mean age was 59 years, 63.7% were male and 53.3% were right eyes; 27% of eyes were pseudophakic; 78% of the total had a confirmed definite PVD, with no PVD in 9.4% and the status uncertain in 12.6%; 9% had a past of concomitant fellow eye RRD. The RD was total in 6.2% and the fovea involved in 49.9%. PVR grade C was present in 7%. 11.1% had myopia of greater than 6 dioptres recorded , visual acuity , lens status , presence of posterior vitreous detachment , refraction , fellow eye RD , extent of RD , foveal sparing , any superior RD , isolated inferior and temporal RD , larger break size , presence of dialysis, GRT, U-tear , presence of round hole, outer leaf break, no break found , superior retinal breaks being the lowest breaks present  Table .Table 2Vp = 0.756.The median duration of female foveal involving RRD was 6 days (IQR 3–13) and for men 6 days (IQR 6–14), The following variables were associated significantly with ocular laterality, with the eye associated with the higher value given in brackets: age (right), visual acuity (left), refraction (left more myopic), fellow eye RD (left), foveal sparing (right), isolated nasal (left) and temporal RD (right), larger break size (right), and finally temporal retinal breaks (right) and nasal retinal breaks being lowest breaks present (left) Table .Table 3VThere were significant interactions between sex and laterality with the age bands 16–30, 31–40, 41–50, 51–60, 61–70, 71–80, >80 shown in Supplementary Table There was no evidence of interaction between sex and laterality between any of the tested variables and all associated variables remained significant.p = 0.009), and similarly for refractive status (p = 0.008). The association of the presence of a PVD and refractive status with the age for male and females is shown in Fig. There was an interaction between age and sex and the presence of PVD, but the association of sex with PVD remained significant (n = 333), U-tear RRD with confirmed PVD (n = 4851), giant retinal tear (GRT) related RRD (n = 186) and dialysis related RRD (n = 257). The male sex predominance varied significantly between the 4 groups being greatest in the GRT group at 78.4%, 71.1% in the dialysis group, 65.3% in the U-tear group and reversed in the round hole RRD group at 44.1% (p < 0.0001). Conversely the right eye predominance was not significantly different between the 4 groups (p = 0.31).To further analyze associations of sex and laterality we studied 4 well known and phenotypically distinct subgroups of RRD, namely round hole RRD with confirmed absent PVD (We also analyzed for the right temporal and left nasal isolated RRD predominance we found. There was no significant predominance of isolated nasal RRD in any of the 4 subgroups between the right and left eyes. There was a significant isolated temporal RRD predominance in right eyes in the round hole and U-tear subgroups, but not in the GRT and dialysis groups (Supplementary Table In this large prospectively entered database study, we confirmed the known higher prevalence of RRD in males and right eyes and discovered several differences in phenotype that have not previously been reported. In particular, we found a greater prevalence of PVD-associated RRD in men, with larger breaks and more extensive and superior involving RD. Interestingly, temporal RD was more common in right eyes with a predominance of temporal breaks being the lowest break present, whilst nasal RD was commoner in left eyes with nasal breaks predominating.The greatest excess of male RRD overall was in the 50–70-year age range, accounting for approximately 65% of cases, whereas after 80 years no clear sex predominance can be detected, consistently with two recent large retrospective studies , 22. It There was a significant difference in vitreous attachment between the sexes, with women having a higher proportion of RRD without a PVD, whilst men had a higher proportion of RRD with PVD in the under 50s, which then became similar. This was also reflected in the types of breaks observed in the two groups. The female predominance of round hole, PVD absent RRD is associated with the higher incidence of myopia in the under 50s, whilst male PVD is more commonly associated with U-tears and RRD, especially in the relatively younger age groups. This could appear in contrast with several studies that have shown a higher prevalence of the more advanced stages of vitreous separation in women at an earlier age in the general population , 31. HowMale RRD more commonly involved the superior retina, was more extensive, less commonly isolated to the inferior and temporal retina, and more commonly foveal involving than in women. We found no evidence that men sought attention less promptly than women, with no significant difference in the duration of visual loss in foveal involved cases and in the proportion of PVR. Whilst the difference in RD distribution and extent partly relates to the predominance of PVD related RRD in men, it may also relate to lower retinal adhesion to the RPE. Interestingly, men more commonly had inferior retinal breaks which may also account for the increased RD extent.Men had a higher proportion of pseudophakic RRD, as reported before . This woAs with sex, the predominance of right eyes was most marked in the 50–70-year age group. It has been suggested that the right eye predominance may be related to ocular dominance and induced myopia , 32. HowThe better presenting vision and lower foveal involvement in right eyes probably relates to ocular dominance and earlier presentation for treatment.p < 0.01, and due to our large sample size, most results had p values considerably less than this level.The main strength of this study is the large number of cases, broadly representative of European populations as suggested by the analysis of pre-operative findings . AlthougFinally, the data entered in the databases is not collected in a standardized manner, however compulsory data fields and classification guidelines will help to reduce variability.In conclusion, sex and, to a lesser extent, laterality affect the phenotype of RRD observed. These differences may support the role of multiple and different pathogenetic mechanisms in RRD development and are also important factors to consider when reporting studies affecting different populations.Rhegmatogenous retinal detachment show a male and right eye predominance.Sex and laterality influence the phenotype of rhegmatogenous retinal detachments in multiple different ways.Sex- and laterality differences give insight into pathogenesis.Sex/laterality differences should be considered in studies with mixed populationsSupplementary Table 1Supplementary Table 2


Because the thermodynamic property is closer to gasoline, advanced biofuels (C ≥ 6) are appealing for replacing non-renewable fossil fuels using biosynthesis method that has presented a promising approach. Synthesizing advanced biofuels (C ≥ 6), in general, requires the expansion of carbon chains from three carbon atoms to more than six carbon atoms. Despite some specific biosynthesis pathways that have been developed in recent years, adequate summary is still lacking on how to obtain an effective metabolic pathway. Review of biosynthesis pathways for expanding carbon chains will be conducive to selecting, optimizing and discovering novel synthetic route to obtain new advanced biofuels. Herein, we first highlighted challenges on expanding carbon chains, followed by presentation of two biosynthesis strategies and review of three different types of biosynthesis pathways of carbon chain expansion for synthesizing advanced biofuels. Finally, we provided an outlook for the introduction of gene-editing technology in the development of new biosynthesis pathways of carbon chain expansion. For example, glycerol-3-phosphate dehydrogenase, glycerol-3-phosphatase from S. cerevisiae and glycerol-3-phosphatase from Klebsiella pneumoniae were introduced into the E. coli cell factory to produce 1, 3-propylene glycol [Geobacillus stearothermophilus was introduced to the cell factory to produce L-alanine. Five foreign genes from different sources were integrated into E. coli cell factory to produce 1,4-butanediol [Generally, obtaining biosynthetic pathways can be divided into three types according to the source: (1) use only the host's own metabolic pathway. For example, the metabolic pathway of  pathway , 8. The e glycol , 10. An tanediol . (3) CreE. coli was designed using only four steps with Acetyl-CoA, α-ketoglutarate, glutamic acid, and succinyl Coenzyme A to achieve a maximum production of 18 g/L of 1,4-butanediol [However, biosynthesis strategies play a fundamental role in the initial exploration of novel biosynthetic pathways. Here, we summarized two biosynthesis strategies based on the above three types of biosynthetic pathways. To achieve advanced biofuels and overcome low efficiency problem of expanding carbon chains for the exploration of biosynthetic pathways, there are two basic biosynthesis strategies to be followed in the synthesis of advanced biofuels. As shown in Fig. tanediol .Fig. 3ThThe second biosynthesis strategy is an evolutionary design (ED) approach based on known metabolic pathways or molecular structures of molecules similar to advanced biofuels Fig. b. This sBased on the above two biosynthetic strategies, the optimal metabolic pathway can be determined to solve the problem of low efficiency to a large extent in the process of extending the carbon chain, such as reducing the intermediate metabolic reaction as much as possible, and reducing the metabolic tributaries producing by-products as much as possible. Through the above strategies, obtaining the metabolic pathway with the fewest reaction steps can reduce the difficulty in the technical level of gene manipulation, for example, it is easier to knock out the by-product generation pathway, more conducive to release the inhibition of product synthesis, the stability of key enzymes in the overexpression synthesis pathway, the calculation of the minimum metabolic network model, the assembly of metabolic pathway, and the dynamic regulation of gene expression.To date, different biosynthetic pathways of expanding carbon chains have been developed based on the above strategies using multi-enzyme expression system to acquire advanced biofuels including C6–C10 alcohols, C6–C18 alkanes, enolates, and C6–C10 olefins [Some natural precursors derived from different intermediates of 2-keto acids metabolic pathways are transformed into advanced biofuels with molecular structure similar to the intermediates based on the ED strategy Table . In thisl-valine), which was then catalyzed by the catalytic reactions of two catalyzing enzymes: IlvC, and IlvD [The biosynthetic pathways for Type I sub-pathway was featured by the addition of two carbon number (+ 2) using pyruvate molecules as the carbon chain extension factor. Various advanced biofuels were obtained using this metabolic pathway via the conversion of interlinked intermediates of the pathway Fig. a. Carbonand IlvD , 20. Furand IlvD , 21, 22.LeuCD [LeuABCD, and some substrates of advanced biofuels, such as KMV, K4MH, K5MH, and KMO, could be produced by the metabolic pathway. Subsequently, the production of some branched alcohols with a range of 6 to 10 carbon atoms could be achieved via oxidation–reduction reactions.The biosynthetic pathways for Type II was characterized by the extension of one carbon number (+ 1) using CoA-dependent molecule as the carbon extension factor. The advanced biofuels produced via this pathway were primarily alcohols with branched chains that were the result of comprehensive interlacement with other inherent pathways: a natural α-keto acid metabolic pathway and a metabolic pathway that shared characteristics of the Type I sub-pathway Fig. b. It is LeuCD , using aLeuCD . SimilarLeuABCD. A distinguishing feature between these two metabolic pathways could be found in the advanced biofuels (C ≥ 6) produced from the Type III sub-pathway as it did not contain any branched chains  were synthesized via a condensation reaction between 2 kV and acetyl-CoA. Based on this strategy, other precursors could produce their corresponding alkane-based alcohols. These precursors include 2-ketoheptanoate , 2-ketooctanoate , and 2-ketononaoate , which, similar to 2 kV, could also be obtained via overexpressing LeuABCD [Clostridium, which possess an acetone–butanol–ethanol (ABE) pathway [The biosynthetic pathways for Type III sub-pathway were also defined by the elongation of one carbon atom number (+ 1) using CoA-dependent molecule as carbon chain expanding factor. The similarity between this sub-pathway and the Type II sub-pathway is that they both leveraged acetyl-CoA as the carbon chain elongation factor, and resulted in a condensation reaction by overexpressing ins Fig. c. The Ty LeuABCD . Then, a LeuABCD . This su pathway , 28. In  pathway –31.E.coli is derived from the Type II sub-pathway and requires two catalytic reactions[LeuABCD and a decarboxylation–reduction reaction involving 2-ketoisovalerate decarboxylase (Kivd) and alcohol dehydrogenase 2 (adh2). Meanwhile, advanced biofuels of unbranched alkane-based alcohols such as n-hexanol [Crt, Ter in tandem, and an exogenous decarboxylation–reduction reaction: kivd-adhE  were simulated using quantum mechanics and protein–substrate complexes to identify the best conformers [A large number of studies have proved that 2-isopropylmalate synthase (IPMS) and acetohydroxyacid synthase (AHAS) are two key enzymes involved in the expansion of carbon chains in the biosynthesis strategy that leverages 2-keto acid intermediates. In recent years, in order to improve the efficiency of expanding carbon chains, different carbon–carbon bond transition states catalyzed by nformers . A divernformers , 36. TheBiosynthesis strategy based on reverse β-oxidation intermediates has been demonstrated to show significant promises for expanding carbon chains to produce a variety of advanced biofuels Table , such asn+2)-acyl-CoA into two main compounds, acetyl-CoA and (Cn)-acetyl CoA intermediates, via a series of catalytic reactions of the four catalyzing enzymes atoB, fadA, fadB, and fadE. A circulation process in which two carbon molecules from the fatty acid pathway are removed through the β-oxidation cycle, followed by the addition of one acetyl-CoA molecule to the tricarboxylic acid (TCA) cycle  have been produced using reverse β-oxidation intermediates. For example, 1-hexanol at a yield of 37 mg/L was obtained by expressing alcohol dehydrogenases and betA , 47 and and betA . Howeverand betA , 50.Nevertheless, a variety of fatty acids and alcohols with various carbon chain could be achieved by this approach due to the interaction between 2-keto acids and the β-oxidation process , 52. Thecyanobacteria can be converted into biodiesel, alcohols, alkanes, and olefins with long carbon chain [S. cerevisiae can also naturally produce a great quantity of acylglycerols, which can be subsequently converted into various advanced biofuels [Biodiesel fuel, as an advanced biofuel, has been proposed as a potential substitute for petroleum-based diesel. Biodiesel fuel is typically produced from fatty acid methyl esters (FAMEs) or fatty acid ethyl esters (FAEEs) via the conversion of plant or animal oils. Decarboxylated derivatives, mainly fatty alcohols, with long aliphatic chains or aromatic hydrocarbon molecules can also be synthesized via the transesterification of acylglycerols using this biosynthesis strategy based on fatty acid intermediates. For example, free fatty acids (FFAs) from on chain , 54, whibiofuels –57. HoweE. coli is first converted into malonyl-CoA under the actions of ATP-dependent-acetyl-CoA carboxylase (AccABCD). The FFA compound bound to an acyl carrier protein (ACP) is then produced through a NADPH-dependent reaction in which malonyl-CoA is converted by a multicomponent fatty acid synthase. These fatty acyl-ACPs are utilized as either carbon chain elongation structures or energy storage molecules. The FFAs are generated from these energy stores, and structural components can be reconstructed by overexpressing thioesterases that cleave fatty acyl-ACPs. Cleavage reaction of fatty acyl-ACP can lead to the accumulation of large amounts of fatty acyl-CoAs because the gene cytoplasmic  thiolase can hydrolyze fatty acyl-ACPs by fatty acid transport and fadDE gene. Finally, the FFAs with long carbon chain are added to some alcohols with short carbon chains between C8 and C18 under the action of different thioesterases  and levels of saturation were also generated based on this biosynthesis pathway using E. coli [Previous literature has demonstrated that yield of fatty alcohols can be increased via systemic improvement of metabolic genes involving fatty acid intermediates. For example, some studies have shown that deletion of  E. coli –62.Alkanes and alkenes are the most important advanced biofuels that are used as substitutes for hydrocarbon fossil fuel such as jet fuels, diesel, and gasoline. With recent advances in system metabolic engineering, synthetic mechanisms of alkanes and alkenes have been investigated in depth to allow for a better understanding of their underlying metabolic pathways Table . There aJE OLS, and CAR/FAR-ADO  that include some advanced biofuels, such as heptadecane, heptadecene, tridecane, pentadecene, pentadecane, heptadecene, pentadecane, and heptadecane, by overexpressing AAR and ADO derived from cyanobacteria strains [Previous studies have shown that pathways that convert FFAs or fatty acid intermediates into alkanes and alkenes can be defined by five diversity-oriented sub-pathways, including AAR-ADO, HEAD-TO-HEAD, OLeTADO Fig. . For AARf alkane . An engi strains .Fig. 7BiMicrococcus luteus, olefin synthesis allows for the expansion of carbon chain via the action of four catalytic enzymes OleA, OleB, OleC, and OleD [Meanwhile, head-to-head condensation reaction can generand OleD . These fand OleD , 76.eTJE was first identified in the bacteria Jeotgalicoccus sp. ATCC 8456 , including multi-domain ketosynthase (KS), acyltransferase (AT), and ketoreductase (KR). The expansion process of carbon chain involves the combination of acyl-ACP with the ACP1 domain to form an acyl-substrate. Then, two carbons from malonyl-CoA are added to the acyl-substrate by the central extension module of KS, AT, and KR. Subsequently, the ACP2 domain and β-keto group are reduced to generate β-hydroxyl, which is then activated by sulfotransferase (ST) via sulfation. Finally, the C-terminal of the thioesterase (TE) domain catalyzes dehydration and decarboxylation reactions to produce the advanced biofuels olefin [ols as the agent responsible for expressing olefin synthase [The sub-pathway OLS pathway was first discovered in marine cyanobacterium s olefin . For exas olefin  and 1,14synthase , 83. Somsynthase .Sfp) [Sfp catalytic enzymes are derived from two bacteria, Mycobacterium marinum [Bacillus subtilis [Photorhabdus luminescens [Sfp from Cyanobacteria in combination with a B. subtilis thioesterase, branched chain α-keto acid dehydrogenase complex, lipase, and β-keto-acyl-ACP synthase III [The engineering sub-pathway CAR/FAR-ADO is dependent on three very important catalytic enzymes: carboxylic acid reductase (CAR), fatty acid reductase (FAR), and phosphopantetheinyl transferase (Sfp) . The CAR marinum  and Bacisubtilis , respectinescens , 89. Afthase III . Some lihase III –93.E. coli to produce many advanced biofuels such as isobutanol at a yield of 22 g/L [In summary, the biosynthesis pathways based on 2-keto acid intermediates, fatty acid intermediates or reverse β-oxidation intermediates could be used to add “ + 1” or “ + 2” carbon molecules to carbon chain. Addition of one-carbon molecule could agitate the whole metabolic network by influencing the process of catalytic reactions Fig. . Therefof 22 g/L , 94. CurIn the aforementioned biometabolic pathways for expanding carbon chain, CoA molecules are first converted to some intermediates via addition of carbon molecule. For example, synthesis of butanol involves the extension of carbon chain using a series of CoA molecules: acetyl-CoA acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase, and butyryl-CoA dehydrogenase . TherefoIn order to overcome the problem of low efficiency, from the early traditional modification techniques such as eliminating the generation pathway of by-products, releasing the synthesis inhibition of products, overexpression of key enzymes in the synthesis pathway, to the development of metabolic response database, and machine learning-based network model calculation, optimal assembly of metabolic pathways, dynamic regulation of gene expression and other new metabolic modification techniques. There are usually the following technical approaches for overcoming low efficiency in carbon chain expansion.Golden Gate assembly techniqueThe creation and optimization of biosynthetic pathways for carbon chain extension first require a breakthrough in DNA fragment assembly technology. DNA fragment assembly technology is an important prerequisite for the creation of biosynthetic pathways for producing biofuel . Traditi(B)Gibson assembly techniqueRestriction endonuclease Type II, which has features that identify and cut the position inconsistencies of double-stranded DNA, is required. This property enables it to cut DNA outside the recognition site, and the sticky ends generated are connected sequentially under the action of ligase and assembled into DNA fragments without the enzyme cleavage site, realizing the seamless connection of multiple fragments. It maybe provide opportunities in creating a flexible, versatile, and data-driven framework to support biofuel research and development in biofoundries , 100.(B)(C)CPEC techniqueNo restriction enzyme is required, but the ends of each DNA fragment contain homologous sequences of 15-20 bp, according to which the assembly is carried out in the order of homologous sequences. The assembly process involves several enzymes: DNA 5' exonuclease, which first cuts out a sticky terminal, which complicates each other according to homology, and polymerase DNA polymerase, which replicates the missing bases. Finally, the sticky end is attached by DNA ligase. This method allows one or more DNA fragments to be inserted into a linearized vector quickly, efficiently and precisely in a predetermined direction, achieving “seamless” assembly . This teE.coli to obtain complete plasmids. In general, CPEC is a simpler, more efficient and economical DNA assembly technology with more extensive applications, but it is not suitable for the assembly of DNA fragments with high GC content. A standard CPEC protocol 67 was used to assemble the three PCR products to produce biofuel [Circular polymerase extension cloning (CPEC) is a short name for circular polymerase extension cloning. The requirements for DNA fragments are similar to Gibson's, and the ends contain homologous sequences. CPEC is a technology to realize the assembly of DNA fragments by PCR: DNA fragments and vectors are denatured and decomposed, and the end homologous sequences complement each other during annealing. The sequences are mutual templates and primers and extend into circular DNA molecules containing gaps under the action of DNA polymerase . The gap biofuel , 106.Single gene regulationGene expression regulation is an important means to optimize biosynthetic pathways and is the core to overcome the low efficiency of target product production. After the pathway is created in the host, it is necessary to regulate the expression intensity of genes at key nodes, including enhancing the product synthesis pathway and cofactor synthesis pathway, or weakening some genes that will lead to non-growth of bacteria and have adverse effects on product production after knockout. According to the technical level, it can be divided into single gene regulation, polygene regulation and gene dynamic regulation.E. coli were constructed and to obtain promoters of different strengths for the regulation of glucose transporter gene galP and glucokinase gene glk [(B)Multi-gene regulationSingle gene regulation is to regulate the expression of a specific gene of metabolic pathway on chromosomes. In the early stage of metabolic engineering, strong promoters are commonly used for single gene regulation, such as IPTG-induced Tac promoter and T7 promoter. However, strong expression of single genes may not be optimal for metabolic pathways. To achieve optimal expression of a single gene, promoter libraries in gene glk , 108. Thgene glk –111.(B)ME.coli realized cooperative expression, and mevalonate production was increased by 7 times using this technique [(C)Gene dynamic regulationSingle-gene regulation can only be operated on the single gene dimension. Even if combined regulation, the library capacity is greatly reduced, and it is difficult to screen out the optimal combination of multiple genes. In the optimization of metabolic pathway, to achieve the efficient production of target biofuel with long-carbon chain, the cooperative expression of multiple genes is often required to achieve the optimization of metabolic pathway. Keasling's team developed a tunable intergenic region (TIGR) library technique . Based oechnique , 116. Isechnique . Based oechnique , 119.(C)S. cerevisiae was engineered to produce and secrete 1-alkenes by manipulation of the fatty acid metabolism, enzyme selection, engineering the electron transfer system and expressing a transporter [Dynamic regulation is one of the most effective strategies in metabolic pathway optimization and an important technique to improve the target product. Although static regulation such as single- and multigene regulation is easy to operate and effective, the intracellular metabolites are dynamic. If the expression level of related genes is too high, the cell resources will be wasted; if the expression level is too low, the metabolic pathway will be restricted, and the yield and output of the final target products will be difficult to improve due to the imbalance of cell metabolism. In order to timely balance the relationship between gene expression and global metabolism required for product synthesis, the introduction of gene dynamic regulation can respond to metabolic signals in real time, and make timely feedback regulation to adapt to changes in cellular metabolism and environment. For example, the nsporter . Furthernsporter –123.E.coli and other microorganism gene editing for producing biofuels [Two-step homologous recombination based on FIp recombinaseIn order to improving microbial synthesis efficiency, through gene editing, exogenous gene insertion, endogenous gene knockout and key gene expression regulation can be realized for expanding carbon chain. Homologous recombination system is the basis of bacterial and fungal genome editing technology , 125. Thbiofuels . Based o(B)Two-step positive and negative screening method based on sacB geneFlp recombinase can recognize short Flippase recognition site sequences and excise the sequences between two flippase recognition targets (FRTS) . CombineE. coli, and is a negative screening marker. Under the guidance of Red homologous recombination system, the fragments containing resistance genes and sacB genes were first integrated into the target site [(C)One-step homologous recombination based on CRISPR/Cas9The sacB gene encodes secreted sucrose fructanase, which can catalyze the hydrolysis of sucrose into glucose and fructose, and synthesize fructose into high molecular weight fructan, which can produce lethal effect on get site , then thE. coli genome and produced biofuels [Genome editing technique, such as CRISPR–Cas, is extensively used in a diverse array of fields, with practical applications in the biological synthesis of new compounds. It is a powerful tool for studying eukaryotic microorganisms and has been reported to be useful for knocking out a specific targeted gene, simultaneously operating multiple genes, disrupting, activating, and repressing genes, and editing the genome in a genetically known or unknown microorganism . Cas9 prbiofuels , 131. ThSaccharomyces cerevisiae and Kluyveromyces marxianus have resulted in enhanced production of bioethanol and 2-phenyl ethanol in these organisms, respectively [The integration of synthetic biology, genome-editing technique like CRISPR/Cas9, and high-throughput sequencing technology, would lend feasibility to expanding carbon chain for the production of new advanced biofuels in industrial-scale applications. For example, some studies have been showed lignocellulosic biofuel production can be improved by CRISPR/Cas9-mediated lignin modification , 133. Fuectively . DesigniOne advantage of carbon chain expansion is that it allows refactoring of any potential catalytic reaction to be performed in a simple, convenient, inexpensive, efficient manner. This technology also lends practicality towards transformation of cellular structures of microorganisms and recombination of metabolic networks, especially in changing mitochondria to improve their synthesis efficiency. For example, substantial increase in production of advanced biofuels had been attributed to the close link between genetic improvement of the whole signaling network and recombination of the whole metabolic flux, such as genome editing of algal species by CRISPR–Cas9 for biofuels .Streptomyces with fine-tuning the regulation [Trichoderma reesei with improving cellulase production [Aspergillus species with improving succinic acid production from renewable biomass [The potential application of this technology is continually being explored in different microorganism platforms, along with bioinformatics and large-scale modeling, for analysis at the genome level, transcriptome level, proteome level, metabolome depth, and fluxome-level. Findings from these studies would contribute to better understanding of the biochemical evolution and functional strain during gene function elucidation and epigenetic regulation in loss-of-function (LOF)  or gain-gulation –142, Trioduction , and Asp biomass , 145 and biomass .The rapid development of biosynthesizing advanced biofuels, coupled with the demand of newly reconstructed engineering pathway for synthesizing novel chemicals and the need to improve efficiency of existing strategies, require strengthening of previous biometabolic pathways and discovery of novel biosynthesis pathways. In this paper, the low efficiency problem of production in expanding carbon chain was presented, followed by the proposal of two biosynthesis strategies RM and ED, and a review of corresponding biosynthetic pathway for producing advanced biofuels. Our findings contribute to future design of novel catalytic routes to maximize the production of target product. In addition, genome editing technique such as the representative CRISPR/Cas9 gene-editing technology will generate a traction for effective resolution of many technical obstacles in discovering new engineering strategy and increasing production of advanced biofuels.