Purpose:Prunella vulgaris (PV), a traditional Chinese medicine, has been used to treat patients with thyroid disease for centuries in China. The purpose of the present study was to investigate its bioactive ingredients and mechanisms against Hashimoto’s thyroiditis (HT) using network pharmacology and molecular docking technology to provide some basis for experimental research.Methods: Ingredients of the PV formula were retrieved from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. Additionally, HT-related genes were retrieved from the UniProt and GeneCards databases. Cytoscape constructed networks for visualization. A protein–protein interaction (PPI) network analysis was constructed, and a PPI network was built using the Search Tool for the Retrieval of Interacting Genes (STRING) database. These key targets of PV were enriched and analyzed by molecular docking verification, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment.Results: The compound–target network included 11 compounds and 66 target genes. Key targets contained Jun proto-oncogene (JUN), hsp90aa1.1 (AKI), mitogen-activated protein kinase 1 (MAPK1), and tumor protein p53 (TP53). The main pathways included the AGE-RAGE signaling pathway, the TNF signaling pathway, the PI3K–Akt signaling pathway, and the mitogen-activated protein kinase signaling pathway. The molecular docking results revealed that the main compound identified in the Prunella vulgaris was luteolin, followed by kaempferol, which had a strong affinity for HT.Conclusion: Molecular docking studies indicated that luteolin and kaempferol were bioactive compounds of PV and might play an essential role in treating HT by regulating multiple signaling pathways. Prunella vulgaris (PV) is a perennial herbaceous plant in the genus Prunella1. It is a Chinese medicine widely used to treat inflammation, eye pain, and headaches   and thyroglobulin antibody (TG-Ab) (PV might exert its anti-inflammatory effects are poorly understood.eadaches . The antne (TCM) . Current (TG-Ab) . HoweverHashimoto’s thyroiditis (HT) is a genetic autoimmune disorder characterized by the destruction of thyroid cells by cell- and antibody-mediated immune responses . In deveAdditionally, the association between vitamin D deficiency, HT pathogenesis, and thyroid hypofunction has been demonstrated in several studies . TherefoNetwork pharmacology, combined with pharmacology and pharmacodynamics, is a novel research field that clarifies numerous compounds’ synergistic effects and underlying mechanisms by analyzing various networks of complex and multilevel interactions . The stuPV were obtained from the Traditional Chinese Medicine Systems Pharmacology Database, the Analysis Platform  database, and subsequent network pharmacology (The active constituents of macology . The TCMmacology . The nammacology . In thismacology .PV were retrieved from both TCMSP databases (http://lsp.nwsuaf.edu.cn/tcmsp.php), including 6,511 drug molecules and nearly 4,000 targets as well as the interaction between them (https://www.uniprot.org).The potential targets for the components of een them . The Unieen them . Gene inhttps://www.genecards.org/) and selection according to the criterion of RiskScore >1, the Therapeutic Target Database , OMIM (https://omim.org/), PharmGkb (https://www.pharmgkb.org/), and DrugBank (https://www.drugbank.ca/) were used to collect information on HT related to target genes. The association of PV with HT was then gathered as the core targets of PV for HT.The GeneCards database (http://www.cytoscape.org/)  . Moreovepe.org/) . In the pe.org/) . The toppe.org/) . The larPV for HT treatment were imported into the STRING database (https://string-db.org/) (The candidate targets of db.org/)  to constdb.org/) . Degree Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were performed in R using the ClusterProfiler package, and p. adjust (FDR) < 0.05 was considered statistically significant .AKI) (PDB ID: P31749) (http://www.rcsb.org/pdb). The protein structure was processed by AutoDock Tools (https://pubchem.ncbi.nlm.nih.gov/) as. sdf. Luteolin and kaempferol were vital active compounds, and AKI and MAPK1 were considered the major targets. Molecular docking of the ligands luteolin and kaempferol to the AKI and MAPK1 protein active sites was carried out using AutoDock Vina 1.1.2. Each docking calculation generated 20 structures, and the molecular docking output was prioritized based on the frequency of the possible ligand-binding site and a free energy score. Finally, the best possible conformations and visualized using Discovery Studio Visualizer 2.5 and PyMOL software .The three-dimensional (3D) molecular structures of hsp90aa1.1 ( P31749)  and mito P31749)  were dowck Tools  to removPV, and the Venn diagram of PV targets is shown in PV containing 11 compounds are shown in JUN), AKI, MAPK1, and tumor protein p53 (TP53) are shown in Of these, 923 were targets of the herbs comprising The herb–compound–target network contained 77 nodes (11 compounds and 66 genes), as shown in PV-related targets, and 17 intersection targets were identified . The relative enrichment analysis showed the following pathways: the AGE-RAGE signaling pathway in diabetic complications, the TNF signaling pathway, the PI3K–Akt signaling pathway, pathways in cancer, the MAPK signaling pathway, and the cancer-associated pathways, as shown in To better understand the function of intersecting genes, we conducted an enrichment analysis of Gene Ontology (GO). The top 10 biological processes mainly an inflammatory response, a cell-to-cell reaction, and a metabolic process are ranked in AKI and MAPK1 proteins have a stronger affinity for HT disease molecules. Luteolin (MOL000006) was considered as the uppermost active ingredient of PV against HT. Meanwhile, there was also a strong association between kaempferol (MOL000422) and AKI with MAPK1. The AKI and MAPK1 protein active pockets showed that the small molecules luteolin and kaempferol have a compact binding pattern  is usually manageable by levothyroxine (L-T4) administration, reducing the thyroid volume and supplementing the lack of hormones. Despite thyroid hormone replacement, some euthyroid patients with HT will continue to experience persistent symptoms that reduce their quality of life. uding HT . In addirophages . This stPV: luteolin, quercetin, beta-sitosterol, kaempferol, stigmasterol, morin, spinasterol, delphinidin, vulgaxanthin-I, poriferasterol monoglucoside_qt, and stigmast-7-enol. Molecular docking showed that the active ingredients, including luteolin and kaempferol, had a good affinity for the hub disease proteins in clinical therapeutics  signaling pathway, and some related to thyroid cancer. The present study identified JUN, AKI, MAPK1, and TP53 hub genes using the PPI network analysis. These results indicated that response . Luteoliresponse . A previresponse . Severalresponse . Luteoliresponse . The keyMeanwhile, the MAPK signaling pathway plays an important role in developing thyroid carcinoma, including cell proliferation and cell survival. The PI3K/AKT signaling pathway plays a key role in regulating the activation of inflammatory response cells and releasing inflammatory transmitters to the chronic inflammatory response in HT . By reguPV and HT is key towards therapeutic discovery and development.The present study included some limitations. First, some further verified experiments need to be conducted. Second, their specific molecular docking methods and locations need to be verified by further experiments. A comprehensive understanding of the key gene target of PV, which indicated that they might play an essential role in treating HT. PV may act against HT mainly through the TNF signaling pathway, the MAPK signaling pathway, and the PI3K–Akt signaling pathway.In summary, the network pharmacology and molecular docking show that luteolin and kaempferol were the main active components of


Although hepatocellular carcinoma (HCC) is developed with various etiologies, protection of hepatocytes seems basically essential to prevent the incidence of HCC. Keratin 8 and keratin 18 (K8/K18) are cytoskeletal intermediate filament proteins that are expressed in hepatocytes. They maintain the cell shape and protect cells under stress conditions. Their protective roles in liver damage have been described in studies of mouse models, and K8/K18 mutation frequency in liver patients. Interestingly, K8/K18 bind to signaling proteins such as transcription factors and protein kinases involved in HCC development. Since K8/K18 are abundant cytoskeletal proteins, K8/K18 binding with the signaling factors can alter the availability of the factors. Herein, we discuss the potential roles of K8/K18 in HCC development. Globally, liver cancer was the third leading cause of cancer-related death in 2020, and about 90% of liver cancers are hepatocellular carcinoma (HCC) ,2. LiverKeratins (Ks) are the intermediate filaments that are expressed in the epithelial cells. More than 50 subtypes of keratin are divided into type I K9-K40) and type II K1-K8, K71-K86) keratins, and type I and type II keratins form heterodimers with different combinations in a cell type- or tissue-specific manner [1-K86 ker-K40 and Most HCC occurs in the environment of chronic liver diseases, meaning that HCC is frequently found in patients with cirrhosis and damaged livers due to a hepatitis viral infection or alcohol abuse ,4,13. HBUnhealthy drinking behavior also causes liver damage, and repetitive liver damage induces cirrhosis and dysfunction in the liver . The conTERT promoter and the amplification of TERT genes in HCC, telomerase is reactivated and contributes to HCC development.It has been revealed that alcohol abuse, smoking, and HBV infection are related to gene mutations in HCC . HCC-relMutations of cell cycle regulators are also found in HCC ,24. In tRB1 (gene of retinoblastoma 1) or CDKN2A (gene of cyclin-dependent kinase inhibitor 2A) are frequently found in HCC lesions, and these mutations disturb the regulation of the retinoblastoma (Rb) pathway [RB1 and CDKN2A result in the loss of their ability in HCC. As such, cancer development is promoted. Similarly, mutations in  pathway ,24,33. B pathway . Through pathway , and the pathway . Consequ pathway . For thiARID1A/B and ARID2, are found in HCC patients [The inactivating mutations of chromatin remodeling factors, such as patients ,24. Chropatients . A defecpatients ,39; thuspatients , and anopatients . Hence, The signaling pathways involved in cell proliferation and/or cell migration are activated by mutations in the Wnt-β–catenin pathway and the Ras–PI3K pathway in HCC patients ,24. The NRAS, KRAS, PIK3CA, and PTEN [2, PIP3, and PIP3 activate downstream proteins, such as PDK and Akt, that eventually induce cell survival and proliferation and downregulate apoptosis [3 to PIP2. Thus, the constitutive activation of Ras family proteins induced by NRas and KRAS mutations may participate in tumor development [PTEN and the activating mutations of PIK3CA [Aberrant activation is also observed in the Ras-PI3K pathway through the gene mutations in and PTEN ,24,48. Rand PTEN . When Rapoptosis . This sielopment ,24,50. If PIK3CA . Higher f PIK3CA . UnregulClinicalTrials.gov. By using the combination of surgical treatment, radiotherapy, and chemotherapy, HCC can be managed, but still it needs more investigations of treatment for patients with advanced stage of HCC.HCC treatment can be divided into surgical treatment, radiotherapy, and chemotherapy. The therapies are adjusted according to tumor stages following the Barcelona clinical liver cancer (BCLC) score ,52. UsuaKeratins are intermediate filament proteins expressed in the epithelial cells. As cytoskeletal proteins, keratins maintain cell shape, support the structure from external mechanical stress, and regulate signaling pathways by interacting with signaling proteins or restricting the availability of signaling proteins ,60. Theyp = 0.179) [p = 0.038), although the physiological functions of the intron variants still need to be determined [The skin-specific keratin 14 mutation K14 R125C was identified in patients with skin diseases . A K18 R= 0.179) . Notablytermined . The anaThe correlation between K8/K18 variants and liver damage has been described in studies of transgenic mice expressing the keratin mutants identified in patients with liver diseases. The studies described K8 R341H and G62C, which are frequently found mutations in the livers of patients . K8 R341Abnormal protein aggregates are easily found inside the cells of some diseases, such as Alzheimer’s disease, Parkinson disease, and liver diseases ,93,94. PThe proteins in inclusion bodies are often hyper-phosphorylated, and keratin phosphorylation may be important for the formation of MBDs ,97. AfteNFE2L2, is mutated in a manner that enhances its function, and these mutations are partly responsible for HCC development [In terms of a relation between MDB formation and signaling pathways, several studies have demonstrated that the BRCA1-mediated signaling pathway and the G1-S cell cycle checkpoint pathway are upregulated in alcoholic hepatitis containing MDBs compared with healthy livers . Furtherelopment ,33 . The PI3K/Akt pathway is one of the well-known signaling pathways in tumorigenesis, since Akt has a critical role in cell proliferation, migration, and anti-apoptosis. A study showed that K8 interacts with Akt under basal conditions and Akt activity was downregulated in K18 glycosylation-deficient transgenic mice (K18 S30/31/49A)  Table 5Table 5. The SAPK pathways can be altered by the disruption of the phosphorylation of K8  Table 5Table 5. The Ras-Raf pathway and protein kinase C (PKC) pathway are possibly affected by K8/K18 . A studyTP53 gene, which encodes p53, are found in HCC patients [In terms of keratin-associated transcription factors, p53 interacts with K8/K18 under basal conditions, and p53 is dissociated from K8/K18 under hyperphosphorylated conditions  Table 5Table 5. patients ,33,131. patients  (Table 5patients . Since Npatients , the dowHCC occurs with complex etiologies, such as genetic alterations and chronic liver damage ,4. Chron


DNA damage repair (DDR) comprises the detection and correction of alterations in the chemical structure of DNA. The dysfunction of the DDR process has been determined to have important implications for tumor carcinogenesis, malignancy progression, treatment resistance, and prognosis assessment. However, the role of the DDR process in gastric cancer (GC) remains to be fully understood. Thus, a total of 2,019 GC samples from our hospital  and 12 public data sets were included in our study. In this study, single-sample gene set enrichment analysis (ssGSEA) was used to generate the DDR pathway activity profiles of 8 DDR sub-pathways and identify a DDR pathway signature by combining the DDR sub-pathway gene sets. The DDR pathway profiling’s impacts on the clinical outcomes, biological functions, genetic variants, immune heterogeneity, and treatment responses were analyzed through multidimensional genomics and clinical data. The results demonstrate that the DDR pathway profiling was clearly distinguished between tumor and normal tissues. The DDR pathway profiling reveals patient-level variations, which may contribute to explaining the high heterogeneity of human GC for the biological features and treatment outcomes. Thus, tumors with low DDR signature scores were independently correlated with shorter overall survival time and significantly associated with mesenchymal, invasion, and metastasis phenotypes. The statistical model integrating this DDR pathway signature with other clinical predictors outperforms each predictor alone for predicting overall survival in discrimination, calibration, and net clinical benefit. Moreover, low DDR signature scores were tightly associated with genome stability, characterized by low tumor mutational burden (TMB) and low fractions of genome alteration. Furthermore, this study confirms that patients with low DDR pathway signature scores might not benefit from adjuvant chemotherapy and a monoclonal antibody directed against programmed cell death-1 ligand 1 (anti-PD1) therapy. These findings highlighted that the DDR pathway profiling confers important implications for patients with GC and provides insights into the specific clinical and molecular features underlying the DDR process, which may help to facilitate clinical management. Gastric cancer (GC) is the sixth most prevalent cancer and the third most common cause of cancer-related death worldwide . Due to Immune checkpoint inhibitors (ICIs), especially the programmed cell death-1 ligand 1 (anti-PD-1) antibody dramatically changed the therapeutic management for patients with advanced GC. However, some limitations exist using this treatment, namely its uncertain curative effects, the low objective response rates (ORRs), several adverse effects, and even drug resistance after the initial patient response –7. ThereDNA damage repair (DDR) consists of the detection of alterations and correction in DNA chemical structure . GeneralHuman GC is a biologically heterogeneous disease , exhibitIn this study, the single-sample gene set enrichment analysis (ssGSEA) was performed to indirectly quantify the DDR pathway activity. Moreover, a novel patient-level DDR pathway profiling approach was developed to explore the DDR pathway activity’s impacts on the clinical outcomes, biological features, genetic variants, immune heterogeneity, and drug responses. The results obtained provide a valuable resource to guide both mechanistic and therapeutic analyses of the role of the DDR process in human GC, which has the promise to facilitate clinical management.® UltraTM RNA Library Prep Kit for Illumina® and were sequenced with Illumina HiSeq 2000. RNA isolation, library construction, and high-throughput mRNA sequencing were performed by Novogene . The data were deposited in the Gene Expression Omnibus (GEO) repository (GSE184336 and GSE179252)  Cancer Hospital in China to construct the HMU-GC cohort . SurgicaE179252) .Gastric cancer gene-expression data sets were systematically searched in GEO and The Cancer Genome Atlas (TCGA) databases. Data sets with missing follow-up data were excluded. A total of 12 public treatment-naive GC cohorts, namely (TCGA-STAD (Stomach Adenocarcinoma), GSE66229/ACRG (Asian Cancer Research Group), GSE15459, GSE57303, GSE34942, GSE38749, GSE29272, GSE84437, GSE26901, GSE26899, GSE13861, GSE28541) were enrolled for further analysis. The basic information for the 12 pubic datasets is shown in ® were processed using the robust multichip average (RMA) algorithm for background correction and normalization through the affy package . Moreover, the DDR gene list also knowledge-based curation of information on specific DNA repair pathways from other published literature  , an onliterature , 30–32. The DDR pathway signature was defined as the sum of the remaining 8 sub-pathways. The GSVA package  was usedThe samples from the patients with survival time lower than three months were excluded, aiming to enhance the robustness of downstream analyses , 35. UniAdditionally, the DDR pathway signature was integrated with other independent prognostic factors to generate a composite nomogram for model visualization and clinical application. The nomogram’s predictive value was compared with the TNM staging system by the C statistic. The performance of the composite model was evaluated by the calibration curve, time-dependent receiver operator characteristic (ROC) analysis, and decision curve analysis (DCA) .p-value < 0.05 were selected as the thresholds to find gene modules significantly associated with the DDR pathway ssGSEA scores.Weighted correlation network analysis (WGCNA) was performed using the WGCNA package, which aims to identify the purity-related gene modules . The scahallmark gene sets” were selected for the enrichment analysis  data from the TCGA-STAD cohort were collected using the TCGAbiolinks package . The GSETo quantify the infiltration level of stromal and immune cells for each GC sample, the stromal and immune scores were calculated using the ESTIMATE algorithm . MCPcouni.e., 5-fluorouracil, cisplatin, oxaliplatin, capecitabine, paclitaxel, docetaxel, irinotecan, and epirubicin), were selected to predict the chemotherapeutic response. Based on the two public drug sensitivity databases, Genomics of Drug Sensitivity in Cancer (GDSC) (50) for each sample. The prediction accuracy was evaluated by 10-fold cross-validation based on each training set. Default values were selected for all parameters, including the combat algorithm for removing batch effect and the mean value for summarizing duplicate gene expression.The most commonly used chemotherapeutic agents in GC treatment (r (GDSC)  and Cancr (GDSC) , the oncr (GDSC)  was implhttp://tide.dfci.harvard.edu/)  . Patientp-value was tested. The p-value < 0.05 was considered statistically significant. The Benjamini-Hochberg method was applied to control the false discovery rate (FDR) for multiple hypothesis testing.Mann-Whitney or Kruskal-Wallis tests were used to study the existing differences between groups for continuous variables. Two-sided Pearson’s chi-squared or Fisher exact tests were used to analyze the categorical data. Associations between continuous variables were tested using Spearman correlation analysis. All the statistical analyses performed were conducted using R software, and a two-sided To investigate the putative contribution of the DDR pathway’s activity in GC, in this study ssGSEA method was used to analyze the activation level of the DDR pathway of samples in the HMU-GC, ACRG, and GSE29272 cohorts. Principal component analysis (PCA) determined that the DDR pathway profiles can distinguish with precision between tumor and normal tissues for GC patients , some proliferation- and metabolism-related  pathways were significantly activated in GC samples with higher DDR pathway ssGSEA scores. However, invasion- and metastasis-related pathways, such as epithelial-mesenchymal transition (EMT), hypoxia, and myogenesis, were significantly enriched in GC samples with lower DDR pathway ssGSEA scores. Furthermore, the activities of some immune-related pathways  were markedly higher in the low-score group than in the high-score group. These findings confirmed that patient-level variation in DDR pathways carried prognostic and biological information, which might be investigated in vivo and at the individual patient level for possible use in the clinical management of GC.Because DDR pathway profiles are closely related to patient prognosis, the biological features associated with the DDR process were analyzed. In this case, the DDR pathway ssGSEA score was correlated with all robustly expressed mRNAs, generating a pre-ranked list sorted by the Spearman correlation coefficient, and performed GSEA  were highly significantly associated with the DDR pathway signature (|R| > 0.4) , were selected for the WGCNA. The cluster dendrogram was constructed according to the optimal soft threshold power of 8  data from the TCGA-STAD dataset, were first analyzed to explore the possible genomic features underlying the DDR pathway-related prognostic signature. A significantly higher TMB was detected in the high DDR signature score group , we alsoi.e., less than 5% of all GC samples), 244 significantly mutated genes were found between the two subgroups   Table S3The CNV data were then investigated, showing distinct chromosomal alteration patterns between the low-risk and high-risk groups in the TCGA cohort  with the DDR pathway signature score. Meanwhile, most immune cells, such as T cells, B cells, neutrophils, and myeloid dendritic cells, also showed a significant negative association with the signature scores. However, cytotoxic T cells were positively correlated to the DDR pathway signature scores. In addition, significant differences for most TME-related cells were detected between the two subgroups (The relationship between the tumor microenvironment (TME) status and the DDR pathway signature to characterize their immune heterogeneity was investigated since GC cases with lower DDR pathway signature scores were markedly enriched in stromal and immune activation pathways. The results demonstrate that both the stromal and immune scores, representing respectively, stromal and immune cell infiltration in tumor tissue were inversely correlated with the signature scores and were significantly higher in the low-score group S3A. Theubgroups .Finally, the prognostic implications of these microenvironment features in GC were also investigated  for each sample. The analyses were performed using, respectively GDSC- and CTRP-derived drug response data   and 4 CTRP-derived compounds . Except for carboplatin, all the other drugs had lower estimated IC50 values in the high DDR score subgroup and a negative correlation with the DDR signature scores. These results suggest resistance to chemotherapy of these drugs in GC patients with low DDR signature scores.Chemotherapy is the main treatment option for patients with advanced GC . Howeverp-value = 0.012) , indicating that the DDR pathway profiles could provide additional information independent of the clinical variables.The current study presents a hypothesis-driven pathway profiling approach in a large GC meta-cohort with follow-up long enough to acquire robust outcomes. A study flow chart is displayed in Most individual sub-pathways (7 of 8) and the identified DDR pathway signature have a statistically significant prognostic association with overall survival, even after adjusting the clinical variables. These findings suggested that the DDR pathway profiles are an excellent resource to improve the prognosis prediction for patients with GC. This study found that GC patients with a high activation level of the DDR pathways often characterized good clinical outcomes. The results were in agreement with a previous study in urothelial carcinoma . Then, ae.g., G2M checkpoint, MYC targets, and oxidative phosphorylation) were significantly activated in tumors with high ssGSEA scores. Notably, TMB and neoantigens showed a positive correlation with the DDR pathway profiles  subtype most benefitted from adjuvant chemotherapy. However, GC patients of the GS subtype are resistant to chemotherapy . MoreoveThe present study demonstrates the specific clinical translational value of patient-level DDR pathway profiles in GC. To the best of our knowledge, this is the first study that investigated the role of DDR pathway activity in a large GC cohort. Compared with previous studies that mainly focused on mutations of a given gene in the DDR pathway , this stin silico outcomes still need in vivo and in vitro validations. In the future, corresponding cell experiments and animal experiments will be performed to determine the influence of the activation level of the DDR pathway in GC, aiming to confirm the outcomes presented in the present study.Although pathway profiling represents a novel approach, this study also has some limitations that need to be mentioned. Thus, the exact meaning of pathway ssGESA score is ambiguous and is not a direct marker of pathway activity. Moreover, there may be a significant discrepancy between DDR gene expression and enzymatic activity, but a high-throughput analysis of enzymatic activity is not yet possible. On the other hand, there are also limitations of the retrospective cohorts, which do not have complete phenotypic data, randomized treatment, or uniform follow-up. Since this study was mainly based on bioinformatics and statistical analyses, the This study identified a novel patient-level DDR pathway profiling approach that revealed distinct DDR pathway clusters and demonstrated that the DDR pathway profiles have broad implications for cancer biology and oncologic patient care of GC. With additional prospective validation in clinical and mechanistic studies, the identified DDR pathway signature could become a powerful tool to be useful in stratifying advanced-stage GC patients toward personalization treatments incorporating chemotherapy and immunotherapy.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The studies involving human participants were reviewed and approved by Institutional Review Board of the Harbin Medical University Cancer Hospital. The patients/participants provided their written informed consent to participate in this study.YX and SL designed this work. SL, YFW, JZ, and XY integrated and analyzed the data. SL, YFW, and JZ wrote this manuscript. SL, YZ, and YMW edited and revised the manuscript. All authors approved this manuscript.This work was supported by funding from the Project Nn10 of Harbin Medical University Cancer Hospital (Grant Number Nn102017–03).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


To explore the main bioactive compounds and investigate the underlying mechanism of Pollen Typhae (PT) against diabetic retinopathy (DR) by network pharmacology and molecular docking analysis.  Bioactive ingredients and the target proteins of PT were obtained from TCMSP, and the related target genes were acquired from the SwissTargetPrediction database. The target genes of DR were obtained from GeneCards, TTD database, DisGeNET database, and DrugBank. The compound-target interaction network was established based on Cytoscape 3.7.2. The protein-protein interaction (PPI) network was constructed via STRING database and Cytoscape 3.7.2. Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were visualized through DAVID database and Bioinformatics. Ingredient-gene-pathway network analysis was conducted to further screen the ingredients, target proteins, and pathways closely related to the biological mechanism on PT for DR, and molecular docking analysis was performed by SYBYL-X 2.1.1 software. Finally, the mechanism and underlying targets of PT in the treatment of DR were predicted.  A total of 8 compounds and 171 intersection targets were obtained based on the online network database. 7 main compounds were screened from compound-target network, and 53 targets including the top six key targets  were further acquired from PPI analysis. The 53 key targets covered 80 signaling pathways, among which PI3K-Akt signaling pathway, focal adhesion, Rap1 signaling pathway, VEGF signaling pathway, and HIF-1 signaling pathway were closely connected with the biological mechanism involved in the alleviation of DR by PT. Ingredient-gene-pathway network shows that AKTI, EGFR, and VEGFA were core genes, kaempferol and isorhamnetin were pivotal ingredients, and VEGF signaling pathway and Rap1 signaling pathway were closely involved in anti-DR. The docking results indicated that five main compounds -5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one) had good binding activity with EGFR and AKT1 targets.  The active ingredients in PT may regulate the levels of inflammatory factors, suppress the oxidative stress, and inhibit the proliferation, migration, and invasion of retinal pericytes by acting on PTGS2, AKT1, VEGFA, MAPK3, TNF, and EGFR targets through VEGF signaling pathway, PI3K-Akt signaling pathway, Rap1 signaling pathway, and HIF-1 signaling pathway to play a therapeutic role in diabetic retinopathy. Diabetic retinopathy (DR), which results from chronic high blood glucose levels and almost occurs in medium and late stage of type 1 and type 2 diabetes, is one of the most serious microvascular complications of diabetes mellitus , 4, whicTraditional Chinese medicine (TCM), as a type of alternative drug, is considered a multicomponent and multitarget treatment for diseases corresponding to complex organic targets. Thus, it is necessary to study pharmacological effects and molecular mechanisms of TCMs. Pollen Typhae (PT) is a classical and vital Chinese medicine with well-defined phytochemicals, which has been used for the treatment of hyperlipidemia , nonprolNetwork pharmacology, an emerged discipline of Bioinformatics, is increasingly applied in TCM research in recent years. This approach is in accordance with the holistic theory that TCM emphasizes the treatment of diseases with multiple components and multiple targets . ConsequIn this study, the bioactive components of PT and the specific target proteins of PT and DR were combined to construct the pharmacological networks of “components-targets-diseases” and PPI. Moreover, several biologic pathways have been proposed to be involved in DR, for example, PI3K-Akt signaling pathways, VEGF signaling pathways, HIF-1 signaling pathways, and Rap1 and TNF signaling pathways. This suggests that it is of great significance to explore the characteristics of ingredients and targets of PT on DR in biological networks and molecular docking analysis. The workflow is summarized in http://old.tcmsp-e.com/tcmsp.php). In order to select the ingredients, which have better pharmacokinetic properties and oral bioavailability in vivo, the ingredients were filtrated by suggested criterion in TCMSP database, and the ingredients whose drug-likeness (DL) ≥0.18 and oral bioavailability (OB) ≥30% were regarded as putative major ingredients and retained [∗ >0 to select the predicted targets.The chemical ingredients of PT were gathered from TCMSP database (retained . Additiohttps://http://www.drugbank.ca/), GeneCards platform (https://www.genecards.org), Therapeutic Target Database (TTD), and DisGeNET database with “diabetic retinopathy” as the keywords [The DR-related targets were searched in the DrugBank  networks were constructed using the STRING database and Cytoscape 3.7.2 software , 20. Thehttps://david.ncifcrf.gov/tools.jsp) setting P < 0.05 as the threshold [http://www.bioinformatics.com.cn/), to explore the key targets and the related pathways [Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) are important methods to screen the key targets and enriched pathways in DAVID database  database. Then the PDB formats of proteins and the compounds were docked and visualized in the SYBYL-X 2.1.1 software, and the docking score was calculated to evaluate the binding degree between the bioactive components and the targets of PT. It is generally believed that the CSCORE >4.0 indicates that the docking molecules have good binding activity with the target.α-L-rhamnosyl(1 ⟶ 2)-β-D-glucoside qt were selected from TCMSP database and 283 related targets of the compounds were screened from SwissTargetPrediction database. In addition, a total of 4077 DR-related targets were identified through the screening of the disease target database after removing the duplicate data. Then, the intersection of 171 targets was obtained.With the values of OB ≥30% and DL ≥0.18 properties, 8 active compounds, (2R)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one, arachidonic acid, isorhamnetin, quercetin, kaempferol, beta-sitosterol, testosterone palmitate, and kaempferol-3-O-In order to identify the interaction relationship between the ingredients and related targets, a compound-target network and topological analysis were made by using Cytoscape 3.7.2 and the degree values of compounds-targets were calculated. The higher the degree is, the closer the relationship between compounds and targets is, and the more important the compounds are in this network. The intersection targets PT and DR were inputted into the STRING database to obtain the PPI network, with 171 nodes and 1649 edges. Cytoscape 3.7.2 was used for merging and calculating topological analysis. The higher the degree is, the more important the targets are in this network. As is shown in P < 0.05, were obtained through inputting the 53 key genes into the DAVID website. Among them, the top ten most significantly enriched GO terms, respectively, were input into Bioinformatics (http://www.bioinformatics.com.cn/) to obtain the GO analysis diagram. As is shown in GO enrichment analysis process consists of biological process (BP), cellular component (CC), and molecular function (MF). A total of GO terms, including 227 biological processes, 27 cellular components, and 55 molecular functions with http://www.bioinformatics.com.cn/), and a bubble diagram was obtained by listing the top 20 signaling pathways according to the enrichment score. As shown in There were 80 KEGG signaling pathways obtained by the 53 key genes which were inputted into the DAVID database. Analysis of the KEGG pathway was carried out via Bioinformatics , a multicomponent and multitarget traditional Chinese medicine (TCM), has been clinically used in the treatment of DR and has a significant effect. The previous pharmacological research demonstrated that the components of PT have good antioxidant and anti-inflammatory effects . TherefoThe PPI network analysis illustrates that gene targets  may be the main targets of PT to DR. The ingredient-gene-pathway network shows that AKT1 and EGFR are the core target genes. AKT1 is a suppressor gene and plays a key role in DR, and pharmacological studies have demonstrated that high glucose promotes retinal endothelial cell migration through activation of AKT1 , 35. TheFunctional and enrichment analysis shows that these pathways  are significantly involved in antioxidative stress and anti-inflammation and suppress the angiogenesis of retina. Study suggested that modulating the PI3K/AKT signaling pathway could ameliorate diabetes-induced proliferative retinopathy , 49. In In summary, this study is aimed at illuminating the therapeutic effect on DR through network pharmacology and molecular docking analysis. We predicted that these active ingredients in PT, including arachidonic acid, isorhamnetin, quercetin, and kaempferol, and core targets, such as PTGS2, AKT1, VEGFA, MAPK3, TNF, and EGFR, mainly intervene in core targets through VEGF signaling pathway, PI3K-Akt signaling pathway, Rap1 signaling pathway, HIF-1 signaling pathway, and other signaling pathways, thereby exerting their roles in the treatment DR.


Saccharomyces cerevisiae can produce isobutanol in negligible amount since it possesses the pathway for its production through valine degradation pathway. Further modifications in the existing pathways made the improvement in isobutanol production in many microbial strains. Fermentation using cost-effective lignocellulosic biomass and an efficient downstream process can yield isobutanol in environment friendly and sustainable manner. The present review describes the various genetic and metabolic engineering practices adopted to improve the isobutanol production in microbial strains and its downstream processing.Fermentation-derived alcohols have gained much attention as an alternate fuel due to its minimal effects on atmosphere. Besides its application as biofuel it is also used as raw material for coating resins, deicing fluid, additives in polishes, etc. Among the liquid alcohol type of fuels, isobutanol has more advantage than ethanol. Isobutanol production is reported in native yeast strains, but the production titer is very low which is about 200 mg/L. In order to improve the production, several genetic and metabolic engineering approaches have been carried out. Genetically engineered organism has been reported to produce maximum of 50 g/L of isobutanol which is far more than the native strain without any modification. In bacteria mostly last two steps in Ehrlich pathway, catalyzed by enzymes ketoisovalerate decarboxylase and alcohol dehydrogenase, are heterologously expressed to improve the production. Native  Isobutanol is also further converted into various other useful chemicals such as isobutyl acrylate, isobutyl acetate [Isobutanol is a four-carbon aliphatic alcohol. The major application of isobutanol as biofuel has urged importance in past few years. Various applications of isobutanol are depicted in  acetate . BioethaIsobutanol can be produced chemically as well as biologically. Petrochemical method of isobutanol production includes mainly two processes – the oxo synthesis and the Reppe synthesis. Oxo synthesis includes hydroformylation of propene to yield aldehyde which is further hydrogenated using catalysts like Co, Rh, or Ru to form corresponding alcohol n-butanol and isobutanol. In Reppe synthesis, propene, carbon monoxide, and water react in the presence of catalyst tertiary ammonium salt or polynuclear iron carbonyl hydrides at high temperature and pressure to form n-butanol and isobutanol . BesidesIn recent years due to the environment concerns like greenhouse gas effect and global warming biological methods are mostly preferred. Gevo and Butamax advanced biofuels are the two companies producing bio-based isobutanol. Various patents-related isobutanol production is depicted in Saccharomyces cerevisiae which uses valine biosynthesis pathway and Ehrlich pathway. However the isobutanol titers obtained by native strains are too low. Therefore metabolic engineering of host cells for improved isobutanol production has been studied. One of the major limitations of yeast cells is that the isobutanol pathway is compartmentalized to cytosol and mitochondria. Studies and reports suggest that confining the entire pathway either to cytosol or to mitochondria improves the isobutanol titer [Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, etc., showed improved isobutanol production [Isobutanol production by native strain is reported in yeast ol titer . Experimol titer ,10. Besioduction .in vitro by using enzymes, substrates, and cofactors. In case of cell free system substrate can be completely used for concerned product but in case of cellular approach substrate can be utilized by other competing pathway to produce by-products. Tolerance of bacteria cell to the product is another problem related to cellular approach but in cell free approach higher concentration of product does not affect the process. Productivity and yield is also higher in case of cell free system. Another advantage of cell free system is that it can operate at broad range of different conditions like pH, temperature etc [Instead of using whole cell, cell free system has been used for isobutanol production. Isobutanol is produced ture etc .2.2.1.Saccharomyces cerevisiae based on response methodology was performed which yield a productivity of 200 mg/L concentration of isobutanol [S. cerevisiae major drawback is that the pathway is distributed in to two compartments half in cytosol and half in mitochondria. Metabolic engineering approaches like over expression and exogenous gene expression were studied which will be discussing in later. In yeast isobutanol is synthesized through Ehrlich pathway by degradation of valine. Isobutanol pathway for yeast is depicted in Researches on isobutanol production by wild type strains are less. The major reason for that are low productivity and also the limitation of genes responsible for isobutanol production in microorganisms. Earlier in 1990s there are few primary reports on trace amounts of isobutanol production detected as part of wine fermentation by few yeast species. The production range varies from 2 mg/L to 174 mg/L . Later oobutanol . In caseS. cerevisiae. Another added advantages of yeast is that no risk of phage contamination, which is common in bacterial fermentation.Tolerance of yeast species toward alcohol makes it a potential candidate for industrial purposes. Cell toxicity and tolerance toward isobutanol is one of major hindrance for production of isobutanol. Yeast is well studied for ethanol production due to its alcohol tolerance and is commercially used organism. Tolerance toward isobutanol is being documented which shows it is capable to grow in more than 2% . This isS. cerevisiae, other reported yeast strain for isobutanol includes Pichia pastoris, Kluyveromyces lactis, and Magnusiomyces magnusii. The wild strain of P. pastoris could produce only very low concentrations of isobutanol. Overexpression of endogenous amino acid biosynthetic pathway of P. pastoris led to an accumulation of 2.22 g/L of isobutanol [K. lactis was studied to understand about the flavored volatile compounds produced. Isobutanol titer of 8.6 mg/L was detected from K. lactis during fermentation [M. magnusii was also reported for isobutanol production of 440 mg/L for a wild type strain. They also developed advanced transformation protocol in which strong constitutive promoter TEF1 cloned and created the reporter system to test promoter strength. These methods also allowed to express heterologous S. cerevisiae ILV2 gene coding for acetolactate synthase which led to advanced isobutanol production of 0.7 g/ L [Besides obutanol . K. lactentation . Recentl0.7 g/ L .Bacterial isobutanol production by wild type strain is rare. Genes responsible for isobutanol production are absent in most of the bacteria. Ketoisovalerate decarboxylase is one such enzyme which is absent in bacteria.Lactococcus lactis is a lactic acid bacterium which harbors Ketoisovalerate decarboxylase (KIVD) gene. In most genetic engineering approaches for bacterial isobutanol production this gene is introduced to bacteria from L. lactis. L. lactis is also being studied for isobutanol production since it contains all the genes. They used native L. lactis, E. coli DH5α which contains a KIVD gene from L. lactis and also combination of both. After fermentation the result showed positive isobutanol production for all three. Maximum production of 0.00173 g/mL was detected for E. coli DH5α. L. lactis showed production of 0.00001 g/mL. The co-culture reported the production of 0.00007 g/mL. This result confirmed L. lactis is a native isobutanol producer. The major disadvantage is the productivity and yield which is too low (Less than 0.2%) [This enzyme converts 2 ketoisovalerate to isobutyraldehyde which is the second last step of isobutanol production. an 0.2%) .Klebsiella pneumonia but this pathway is dormant in wild type strain. K. pneumonia is a well-known 2,3-butanediol producer. 2,3-butanediol and isobutanol is produced from pyruvate by condensation to yield α acetolactate. Inactivation of α acetolactate decarboxylase has been investigated which resulted in conversion of α-acetolactate toward valine pathway which finally leads to isobutanol synthesis. Further genetic modification was also done which increased the isobutanol concentration [Isobutanol synthesis pathway is reported in ntration .2.2.2.2.1.L. lactis is the only commonly known organism which contains this KIVD enzyme. KIVD gene from L. lactis is broadly being studied for isobutanol production. The last step in Ehrlich pathway is the conversion of isobutyraldehyde to isobutanol aided by enzyme alcohol dehydrogenase (ADH). Alcohol dehydrogenase from different species has been studied to understand the most favorable one for isobutanol production which efficiently converts isobutyraldehyde to isobutanol. Besides introduction of last two genes various other modifications which include overexpression, inactivation of competent pathway, and genesis are also done to improve isobutanol production depending on the host studied. The improvement of process conditions also further improves the isobutanol yield [Isobutanol production by metabolic engineering of microorganisms has been well studied and shed a light into the various genes and its rate limiting factors that are associated with the production process. The common method adopted for bacteria is the introduction of last two step of Ehrlich pathway. The second last steps catalyze the conversion of ketoisovalerate to isobutyraldehyde by ketoisovalerate decarboxylase (KIVD). Most of bacteria lack this gene which is commonly found in plant, yeast, and fungi. ol yield .E. coli strain for isobutanol production. Here he introduced last two steps of Ehrlich pathway into E. coli. He studied five different KIVDs, three from S. cerevisiae , KIVD from L. lactis and PDC from Clostridium acetobutylicum. The alcohol dehydrogenase enzyme was taken from S. cerevisiae. All the introduced genes were overexpressed to study the isobutanol production. The most efficient gene in E. coli was found to be KIVD from L. lactis. Beside the introduction of last two steps they try to increase keto acid flux by overexpression of ilvIHCD gene under control of PLlacO1 promoter. This led to a ≈ 5 fold increase as compared to gene without overexpression of ilvIHCD gene. Further isobutanol improvement was done by deleting gene that contributes by-product formation so that availability of pyruvate is increased. Instead of ilvIH of E. coli they introduced Als gene from Bacillus subtilis. The combined effect of all these manipulation lead to a final titer of 22 g/L of isobutanol production [Atsumi et al. were the first to carry out genetic engineering in oduction .Aerobic isobutanol has been widely been studied since anaerobic production has an imbalance in cofactor utilization. In isobutanol production pathway two enzymes namely ketol acid reductoisomerase and alcohol dehydrogenase require nicotinamide dinucleotide phosphate (NADPH). Glycolysis produces only nictoniamidedinuleotide (NADH). In aerobic fermentation cell utilizes the NADPH produced by tricarboxylic acid cycle and pentose phosphate pathway. Bastion et al. have worked out two approaches to compete the cofactor imbalance. First approach uses the overexpression of pyridine nucleotide transhydrogenase PntAB to regenerate NADPH. Overexpression of transhydrogenase gene is one possible strategy to increase the availability of NADPH but the implementation of this in other microorganisms may not always shift the hydride ion in proper direction. Hence the second approach is found to be more effective. The second approach is shifting the NADPH-dependent pathway to NADH-dependent pathway. First they reduce the NADPH dependency partially which led to an increase of 2–3 fold as compared to NADPH dependent pathway. Finally by completely removing NADPH dependency they received isobutanol titer of 13.4 g/L which is 100% theoretical yield ,25.Similar studies were done by Aiqin et al., in their experiment they tackled the cofactor imbalance by overexpression of plasmid containing transhydrogenase gene and NAD kinase gene (pntAB and yfjB gene) and chromosomal modulation of transhydrogenase gene and NAD kinase gene. Plasmid overexpression showed only little effect on the production. The combinational chromosomal modulation of both the genes showed 50 and 30% increase in isobutanol titer and yield. Ultimately it was concluded from their study to have a positive effect on chromosomal modulation than using plasmid. The NADPH supply was increased by this method .E. coli; here they addressed the disadvantages due to plasmid instability and antibiotic to maintain selection pressure. Other problem which they addressed is same as above which is cofactor imbalance. They constructed E. coli strain which has chromosomally integrated isobutanol pathway rather than plasmid insertion. The enzyme for this pathway was selected based on cofactor preference. In that study enzyme which prefers NADH was selected as a cofactor instead of NADPH. They also diverted the pyruvate flux toward isobutanol production by deleting the by-product formation [Another study related to this in 2019 Deb et al. used ormation .From all above possible and effective solution for cofactor imbalance and to produce isobutanol titer include – chromosomal integration of pathway by selecting the appropriate cofactor and chromosomal overexpression of pntAB and NAD kinase for efficient NADPH production, shifting the cofactor from NADPH to NADH and deleting or inactivating the competing pathway to divert pyruvate flux toward isobutanol production.E. coli is adopting Entner–Doudoroff (ED) pathway. Glucose degradation uses normally Embden–Meyerhof (EM) pathway to produce isobutanol. However recently it is reported that Entner–Doudoroff pathway is being attempted for isobutanol production. ED pathway is notable in Gram negative bacteria, few Gram positive bacteria and archaea. In case of EM pathway which acquire energy from glucose uses 1 mol of glucose to produce 2 mol of pyruvate. In this process 2 ATP and 2 NADH is being produced. While in ED pathway 1 mol of glucose metabolized to 2 mol of pyruvate and produces 1 mol ATP, NADH and NADPH. ED pathway requires only few enzymes as compared to EM pathway. Isobutanol production from pyruvate requires 2 mol of NADPH. Therefore on that basis researcher constructed this ED pathway in E. coli due to redox balance. An active ED pathway constructed by suppressing the negative repressor of the pathway and genes of initial reaction of EM and pentose phosphate pathway (PPP). They also inactivated genes concerning organic acid. All together this improved isobutanol production. They obtained a final titer of 15 g/L of isobutanol [Another metabolic engineering method by obutanol ,29.E. coli was reported to be 50 g/L in 72 hrs and they have used constant product removal to tackle the toxicity of the product. The strain used was E. coli JCL 260 [E. coli isobutanol production include utilization of acetate to produce isobutanol. Advantages include no limiting factors as in case of glucose as carbon source [E. coli strain. The major genes responsible for growth on cellobionic acid are 6-phospho-beta-glucosidase (AscB) gene. Cellobionic acid broken to glucose 6 phosphate and gluconate further converted to isobutanol. The major drawback of the process is the cost of cellobionic acid is high. In order to reduce the cost waste containing cellobionic acid could be preferred [The highest isobutanol production in  JCL 260 . Variousn source . Cellobireferred .Corynebacterium glutamicum, Bacillus species, Shimwellia blattae, Cyanobacteria, Acetogenic bacteria, L. lactis, Geobacillus thermoglucosidasius, Enterobacter aerogenes, Clostridium ljungdahlii, Zymomonas mobilis, Ralstonia eutropha, Clostridium cellulolyticum, Pseudomonas Putida. Methodology is similar as that which is adopted in E. Coli in most of the reports.Various other organisms also reported for isobutanol production by metabolic engineering approach which include Corynebacterium glutamicum is a gram positive rod-shaped non-spore forming bacteria. The application of this organism in industries has widely improved in recent years due to its tolerance and more knowledge on metabolic engineering approaches. The cellular machinery of C. glutamicum is ample for amino acid production further which aid alcohol production. Extensive understanding of the genetic level modification opened up its application in divergent industrial process. Isobutanol production is being studied by various scientists using C. glutamicum. Initial studies of isobutanol production by C. glutamicum harbors 4.9 g/L . Further researcher has gradually improved the production titer in C. glutamicum. Different metabolic engineering methodology to improve yield include 1) Inactivated L-lactate and malate dehydrogenase, inserted KIVD gene from L. lactis, ADH2 from S. cerevisiae and pntAB from E. coli. Further chromosomally integrated adhA was used for improved production. Final isobutanol production of 175 mM was obtained . 2) Introduction of ED pathway which enhances glucose consumption and productivity. Here they used enzymes which prefer NADH rather than NADPH. They also inactivated succinate by production. Overall isobutanol production of 280 Mm was obtained in 24 hrs . 3) Disruption of ppc gene, encoding phosphoenolpyruvate carboxylase, NADH preferred acetohydroxylacidisomeroreductase (AHAIR) was selected instead of NADPH dependent, overexpressing select glycolytic gene. Beside all this they continuously extracted isobutanol based on oleyl alcohol method. Final volumetric production of 981 mM was obtained from C. glutamicum 4) C. glutamicum engineered to use hemicellulose fraction. Integrated genes required for D-xylose and L-arabinose utilization. Isobutanol of 7.2 mM was obtained . 4.9 g/L . Furtherobtained . 2) Intrn 24 hrs . 3) Disrutamicum 4) C. globtained .Bacillus subtilis is also an appropriate host for isobutanol production due to its tolerance and extensive substrate utilization ability. Isobutanol biosynthesis occurs by 2 ketoisovalerate pathway with addition of heterologous ketoacid decarboxylase and alcohol dehydrogenase. Initial study on B. subtilis produced 0.6 g/L [ 0.6 g/L . Later i 0.6 g/L .Shimwellia blattae, Cyanobacteria, Acetogenic bacteria, L. lactis, Geobacillus thermoglucosidasius, Enterobacter aerogenes, Clostridium ljungdahlii, Zymomonas mobilis, Ralstonia eutropha, Clostridium cellulolyticum, Pseudomonas putida. Mostly last two steps were introduced heterologously that is KIVD and ADH enzyme. Further cofactor is being balanced by engineering approach. Finally media optimization is also been done [All other bacterial reports are also similar which include, een done . All tog2.2.2.S. cerevisiae will produce higher alcohols like isobutanol in small amount through valine biosynthesis and Ehrlich pathway.In microbial isobutanol production major hindrance is they have to introduce heterologously the genes for isobutanol production. Most bacteria lack ketoisovalerate decarboxylase (KIVD) which aids the conversion of 2 ketoisovalerate to isobutyraldehyde. The yeast has a well-established isobutanol pathway. In addition yeast utilizes efficiently lignocellulose biomass and also can with stand harsh conditions. Yeast especially S. cerevisiae isobutanol pathway is distributed in mitochondria and cytosol. Initial steps in isobutanol production which include valine biosynthesis occur in mitochondria and the rest so called Ehrlich pathway is confined to cytosol. Pyruvate produced through glycolysis are transferred into the mitochondria through the mitochondrial pyruvate complex [In  complex .The pyruThe proceeding reaction of Ehrlich pathway for isobutanol occurs in cytosol. BAT 2 present in cytosol by its transaminase activity convert valine to 2 ketoisovalerate (KIV). 2-ketoisovalerate in cytosol gets converted to isobutyraldehyde by pyruvate decarboxylase. Genes responsible for this conversion include PDC1, PDC5, PDC6, ARO10, THI3. Formed isobutyraldehyde is reduced to isobutanol by alcohol dehydrogenase enzyme which require NADPH as cofactor .Different techniques were evaluated by scientists across the globe to understand the effect of isobutanol production by yeast. First three gene starting from pyruvate  and BAT2 were over expressed. Overexpression of these three genes increased isobutanol production from 0.16 to 0.97 mg/g of glucose . In anotS. cerevisiae was engineered to utilize xylose. For efficient utilization of xylose by S. cerevisiae xylose isomerase, transaldolase and xylulokinase genes were overexpressed. In this study over expression of KIVD, ARO10 and ADH2 were carried out in which all the genes for isobutanol production were localized cytosolically. Final titer of 1.36 mg/L was obtained [Rather than overexpressing another approach applied include translocation of isobutanol biosynthesis pathway entirely to cytosol. In addition to the translocation they also overexpressed KIVD, ARO10, and alcohol dehydrogenase .Final yield of 0.63 g/L was obtained . Insteadobtained . Insteadobtained .S. Cerevisiae to understand their role in isobutanol production. Heterologous expression of PPC improved the NADPH supply which is needed for isobutanol synthesis and also thus improved its production. Whereas heterologously expressed enzyme in ED pathway has no effect on isobutanol synthesis but have a role in improving the growth rate [Mutagenesis is another method adopted for increasing isobutanol. Ethyl methyl sulfonate mutageneses with adaptive laboratory evolution for improving tolerance have done. In addition to this they also overexpressed ILV3, ILV2, ILV5,and ARO which resulted in improved production of 4 g/L . As we dwth rate .Along with mitochondrial localization of pathway improving the mitochondrial pool of pyruvate availability have a positive result in isobutanol production. Mitochondrial pyruvate carriers (MPC) which include mpc1, mpc2, and mpc3 were overexpressed in various combinations. Overexpression of mpc1 and mpc 3 improved isobutanol production. Improvement of 22 fold with an isobutanol titer of 330.9 mg/L from 20 g glucose was obtained .These are the different approaches used in yeast for improving isobutanol production. In most of the studies in yeast over expression of genes in isobutanol pathway along with localization of the pathway either to mitochondria or toward cytosol is attempted. In most of the cases they also block the competing pathway to redirect the entire pyruvate flux toward isobutanol synthesis. Many commercial companies like Gevo located in Douglas County, Colorado, use yeast for isobutanol production.They claim to have theoretical yield of 97.5%. They use crab positive and crab negative yeast with added product removal system. They engineered strain by overexpressing isobutanol pathway and also by deleting PDC gene. Yeast is a potential host for isobutanol production. Production profile of isobutanol from different microorganisms is depicted in Different strain improvement strategies used for bacteria and yeast cell are shown in 2.3.S. Cerevisiae) on methacrylate resin. Another enzyme FDH required for NADH recycle is added to solution. Substrate used was ketoisovalerate. Product conversion of 55% was achieved by this method [Besides the whole cell method for isobutanol production, an alternative strategy includes cell free bio-production system. In cell free system, substrate, enzymes and cofactors are added together and is converted into corresponding product. This originates from so called biocatalyst field. Comparison of whole cell based and cell free approach is shown in s method .Figure Similar type of experiment with cell free system was carried out by another group of researcher in which they received a yield of 95% and isobutanol titer of 275 g/L within 5 days. This was the higher titer reported till date. Here they used a bioreactor with the enzymes that are tolerant to isobutanol. The production rate is high but the major complication with cell free approach is the cost of enzyme and cofactor. Once we can replace it with cheaper cofactor and enzymes this process open up a broad area for commercial production of biofuel using cell free system .2.4.Earlier biofuel industries focus on the production based on first generation biofuel. This will lead to a competition with animal feed and food industries. To avoid this later lignocellulosic biomass like plant and crop waste was selected as feedstock for biofuel production. Lignocellulosic biomass is one of the most available renewable wastes that can be utilized. Lignocellulosic biomass is made up of polysaccharides (cellulose and hemicellulose) and lignin. Due to its complex structure lignocellulose material should undergo pretreatment using chemical so as to remove lignin and followed by hydrolysis step in which enzyme added to break cellulose and hemicellulose into sugar monomer. Final fermentation using this result in biofuel production .Instead of separate hydrolysis and fermentation, simultaneous saccharification and fermentation (SSF) is a process which combines hydrolysis and fermentation via single step. Most industries prefer simultaneous saccharification and fermentation due to lower cost, less inhibitory compounds and also low risk of contamination. Concern regarding simultaneous saccharification and fermentation include optimum pH since the condition might be different for hydrolysis and fermentation. Therefore there is a necessity to find an equilibrium where both the process works efficiently .Nowadays consolidated bioprocessing(CBP) is being so as to reduce the cost of pretreatment and hydrolysis step. Consolidated bioprocessing uses single step approach in which enzyme production, biomass hydrolysis, and fermentation are accomplished in a single process step by lignocellulolytic microorganisms.Clostridium thermocellum for isobutanol production. Metabolically engineered C. thermocellum able to utilize cellulose and can produce isobutanol. C. thermocellum is a well known cellulose utilizer which is used for CBP, therefore they engineered the genes responsible for isobutanol production and the plasmid was chromosomally integrated into C. thermocellum. Final titer of 5.4 g/L isobutanol obtained from cellulose with 75 hrs which correspond to theoretical yield of 41% [There are only few reports on isobutanol production using lignocellulosic biomass. There is a large scope for isobutanol using lignocellulose material. Consolidated bioprocessing was experimented using d of 41% . Overall2.5.In situ removal of n-butanol has been reported but for isobutanol there are only lower reports as compared to n-butanol. For n-butanol various techniques like gas stripping, pervaporation, and adsorption have been evaluated. Isobutanol due to its lower concentration in fermentation media, energy consumption is high for its separation process .One method of isobutanol purification and removal include rectification. Isobutanol containing fermentation broth is passed through different rectification tower. Finally the isobutanol with little remaining water is separated by using a cooler and decanter. The immiscible isobutanol-rich phase is refluxed to isobutanol stripper and is thus separated .Enterobacter aerogens. They used PDMS membrane casted on polyvinylidene fluoride for pervaporation. The condensate contained 55–226 g/m2h of isobutanol [Pervaporation is a process in which compounds can be separated based on potential difference across the membrane. Based on the affinity of membrane, molecules having affinity toward membrane will adsorb and diffuses while the others with low affinity is being retained. For isobutanol separation using this method polydimethylsiloxane (PDMS) membrane is used to reduce the toxicity. Fermentation coupled with pervaporation was done using obutanol .Gas stripping is also an effective recovery strategy. In gas stripping feed is injected toward stripping jar and hot gas is supplied. Volatile difference of the compound is used to separate the solvents from fermentation media. For isobutanol separation, coupled gas stripping and fermentation carried out which produced 50 g/L of isobutanol . IsobutaSalting out extraction is another methodology in which organic solvent and salt is used for separation. Salting out is already known technique for n butanol. In salt out extraction requires low amount of salt and is more efficient separation. Different salts were tried out for isobutanol separation and potassium pyrophosphate proved to be the best one. Among different extractants 2-ethyl 1 hexanol served as best host. Isobutanol recovery of 100% was achieved by this method .Vacuum evaporation is also a separation technique used commonly. This process uses vacuum and heat to vaporize the compound at a reduced pressure at lower boiling point. This method has been experimented for separation of isobutanol. Isobutanol of 15 g/L occurred at ≤ 34.3° . Various3.Recent advances and studies on isobutanol production in microorganisms open up a platform for successful production of isobutanol. Bacterial and yeast strains have been studied for isobutanol production. Due to lack of a proper pathway and also the genes responsible for isobutanol production, there is the major hindrance in bacterial cell to produce this alcohol. This problem was addressed by genetic engineering approach in which isobutanol pathway was heterologously introduced into bacterial cell. NADPH availability is another factor responsible for lower production which was addressed by using the enzyme which requires NADH as cofactor instead of NADPH. Another strategy to address this issue includes improving NADPH production by overexpressing pyridine nucleotide transhydrogenase and also chromosomal integration of the genes.Yeast cells have isobutanol production pathway which is confined to mitochondria and cytosol. Strategies used to improve isobutanol production include overexpression of genes involved in this pathway, localization of the pathway either to cytosol or mitochondria, blocking the competitive pathway to obstruct the by-product formation and also improved NADPH availability.In yeast and bacterial cell major limitation include tolerance of microorganism to the product due to the product toxicity. This can be addressed both by using a tolerant strain and through genetic engineering approach the tolerance could be improved. Improving the microbial phenotype by adopting methods like adaptive evolution could open up future directions to improve the production of isobutanol by reducing the product toxicity. Another approach is the integrated removal of the product as and when produced.Addition to all these, for an environmental friendly and sustainable production of isobutanol, there is a need to develop efficient methods for the utilization of cheap raw materials such as lignocellulose biomass or other industrial and municipal wastes. There are only a few studies are reported for microbial isobutanol production in this direction. Hence there exists a wide scope for research in this area.Isobutanol production urged importance few years ago, but the commercial production of isobutanol is low mainly due to the low productivity issues due to various reasons like cell toxicity, scarcity of NADPH availability which has been addressed in various studies as mentioned above. Based on the available knowledge, engineering of an effective strain and utilization of lignocellulosic waste as substrate helps to open up a wide possibility for commercial production of isobutanol.


Radix Astragali and Semen Lepidii (HQ-TLZ) is a commonly used herbal medicine combination for treatment of heart failure, which has a good clinical effect. However, its active components and mechanism of action are not clear, which limits its clinical application and development. In this study, we explored the mechanism of action of HQ-TLZ in the treatment of heart failure based on network pharmacology. We obtained 11 active ingredients and 109 targets from the TCMSP database and SwissTargetPrediction database. Next, we constructed the action network and carried out enrichment analysis. The results showed that HQ-TLZ treatment of heart failure is primarily achieved by regulating the insulin resistance, erbB signaling pathway, PI3K-Akt signaling pathway, and VEGF signaling pathway. After inverse targeting, molecular docking, and literature search, we determined that the equivalent molecular groups of HQ-TLZ in the treatment of heart failure were quercetin and kaempferol. Based on network pharmacology, we reveal the mechanism of action of HQ-TLZ in the treatment of heart failure to a certain extent. At the same time, we determined the composition of the equivalent molecular group. This provides a bridge for the consistency evaluation of natural herbs and molecular compounds, which is beneficial to the development of novel drugs and further research. Heart failure (HF), the ultimate cause of most cardiovascular diseases, is the result of the filling or ejection of damaged ventricles caused by various cardiac structural or functional disorders . CardiacIn 2008, a British pharmacologist Hopkins first proposed the concept of network pharmacology. Network pharmacology is a combination of bioinformatics, multimodal pharmacology, network data analysis, and computer technology to explore the association between drugs and diseases through the analysis of the “drug-targets-disease” interaction network . TherefoEquivalent molecular cluster is a new concept put forward by our research group in order to find a means of modernization of traditional Chinese medicine . An equihttp://tcmspw.com/tcmsp.php) is an open and comprehensive database of Chinese medicine ingredients and action targets. In clinical treatment, traditional Chinese herbals enter the body by oral administration. Oral bioavailability (OB) and drug-likeness (DL), two ADME-related models, are the main variables affecting the absorption of drugs from the gastrointestinal tract. Therefore, we screened bioactive components under the conditions of OB ≥30% and DL ≥ 0.18. PubChem (https://pubchem.ncbi.nlm.nih.gov/) is a chemical database module intended to promote small molecular data resources for public use. We use it to get compound SMILE expression. Then, we used the SwissTargetPrediction database (http://www.swisstargetprediction.ch/) to predict the target of the active ingredient, and we selected the probability greater than 0.5 as the target of the active ingredient of HQ-TLZ.The Chinese medicine systematic pharmacology database (TCMSP) (https://www.genecards.org/) to retrieve the heart failure targets.We take “heart failure” as keywords in Genecards database search  method. A cube of 20 Å around the binding site in the kinase domain was defined as the docking site. The number of modes was set to 20, and the exhaustiveness was set to 8. Other parameters were set to default. At last, Vina was utilized to connect the active ingredients with the target protein in turn, and Affinity was extracted. PyMol was utilized to analyze and plot the results. AutoDock Vina uses semiflexible molecular docking; that is, the pharmacophore is flexible while the protein remains rigid during the docking. The docking results are evaluated by a semiempirical free-energy function.After TCMSP retrieval, ADME parameter screening, and SwissTargetPrediction prediction target, 9 active components of HQ were obtained and 102 active targets were identified. Similarly, we obtained 6 active components and 102 target sites of TLZ. After summing up the obtained compounds and action targets and removing the duplicated values, 11 active components and 109 action targets of HQ-TLZ were finally obtained.We retrieved 13,194 heart failure targets (both protein and RNA targets) from the Genecards database. We intersected the results of the two and obtained 102 direct-action targets.We built the network diagram using Cytoscape's Clustermaker capabilities, as shown in P value less than 0.05. Among these pathways, the insulin resistance, erbB signaling pathway, PI3K-Akt signaling pathway, and VEGF signaling pathway are closely related to this study. We show the top 20 pathways for enrichment results, detailed in KEGG analysis showed that the key targets of HQ-TLZ in the treatment of heart failure were enriched in 68 pathways, and 50 of them had P < 0.05. We screened out the top 25 of GO notes based on P value, count, and pop hits, as shown in GO functional enrichment analysis showed that there were 247 GO annotations with After analyzing KEGG signaling pathways, we got the 24 targets associated with heart failure. Among them, we obtained 6 key targets through the screening of protein location and degree, named PIK3CG, AKT1, PIK3R1, GSK3B, CAMK2B, and IGF1R.After network screening, we obtained two key compounds, quercetin and kaempferol.AutoDock Vina evaluates the binding ability of small molecules to proteins mainly by affinity. Affinity less than 0 indicates that the ligand can spontaneously bind to the receptor, and the smaller the value is, the higher the affinity is and the easier it is for the active component to bind to the receptor. The docking results and affinity are shown in Treatment for heart failure has evolved in three stages. From the anatomical stage of cardiac strengthening and diuresis, to the hemodynamic stage of cardiac strengthening, diuresis, and vasodilation, and finally, to the present neuroendocrine stage, people have gradually realized that the essence of heart failure is ventricular remodeling. Overcompensation and persistence of neuroendocrine will be an adverse prognostic factor. Therefore, reversing ventricular remodeling and regulating neuroendocrine system are the cornerstone of the treatment of heart failure . Owing tWe know that systemic insulin resistance is a risk factor for heart failure and is independent of coronary artery disease . StudiesThe PI3K-Akt signaling pathway is one of the main signaling cascade pathways downstream of IGF1R. On binding to its ligand, insulin and insulin-like growth factor-1 (IGF-1) receptors undergo autophosphorylation, which increases their tyrosine kinase activities. Tyrosine phosphorylation and activation of the docking proteins insulin receptor substrates 1 and 2 (IRS1/2) engages regulatory subunits of the phosphatidylinositol-3-kinase (PI3K) to regulate the PI3K-Akt signaling pathway .Inhibiting the PI3K-Akt signaling pathway has been shown to improve cardiac hemodynamic impairment and fibrosis . MeanwhiThe ErbB signaling pathway is regulated by the widely expressed signaling molecule neuroregulatory protein-1 (NRG-1). NRG-1/ErbB signaling is important for maintenance of cardiac function in adult organisms. Various mechanisms are believed to be involved in this process, including promotion of cardiac myocyte survival, improvement of sarcomeric structure and cell-cell adhesion, and maintenance of Ca2+ homeostasis . MeanwhiVascular endothelial growth factor (VEGF) plays an important role in mediating normal cardiac function by maintaining vascular homeostasis. VEGF maintains vascular homeostasis mainly through the following mechanisms: improve the sensitivity of blood vessels to nerve response, improve the permeability of blood vessels, promote the generation and stability of new blood vessels, recruit stem cells, and promote their homing . The resArtemisia annua offers the functions of cooling and detoxifying blood, eliminating osteopyrexia and fever, freeing from summer heat, preventing recurrence of malaria fevers, and removing jaundice. But, from the perspective of modern science, Tu Youyou discovered that artemisinin in Artemisia annua can kill malaria parasites [Artemisia annua for treating malaria. Even so, research on other traditional Chinese medicines is not as clear as that on Artemisia annua. Therefore, we decided to first look for the equivalent molecular group of Chinese medicine to treat a single disease. After component screening, target prediction, enrichment analysis, and inverse targeting, we concluded that the equivalent molecular groups of HQ-TLZ in the treatment of heart failure were quercetin and kaempferol.Traditional medicine (TM) is being used more frequently all over the world. However, most often, these are choices made by the patient. These medical traditions have a unique understanding of physiology, pathogenesis, pharmacology, and pharmaceuticals, which are different from Western biomedicine . These darasites . We can Molecular docking results showed that quercetin and kaempferol could spontaneously bind to key targets in the pathway. When kaempferol and quercetin bind to these proteins, the proteins lose the chance to bind to other compounds and are expressed in an inhibited state. Considering the location of key targets in these pathways, we hypothesized that quercetin and kaempferol could inhibit these four pathways to some extent. To understand the relationship between quercetin and kaempferol and these pathways, we searched the relevant literature. The retrieval results were in line with our prediction.Quercetin significantly attenuated EGF and TGF-a-induced growth and phosphorylation of ErbB2, ErbB3, cRaf, MAPK kinase 1/2 (MEK1/2), MAPK, Elk1, and Akt1 . At the Kaempferol can inhibit the VEGF signaling pathway to play an antiangiogenesis role and, thus, play a certain role as an antitumor agent and in reducing diabetic retinopathy , 30. SimFrom the abovementioned results, the effects of quercetin and kaempferol on the ErbB signaling pathway and VEGF signaling pathway are not conducive to the treatment of heart failure. However, its role in the insulin resistance and PI3K-Akt signaling pathway is positive. It seems difficult to show what the combined effects of quercetin and kaempferol are. Then, we looked at the relationship between quercetin and kaempferol with heart failure.Our literature search results indicate that there are no clear studies about quercetin and kaempferol with heart failure, but there are experiments showing that both quercetin and kaempferol can inhibit angiotensin-II-induced ventricular remodeling , 36. ThiWe know that heart failure has the same risk factors as cancer. Also, patients with heart failure have a higher rate of cancer than healthy control populations . It may There are still some shortcomings in this study. Due to the limitations of network pharmacology, it is difficult to analyze the dosage of compounds. More preparation is needed in the next step of consistency research.In conclusion, this study has found the potential mechanism of HQ-TLZ in the treatment of heart failure based on the network pharmacology method, which is consistent with the mechanism of action of multicomponent and multitarget of traditional Chinese medicine. At the same time, it is reasonable to assume that the equivalent molecular group of HQ-TLZ is the collection of quercetin and kaempferol. This provides a bridge for the consistency evaluation of natural herbs and molecular compounds, which is beneficial to the development of novel drugs and further research.