The latest meta-analysis of genome-wide linkage studies (GWLS) identified nine cytogenetic locations suggestive of a linkage with diabetic nephropathy (DN) due to type 1 diabetes mellitus (T1DM) and seven locations due to type 2 diabetes mellitus (T2DM). In order to gain biological insight about the functional role of the genes located in these regions and to prioritize the most significant genetic loci for further research, we conducted a gene ontology analysis with an over representation test for the functional annotation of the protein coding genes. Protein analysis through evolutionary relationships (PANTHER) version 16.0 software and Cytoscape with the relevant plugins were used for the gene ontology analysis, and the overrepresentation test and STRING database were used for the construction of the protein network. The findings of the over-representation test highlight the contribution of immune related molecules like immunoglobulins, cytokines, and chemokines with regard to the most overrepresented protein classes, whereas the most enriched signaling pathways include the VEGF signaling pathway, the Cadherin pathway, the Wnt pathway, the angiogenesis pathway, the p38 MAPK pathway, and the EGF receptor signaling pathway. The common section of T1DM and T2DM results include the significant over representation of immune related molecules, and the Cadherin and Wnt signaling pathways that could constitute potential therapeutic targets for the treatment of DN, irrespective of the type of diabetes. The genetic background of diabetic nephropathy (DN) remains obscure, although unquestionable ,2,3. ManThree genome wide linkage studies (GWLS) were conducted in probands with DN due to T1DM ,10,11. TSeveral GWLS have also been conducted for the genetic dissection of DN due to T2DM. Strong and suggestive evidence for a linkage with DN was detected in 6p and 7p, respectively . Bowden However, the findings of the individual genetic linkage studies have been inconclusive. In an effort to produce more consistent evidence, a meta-analysis of GWLS was performed . With reGWLS often produce many cytogenetic locations as suggestive for linkage with a trait or a disease harboring hundreds or even thousands of genetic loci. The next step is the prioritization of the most promising genes for biological validation ,17. BecaIn order to prioritize the most relevant genes among the genes harbored in specific locations identified by a meta-analysis of GWLS, we performed both gene ontology and enrichment analysis, as well as protein network analysis, for the identification of the key genes in DN secondary to T1DM or T2DM exclusively.In the nine cytogenetic locations that were revealed to be significant in the T1DM subgroup meta-analysis of GWLS , 3500 genetic loci were harbored, out of which 1827 loci were protein coding genes . In the In T1DM and regarding the principal molecular functions of the genes, most of the genes are involved in the binding process, catalytic activity, molecular function regulation, and transporter and molecular transducer activity. With regard to the principal biological processes, most of the genes are implicated in a cellular and metabolic process, in biological regulation, in response to stimulus, and in localization. Regarding the principal cellular components, most gene products are localized in a cellular anatomical entity, intracellularly, and in a protein-containing complex. The five protein classes with the most genes are a metabolite interconversion enzyme, a protein modifying enzyme, a gene-specific transcriptional regulator, a transmembrane signal receptor, and a nucleic acid metabolism protein. The majority of the genes are involved in certain signaling pathways, like angiogenesis, integrin signaling pathway, inflammation mediated by chemokine and cytokine signaling pathway, Gonadotropin-releasing hormone receptor pathway, and EGF receptor signaling pathway  Figures.In T2DM, the principal molecular functions, biological processes, and cellular components of the genes are identical to these of T1DM. Regarding the five protein classes with the most genes, these are a metabolite interconversion enzyme, a protein modifying enzyme, transporters, cell adhesion molecules, and gene-specific transcriptional regulators. The signaling pathways with the most of the genes involved are the Wnt signaling pathway, cadherin signaling pathway, EGF receptor signaling pathway, gonadotropin-releasing hormone receptor pathway, and angiogenesis  Figures.An overrepresentation test with PANTHER v16.0 followed the GO analysis. In T1DM, the top five most enriched biological processes are heterocycle biosynthetic process, biosynthetic process, aromatic compound biosynthetic process, organic substance biosynthetic process, and transcription by RNA polymerase II. The top five molecular functions are the RNA polymerase II cis-regulatory region sequence-specific DNA binding and, more specifically, the cis-regulatory region sequence-specific DNA binding, the transcription regulatory region sequence-specific DNA binding, the regulatory region nucleic acid binding, and the nucleic acid binding. Regarding the most enriched cellular components, these include a secretory granule, a secretory vesicle, a membrane-bounded organelle, an organelle, and an intracellular organelle. With regard to the top five statistically significant enriched protein classes, these are the immunoglobulin superfamily cell adhesion molecule, the immunoglobulin receptor superfamily, the C2H2 zinc finger transcription factor, the zinc finger transcription factor, and the serine protease. Regarding the most enriched signaling pathways, the VEGF signaling pathway, the Cadherin signaling pathway, the angiogenesis pathway, the p38 MAPK pathway, and the EGF receptor signaling pathway .In T2DM, the most enriched biological processes include biological adhesion, cell adhesion, peptidyl-tyrosine modification, cellular response to stimulus, and response to a molecule of bacterial origin. The most overrepresented molecular functions include the chloride transmembrane transporter activity and, more specifically, inorganic anion transmembrane transporter activity and anion transmembrane transporter activity, while the most enriched cellular components include integral and intrinsic components of the plasma membrane. The most overrepresented protein classes include cadherins, cell adhesion molecules, chemokines, cytokines, and intercellular signal molecules. Lastly, the most enriched pathways include those of the Cadherin and Wnt signaling pathways .p-value was 6.78 × 10−12, which means that the network had significantly more interactions than expected.Regarding T1DM, in order to further elucidate the function of the 1827 genes, we constructed a PPI network that includes 1779 nodes and 3761 edges, with an average node degree of 4.23 by using the STRING database and Cytoscape software . The PPIp-value was <1.0 × 10−16, which means that the network had significantly more interactions than expected.Regarding T2DM, in order to elucidate further the function of the 1211 genes, we constructed a PPI network that included 1197 nodes and 1313 edges, with an average node degree of 2.19, by using the STRING database and Cytoscape software . The PPIIL6, ACTB, MAPK1, RAC1, CYCS, CXCL8 (IL8), SNRPD3, CSF2, RPL9, and HSPA4 , immunoglobulin M (IgM), and complement deposition are common findings in the kidneys of patients with DN, as they have been depicted by immunofluorescence staining . It has nt of DN ,24,25. PIn addition, one of the most over-represented pathways is angiogenesis. Indeed, experimental data indicate that VEGF is responsible for the increase of neovascularization observed in patients with DN, which is correlated with the expression of VEGF and angiopoietin . VEGF isOne of the most significantly enriched signaling pathways either in T1DM or T2DM is also the Cadherin pathway. This finding confirms the results of our previous in silico study, which is referred to DN secondary to both types of diabetes, T1DM and T2DM, and demonstrated the contribution of Wnt and Cadherin signaling pathways in the pathogenesis of DN . PreviouOne more finding of the present bioinformatics study with regard to T2DM analysis confirms the results of our previous in silico analysis about the significant enrichment of the Wnt signaling pathway . PreviouThe EGF receptor signaling pathway is also one of the most enriched pathways in DN due to T1DM. EGF is the most well studied ligand of EGFR which is expressed throughout the kidney epithelium and interstitium . EGF couOne new or unconventional pathway linked to T1DM-DN or T2DM-DN was the gonadotropin-releasing hormone receptor pathway. However, after extensive literature research, we found that this pathway controls miRNAs that are upregulated after hemodialysis. Therefore, the authors concluded that hemodialysis alters the circulating miRNAs and this alteration affects gonadotropin-releasing hormone receptor, cell cycle, and cell pluripotency-related pathways, which are associated with subfertility and increased risk for cancer development, conditions that have been associated with hemodialysis .IL6 and CXCL8. It is interesting that many of the hub genes are harbored in chromosome 5q, which was one of the common significant regions in both T1DM and T2DM in the meta-analysis whose data we used in the present study.Novel hub genes, genes with the most interactions in the protein networks, we noticed in both T1DM-DN and T2DM-DN protein networks. In the case of T1DM, all the hub genes were novel findings. We searched in the literature for genetic association studies regarding these genes, but we did not find any studies. On the other hand, in the T2DM-DN protein network, the hub genes include two genes already examined in the context of genetic association studies, It is noteworthy to mention that MODY and ketosis-prone T2DM were not included in this analysis because there was not a sufficient number of linkage studies regarding MODY in order to be included in the meta-analysis. Future studies should also investigate the role of the underlying genetics of these types of diabetes, although the available studies are less common in the literature than the studies regarding the T1DM and T2DM, and thus it is not likely to change the results of the current study.Last, but not least, the importance and the novelty of this study regards the methodological approach used, because it offers a new approach in the interpretation of the results of genetic linkage studies. This approach is more common in the case of the results derived from genetic association studies. In the context of the present study, we extended the use of genetic ontology analysis and protein network analysis in the results of linkage studies of diabetic nephropathy. It is interesting that we replicated the results from other methodological approaches. This convergence strengthens our results and constitutes further evidence for the credibility of our conclusions.https://genome.ucsc.edu/, accessed on 13 October 2021) and, more particularly, the assembly December 2013 (GRCh38/hg38) [The data are derived from the most recent meta-analysis of GWLS in DN . DN was 38/hg38) . The afo38/hg38) .p value threshold of <0.05 was used.In order to delineate the biological role of the genes harbored in the locations identified from the meta-analysis, we focused on the role of the protein coding genes. For this purpose, the PANTHER functional classification system was used ,56. Stathttps://string-db.org, accessed on 13 October 2021) database was used to construct the protein network for the protein coding genes located in the locations identified from the meta-analysis of GWLS [https://cytoscape.org/index.html, accessed on 13 October 2021) was used for PPI network visualization [The STRING  ( of GWLS . The minlization . The Cytlization .Taken together, in the present study, we analyzed in silico the findings of a meta-analysis of genome-wide linkage studies and prioritized the most probable candidate genes involved in DN in the context of T1DM or T2DM. The significance of our findings rests on the identification of immune-related molecules, as well as the importance of Cadherin and Wnt signaling pathways. Our findings may indicate potential biomarkers or therapeutic targets for DN per se, irrespective of the type of underlying diabetes.


Acute coronary syndrome can present with atypical symptoms. Hiccups, generally considered benign and self-limiting, can be an indicator of myocardial ischemia if persistent. We present the case of a 62-year-old gentleman with a past medical history significant for hypertension, type II diabetes mellitus, and ischemic stroke who presented with persistent hiccups. Coronary angiogram revealed severe triple vessel disease and he underwent coronary artery bypass graft surgery, following which his hiccups resolved. There are very few cases that report the association of hiccups and myocardial ischemia. To our knowledge, this is the first reported case in which hiccups were a part of the primary symptoms associated with severe triple vessel coronary artery disease. This could be due to irritation of the phrenic nerve from the infarcted myocardium resulting in activation of the hiccup reflex arc. Our case highlights the association between these two common entities and stresses the importance of having a high index of suspicion, especially among high-risk and elderly patients. Acute coronary syndrome (ACS) can manifest with atypical symptoms, especially among women and the elderly -3. HiccuA 62-year-old gentleman with a history of hypertension, type II diabetes mellitus, and cerebrovascular accident presented to the emergency department (ED) with persistent hiccups for one week. He was seen by his primary care physician five days prior to the presentation for his hiccups and was given baclofen and omeprazole. He reported that his hiccups were persistent despite taking these medications, which prompted him to receive a further evaluation in the ED. He also endorsed progressively worsening shortness of breath and intermittent, burning epigastric pain for the past week.Upon presentation, he was afebrile with a heart rate of 79 beats per minute and blood pressure of 174/104 mmHg. His physical examination was unremarkable. His chest X-ray was normal, and an electrocardiogram (ECG) revealed normal sinus rhythm with a Q-wave and T-wave inversion in lead aVL  Figure , 60-70% Helicobacter pylori, esophageal tumor, etc.), and occasionally myocardial ischemia [Hiccups can be associated with a wide range of etiologies and are usually self-limited and resolve spontaneously without intervention. However, in some instances, hiccups can be persistent or intractable. Such cases have been reported in association with disease or injury of the central nervous system , procedures and medications , gastrointestinal disturbance  led us to further evaluate his persistent hiccups for myocardial ischemia. Given that ACS is often viewed as a must-not-miss diagnosis, this case stresses the importance of ACS workup in any patient presenting with persistent hiccups, especially those who are more likely to have an atypical presentation or “anginal equivalents”  ,14, suchHiccups, though generally benign and self-limiting, may run a more protracted course. In such instances, persistent hiccups, though rare, can occasionally be indicative of a common serious pathology, such as ACS, particularly involving the RCA and inferior myocardial wall. Given significant morbidity and mortality associated with ACS, it would be prudent to further evaluate persistent hiccups for ACS, especially among elderly patients and those with pertinent risk factors. Hiccups should resolve on treating the underlying etiology, such as treatment of myocardial ischemia.


AKT1, IL6, MAPK1, TP53, and VEGFA may be one of the important mechanisms. This study provides a theoretical basis for further study of the material basis and molecular mechanism of SZRD in the treatment of PDSD.This study is aimed at exploring the possible mechanism of action of the Suanzaoren decoction (SZRD) in the treatment of Parkinson's disease with sleep disorder (PDSD) based on network pharmacology and molecular docking. Traditional Chinese Medicine Systems Pharmacology (TCMSP) was used to screen the bioactive components and targets of SZRD, and their targets were standardized using the UniProt platform. The disease targets of “Parkinson's disease (PD)” and “Sleep disorder (SD)” were collected by OMIM, GeneCards, and DisGeNET databases. Thereafter, the protein-protein interaction (PPI) network was constructed using the STRING platform and visualized by Cytoscape (3.7.2) software. Then, the DAVID platform was used to analyze the Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. Cytoscape (3.7.2) software was also used to construct the network of the “herb-component-target-pathway.” The core active ingredients and core action targets of the drug were verified by molecular docking using AutoDock software. A total of 135 Chinese herbal components and 41 corresponding targets were predicted for the treatment of PDSD using SZRD. Fifteen important signaling pathways were screened, such as the cancer pathway, TNF signaling pathway, PI3K-AKT signaling pathway, HIF-1 signaling pathway, and Toll-like receptor signaling pathway. The results of molecular docking showed that the main active compounds could bind to the representative targets and exhibit good affinity. This study revealed that SZRD has the characteristics and advantages of “multicomponent, multitarget, and multipathway” in the treatment of PDSD; among these, the combination of the main active components of quercetin and kaempferol with the key targets of  Semen ziziphi spinosae, Glycyrrhiza glabra, Rhizoma anemarrhenae, Poria cocos, and Rhizoma chuanxiong [Parkinson's disease (PD) is a common neurodegenerative disease in middle-aged and elderly individuals. Clinically, symptoms include classic motor symptoms—tremor, myotonia, bradykinesia, and postural imbalance—as well as nonmotor symptoms—sleep disorders, smell disorders, autonomic nervous dysfunction, and cognitive and mental disorders . Sleep duanxiong . It has uanxiong –12. PD buanxiong . Clinicauanxiong –16. Howehttps://tcmspw.com/tcmsp.php) [https://www.uniprot.org/) [Application analysis platform and database system pharmacology of Chinese medicine   were usemsp.php) , 19. TCMot.org/) , which whttps://omim.org/search/advanced/) [https://www.genecards.org/) [https://www.disgenet.org/) [The targets related to the PD and SD were obtained through retrieving the OMIM (vanced/) , GeneCards.org/) , and Diset.org/)  using thhttp://bioinfogp.cnb.csic.es/tools/venny/index.html) was used to map “Parkinson's diseases,” “Sleep disorder,” and “SZRD” targets and to get the intersection targets.The online Venny 2.1 mapping platform (https://string-db.org/cgi/input) [The intersection targets were imported into the STRING database (i/input) . A confii/input)  to realihttps://david.ncifcrf.gov/tools.jsp) [https://www.bioinformatics.com.cn/) [For the screened core targets, the DAVID data platform (ols.jsp)  was usedcom.cn/)  to visuaCytoscape 3.7.2 software was used to construct the network of “herb-component-target-pathway” (H-C-T-P). This “H-C-T-P” network together with the screened main active ingredients, core targets, and concentrated main signal pathways was used to systematically analyze the possible mechanism of SZRD in the treatment of PDSD.https://www.rcsb.org/) [The 2D structures of the potential active ingredients in SZRD were downloaded from the TCMSP database while the 3D structure of the PDSD docking targets (top 5 of degree in PPI network) treated by SZRD was downloaded from the Worldwide Protein Data Bank (PDB) database (sb.org/) . They wesb.org/)  for hydrsb.org/) .Semen ziziphi spinosae, 15 in Poria cocos, 15 in Rhizoma anemarrhenae, 7 in Rhizoma chuanxiong, and 92 in Glycyrrhiza glabra. The results showed that these different drugs contained common active ingredients, namely A, B, and C as shown in By searching the TCMSP database, 135 different active ingredients of SZRD were screened, including 9 in Cytoscape 3.7.2 software was used to screen the active ingredients with a degree ≥ 20 in SZRD. As shown in Semen ziziphi spinosae, 16 in Poria cocos, 90 in Rhizoma anemarrhenae, 23 in Rhizoma chuanxiong, and 193 in Glycyrrhiza glabra. The data of potential active ingredients and potential targets of SZRD in the treatment of PDSD were imported into Cytoscape 3.7.2 software to obtain a diagram of the traditional Chinese medicine component-target network  C, homocysteine (Hcy), and dopamine levels in the striatum caused by PD itself –34.Semen ziziphi spinosae that nourishes the blood and liver and gives peace of mind. Rhizoma anemarrhenae nourishes Yin and clears the heat, and when the evil heat is gone, the healthy Qi is restored. “Poria cocos calms the heart and tranquilizes the mind.” Moreover, the theory of the properties of drugs says it is “good at calming the mind.” Rhizoma anemarrhenae and Poria cocos are used to help Semen ziziphi spinosae calm the mind from vexation. The nature of Rhizoma chuanxiong is scattered, “Qi medicine in the blood,” and is introduced into the liver meridian. Furthermore, this regulates Qi activity to help the liver recover its ability to dredge and release Qi. Rihua Zi medicine said that it “cures all wind, all Qi, all strain, all blood, and replenish five kinds of weakness.” Rhizoma chuanxiong is used in combination with King medicine, which has a magical effect to help sanguification and liver recuperation. Then, Glycyrrhiza glabra is used to harmonize the various drugs. All the drugs work together, to calm the mind in addition to the effect of vexation.There is no related record of PDSD in traditional Chinese medicine classes, but according to its related clinical symptoms, it can be classified as a combination of “fibrillation” and “insomnia.” SZRD is a classic prescription, with tranquilizing properties, mainly treating “deficiency of liver and blood, deficiency of heat, and internal disturbance.” This prescription uses a lot of Based on the network pharmacological research method, a total of 17 major active components of SZRD, which include quercetin, kaempferol, visistin, 7-methoxy-2-methyl isoflavone, naringenin, anhydroicaritin, formononetin, stigmasterol, licochalcone A, and isorhamnetin, in the treatment of PDSD were obtained.2O2-induced PD model PC-12 cells, quercetin treatment increased cell viability, reduced mutagenesis of antioxidant enzymes, and reduced apoptosis of cells and hippocampal neurons [Quercetin, kaempferol, and isorhamnetin are flavonoids which can scavenge reactive oxygen free radicals and achieve antioxidant and neuroprotective effects –38. In t neurons . In addi neurons . Filomen neurons . Moreove neurons . We also neurons . At the  neurons –46.AKT1, IL6, MAPK3, TP53, and VEGFA multiple key targets, cell aging, adhesion, regeneration, and angiogenesis, leukocyte differentiation is closely related to the process of cell metabolism. Recent studies have shown that PI3K-Akt, MAPK, and FOXO are all involved in the process of human cell apoptosis [α and TNF-β) play a role in promoting sleep [β (IL-1β) and TNF-α can directly act on central sleep-wake neurons, causing increased nonrapid eye movement (NREM) sleep and regulating the pathologic circadian rhythm [β and TNF-α, as sleep-regulating cytokines, can induce the production of each other [α, IL-6, and IL-1β, thus playing a role in regulating sleep [VEGF can play a protective role in PD through mechanisms such as antioxidant stress, and the overexpression of VEGF can promote the proliferation and differentiation of neurons and reduce MPTP-induced substantia nigra cell injury [According to the GO and KEGG pathway enrichment analysis results, we speculated that the mechanism of SZRD in the treatment of PDSD may be mainly related to TNF, PI3K-Akt, MAPK, HIF-1, Toll-like receptors, and FoxO-associated signaling pathways. Such as involving poptosis –49. AKT1poptosis . AKT1 ispoptosis . The PI3poptosis . Studiesng sleep . Elevatin rhythm , 57. IL-ch other . After tng sleep . Mitogenng sleep . Studiesng sleep . It was ng sleep –64. The ng sleep , 66. Foxng sleep . After png sleep . For exang sleep . HIF-1 al injury . The expl injury .In this study, we used the network pharmacology research method to predict the chemical composition, target, and signal pathways at multiple levels. The prediction results were verified by molecular docking technology. The results show that SZRD plays an important role in the treatment of sleep disorders associated with PD through “multicomponent, multitarget, and multipathway,” which provides a new theoretical basis for further experimental research and clinical treatment.


Fruit thinning is a cultivation technique that is widely applied in horticulture in order to obtain high-quality horticultural crops. This practice results in the discarding of a large number of thinned unripe fruits in orchards each year, which produces a great waste of agricultural resources and causes soil pollution that may be an important reservoir for pest and plant diseases. Current studies showed that bioactive compounds such as polyphenols, organic acids, monosaccharides and starches are present in unripe fruits. Therefore, we reviewed the bioactive components obtained from thinned unripe fruits, their revalorization for the food industry, their beneficial effects for human health and the methods for obtaining these components. We also performed a calculation of the costs and benefits of obtaining these bioactive compounds, and we proposed future research directions. This review provides a reference for the effective utilization and industrial development of thinned unripe fruits obtained from horticultural crops. Furthermore, revalorizing the waste from this cultural practice may increase the economic benefits and relieve the environmental stress. Thinning is the removal of individual fruits after fruit set ,2. Many Therefore, many horticultural crops, such as grapes, apples, peaches, pears, pomegranates, citruses, and cherries, need fruit thinning in their crop management strategies . Due to Thinning fruit improves the economic value of horticultural crops. However, the careless handling of thinned unripe fruits will cause variable harm to the soil, plants and environment. The traditional utilization of some discarded unripe fruits could reduce the toxicity of the soil environment. Some farmers use unripe thinned fruits as poultry feed . In the In recent years, many researchers have found bioactive compounds, such as polyphenols, organic acids, monosaccharides and starches. These are important parts of unripe fruits, and are potential beneficial resources in many industries such as human health, food preservation, functional foods, and industrial materials  25,26,2,29,30,31Thinned unripe fruits obtained from horticultural crops are rich in various active substances. However, traditional utilization was not employed by farmers, and this may detrimentally impact the biological and physico-chemical components of orchard soils. Therefore, the present review summarized the bioactive compounds contained in discarded unripe fruits, the beneficial health effects derived from the utilization of these bioactive substances, and methods for obtaining these compounds from thinned unripe fruits. After that, we roughly calculated the costs and benefits of obtaining bioactive compounds from discarded unripe fruits. This review has concluded and summarized the development and utilization of agricultural resources such as thinned unripe fruits from horticultural crops, and proposes issues for future research directions.During the growth and development of horticultural crops, the content of phenolic compounds in fruits decreases as fruit weight continues to increase because these compounds are mostly synthetized in fruit skins. Thereby, phenolic compound content in some unripe fruits is higher than in mature fruits , this haDuring the growth and development of unripe fruits in certain fruit species, the content and profile of phenolic compounds are strongly affected by multiple factors. Labbé et al.  researchAs mentioned above, phenolic compounds is a generic term that contains multiple hydroxyphenol compounds. Therefore, the composition of phenolic compounds is very complex, and their value does not always depend on total phenol content. Several researchers studied the phenolic composition of different unripe fruits in depth.p-coumaric acid, quercetin, and resveratrol. Redondo et al. .,63.62,63Musa paradisiaca achieved the same effect because of its ability to adsorb blood sugar, and Bhinge et al. [Bacillus cereus, Escherichia coli, Listeria monocytogenes, Salmonella Typhimurium, and Staphylococcus aureus. Therefore, the juice from unripe grape contained antibacterial properties and could be considered a functional food as well [An extract from unripe kiwi fruits was obtained by Abe et al.  and may e et al.  verifiede et al. . In addie et al. ,73, and  as well . Additio as well . However as well ,77.Bioactive compounds from unripe fruits are variable, and if we only study the various effects of the unripe fruits as a whole, then many impurities may be obtained from the extraction reactions. Therefore, it is possible that the functional components of the unripe fruits cannot be used in a targeted manner. The commercialization of bioactive compounds may improve the utilization rate of unripe fruits by farmers. Consequently, the extraction of these components still needs further research. Some researchers simply extracted polyphenols, polysaccharides, organic acids, starches and other high-added-value components.During plant growth and reproduction, polyphenols have a protective effect against pathogenic bacteria and have important antioxidant activities and their consumption is beneficial to human health ,79. ThesYue et al.  extracteWith the continued research on polyphenols from unripe fruits, researchers are discovering other various study topics and different application aspects in addition to the antioxidant activity of unripe fruit polyphenols. Choi et al.  showed tCtenopharyngodon idellus) surimi quality during cold storage at 4 °C. The research displayed that adding polyphenols from unripe apples to grass carp may have a dramatic effect on slowing down lipid oxidation and lead to a degradation of soluble myofibrillar proteins. Other interesting results from this report are that polyphenols from unripe apples also protected the functional characteristics of grass carp during the refrigeration process. They also increased the surface hydrophobicity and reduced their emulsification activity and stability. Among the studied phenolic compounds, chlorogenic acid played a major role in the aforementioned beneficial effects. It is precisely because polyphenols from unripe fruits have antioxidant activities and inhibitory effects on many enzymes that these biological properties give unripe fruit polyphenols a varied range of utilization. Sun et al.  studied Staphylococcus aureus and Escherichia coli, which are generally present in wounds and showed the potential application for wound bacteriostasis [Owing to the antioxidant effect of polyphenols from unripe apples, a positive effect on the preservation of food and significant improvement in the antioxidant and bacteriostatic abilities of the chitosan film were observed. The author also tried to add polyphenols from unripe apples to edible packaging materials. The thickness, density, swelling, solubility, and opacity of the bioactive chitosan film were increased because of the addition of polyphenols extracted from unripe apples, but at the same time, there are some negative effects on the film, such as the water content, moisture permeability, and mechanical properties . Nisar eiostasis . Additioiostasis .Escherichia coli, Listeria monocytogenes, Salmonella typhimurium and Staphylococcus aureus [Unripe fruits are rich in polyphenols and good for human health, so their use as functional foods has become more popular in recent years. The transformation of polyphenols from unripe apples into functional foods is a very interesting topic currently, and it has promoted the development and utilization of discarded unripe fruits. Functional food such as industrially processed or natural food that when consumed regularly at efficacious levels has potentially positive effects on health beyond basic nutrition . Verjuics aureus . Therefos aureus . HoweverThe development of unripe fruits has a very positive effect on human health and the environment. Although polyphenols from unripe fruits may be used as functional foods, the taste of polyphenolic substances in unripe fruit is bitter, there are negative effects on the sensory quality of food. The addition of unripe fruits may also change the composition of polyphenolic substances. Bucalossi et al.  investigIn unripe fruits, with the exception of polyphenols, there are many other bioactive compounds in unripe fruits with high value added, which also have antioxidant activity, free radical-scavenging activity, and many other useful functions.Unripe banana fruits contain a large amount of starch, rich in carbohydrates that are not digested and absorbed by the human stomach and small intestine. Due to the presence of resistant starch, there is a low response in postprandial blood glucose. Long-term diet intervention on the body increases insulin sensitivity, and in vitro colonic fermentation produces more short-chain fatty acids. Unripe bananas have significant effects in improving diabetes, obesity and other diseases . Sardá eMany researchers characterized the polysaccharides of unripe apples and some research examined its development in recent years due to its potential functional value. Dou et al.  characteUnripe fruits contain important organic acids, and it is possible to produce unripe fruit juices or powders as natural acidifier condiments . Some stw/w reduction in sugars by acid hydrolysis, and Saccharomyces cerevisiae was screened for ethanol conversion efficiency. On the basis of optimizing the conditions of fermentation, 35.5 g/L ethanol was produced. In this condition, unripe banana peels were also a potential resource for ethanol production. Waghmare et al.  made a p2 during wine ageing. Considering that unripe fruits lack phenolic maturity, and bring an excess of astringency and herbaceous aromas, Junior et al. [S. bacillaris can be used to increase glycerol content and malic acid degradation, thereby balancing the lack of palate fullness caused by low alcohol content and masking ‘vegetative’ odors. Global warming is one of the greatest issues in the wine industry because wines have an increasingly high alcoholic content due to the high accumulation of soluble solids in berries. Certain studies showed that unripe grapes could be used to produce low-alcohol wines or reduce the alcohol content of the wine by mixing unripe grapes with grapes that had reached harvest maturity ,113. “Dor et al.  found thSucrose and glucose are the main sugars in unripe peach fruits, and citric acid is the main organic acid. Compared with mature fruits, unripe fruits have higher contents of organic acids and a lower sugar content than ripe fruits, which may be helpful to the formation of carbon materials . For thiAfter unripe bananas were made into banana powder, Martínez et al.  producedBased on the published research, the utilization of unripe fruits involves different fields such as medicine, food preservation, functional foods, industrial materials, and environmental care. Therefore, the use of unripe fruits at this stage is extremely important. Several wastes are produced during horticultural production and we must identify an economical method to recycle agricultural waste. The collection of bioactive components from unripe fruits is of wide importance, and one critical step is to maximize the value of their extraction using different methodologies, which is discussed in the following sections.It has been described above that unripe fruits are one source of polyphenolic compounds. According to many experiments, because of the important effects on human health and food preservation, polyphenols of unripe fruit may have the potential to replace traditional chemical inhibitors and preservatives. The development of polyphenols from unripe fruits would greatly impact environmental resources. Present extraction methods of phenolic compounds include solvent extraction , subcrit® L). Subsequently, Zheng [To improve the extraction efficiency of plant polyphenols, years ago, researchers introduced carbohydrate hydrolases  to release complex polyphenols from cell walls  125,126126,127. y, Zheng  enhancedy, Zheng . The totManilkara zapota) fruits were also isolated using solvent extraction through gel filtration media (Sephadex LH-20) [Shojaee-Aliabadi et al.  extractex LH-20) . Yue et x LH-20)  obtainedx LH-20) . In recex LH-20)  obtainedIn terms of the purity of extracted polyphenols, some studies elucidated that the use of macroporous resins is generally recommended because they possess suitable adsorption and desorption capabilities for polyphenol extraction . The X-5In the development of unripe fruits, few studies were performed on the extraction and purification of non-phenolic bioactive compounds. Due to the rich fiber content in unripe banana fruits, fiber powder from unripe banana was obtained by Rodriguez-Ambriz et al.  via a seAs mentioned above, the polysaccharides in unripe fruits have beneficial effects on the human body for example as antioxidant, so the extraction of polysaccharides is also worthy of further study . Dou et As mentioned in the previous sections, thinned unripe fruits are generally discarded in the orchard, which generates much waste. Some researchers successively characterized the bioactive components of unripe fruits. These studies were performed to extract high-added-value components from unripe fruits for use in different fields such as in the cosmetic, pharmaceutical, food, and plastic industries or in energy production. The development of a fledgling industry of unripe fruit management could generate significant economic benefits in addition to the ecological benefits involved in the utilization of agricultural waste. Therefore, the discussion of costs and benefits is an inevitable topic in the process of unripe fruit management.We will now take into account the report published by Sun et al.  that it In the course of the experiment, 0.534 g chlorogenic acid, 0.382 g epicatechin, 39.54 mg hypericin, and 0.512 g phlorizin were obtained from 100 g fresh unripe apple fruit, and all with a purity higher than 95%. We calculated the outputs according to the lowest price of the corresponding reagent published by the official website of the Sigma-Aldrich company. The total income produced from unripe fruit waste management was $1608.76, as shown in In addition, unripe fruits may also be used as food additives to replace the original materials to develop functional foods, such as unripe banana flour instead of wheat flour to make bread slices , and cakIn summary, the thinned unripe fruits from horticultural crops are indeed a potential agricultural resource. Thinned fruit bioactive components, such as polyphenols, polysaccharides and organic acids, may be used in the food preservation, food additives, medicine, materials, cosmetics and other industries. However, the development of unripe fruits faces many challenges simultaneously.Since unripe fruits are still growing, their bioactive components undergo different changes before thinning. The monitoring and recording of some physico-chemical parameters may be performed to establish a database of the bioactive components at different ripening stages of horticultural fruits. This measurement would help orchard coordination choose a suitable thinning date to maximize the content of bioactive compounds from thinned fruits without affecting the potential productivity of the orchard. Most of the collected thinned fruits are fallen fruits in soils, which presents serious problems in quality such as rotting, dehydration and the presence of insects. With the frequent use of inorganic fertilizers and pesticides, heavy metal and pesticide pollution of unripe fruits is inevitable. Therefore, collection, storage, preservation, and grading after thinning are equally important. Toxicity detection is also an essential link before unripe fruit are used as raw materials for semi-industrial and industrial purposes. How to reduce or eliminate potential harm from unripe fruit to health is one of the main challenges we must face in the future. As mentioned above, the blend fermentation of unripe grapes and wine grapes can solve the disadvantage of “high sugar and low acid” caused by global warming. However, the addition of unripe fruit in wines may bring unpleasant green tastes to the wines. The removal of green tastes is a problem that needs to be solved in this process. In addition, different wine-making materials have different sugar and acid ratios, so the optimal amount of unripe fruit is often adjusted according to the quality of wine grapes.Among the unripe fruits of many horticultural crops, unripe apples are the most investigated. Based on these studies, phenolic compounds are the most abundant bioactive components of unripe fruits. Although the polyphenol content in unripe fruits is higher than mature fruits, the proportion of total phenols extracted from unripe fruits is small. In addition, many resources are wasted in the extraction of monomeric phenols from unripe fruits. Therefore, future development and utilization of unripe fruits resources may resort to the “biorefinery” concept to fully extract and use high-value-added by-products from thinned fruits. Arevalo-Gallegos et al.  defines This review summarized the comprehensive utilization of thinned unripe fruits from horticultural crops, especially apples, grapes, bananas, peaches, mangos, and kiwis. Thinned unripe fruits are often abandoned in orchards and cannot be optimally utilized. These unripe fruits are rich in several bioactive compounds, such as polyphenols, polysaccharides and organic acids. These compounds prevent oxidation, inhibit bacteria, reduce blood sugar, improve obesity, and possess emulsifying, thermoplastic, and stability properties. Increasingly, researchers have begun to extract high-value-added components from unripe fruits to generate a wide range of by-products for use in many industries such as medicine, food preservation, and polymer materials. Therefore, unripe fruits have great potential in the revalorization of agricultural wastes, which could improve economic benefits for farmers. Solving the environmental pressure of unripe fruits may also create greater economic benefits for agricultural workers.


Astragalus mongholicus  and Curcuma phaeocaulis Valeton  can slow the onset and progression of GC, the mechanism by which AM-CPV works in the treatment of GC is uncertain.  Gastric carcinoma (GC) is a kind of digestive tract tumor that is highly malignant and has a very poor prognosis. Although both  The traditional Chinese medicine network databases TCMSP, TCMID, and ETCM were used to identify the key functional components and associated targets of AM and CPV. To establish a theoretical foundation, the development of gastric cancer (GC) was predicted utilizing a GEO gene chip and TCGA difference analysis mixed with network pharmacology. A herbal-ingredient-target network and a core target-signal pathway network were created using GO and KEGG enrichment analyses. The molecular docking method was used to evaluate seventeen main targets and their compounds.  Cell activity, reactive oxygen species modification, metabolic regulation, and systemic immune activation may all be involved in the action mechanism of the AM-CPV drug-pair in the treatment of GC. It inhibits the calcium signaling route, the AGE-RAGE signaling system, the cAMP signaling pathway, the PI3K-Akt signaling network, and the MAPK signaling pathway, slowing the progression of GC. The number of inflammatory substances in the tumor microenvironment is reduced, GC cell proliferation is deprived, apoptosis is promoted, and GC progression is retarded through controlling the IL-17 signaling route, TNF signaling pathway, and other inflammation-related pathways.  The AM-CPV pharmaceutical combination regulates GC treatment via a multitarget, component, and signal pathway with a cooperative and bidirectional regulatory mechanism. Its active constituents may treat GC by regulating the expression of STAT1, MMP9, IL6, HSP90AA1, JUN, CCL2, IFNG, CXCL8, and other targets, as well as activating or inhibiting immune-inflammatory and cancer signaling pathways. Gastric carcinoma (GC) is one of the five most common cancers in the world. At present, about 1.03 million patients suffer from GC . CellulaIt is because of the multitarget, multipathway, and multilink feature of TCM in the treatment of GC that makes it a major obstacle to the exploration of its therapeutic mechanism . How to The concept of traditional Chinese medicine has developed and inherits for thousands of years, having formed its unique theoretical basis and characteristics of herb use. In terms of compatibility, two or more herbs are often selected for synergism and toxicity reduction . HoweverThe TCM system pharmacology database and analysis platform (TCMSP) was used to identify the active ingredients of AM and CPV. The main active ingredients were then selected according to the optimal toxicologic ADME rules reported in the literature . If the compounds did not meet the screening criteria or does not exist in the TCMSP database, it would be supplemented from the TCMID and the active components of AM-CPV drug-pair reported in the related literature were included in , 12. TheThe active components of a drug performance-related biological functions through the relevant targets. In addition to obtaining the targets of the core functional ingredients of AM and CPV directly from the TCMSP database, the core active components reported in TCMID database and related literature were identified by using the information of small molecular structure. Swiss Target Prediction and STITCH identified a large number of possible targets for gastric carcinoma. According to Uniprot, the names of the collected target about AM and CPV drug-pairs were standardized by Perl.P < 0.05 and |log2FC| ≥ 1 were examined significantly differentially expressed and GC-related targets. In addition, GC-related disease targets were extracted in the TCGA database, combined with the TCGA and GEO different analysis results to eliminate repeated disease targets and establish the disease target database of GC. Then, it was checked by the intersection of different genes in the two databases.Micro array data of expression of mRNAs between the GC and normal samples were screened from the GEO database. Series: GSE27342, GSE33335, GSE56807, GSE79973, GSE81948, and GSE103236. Sva and Limma of R4.1.0 were utilized to carry out joint analysis of multiple chips and correct data batches . The Venn map was drawn by Venn of Rmur4.1.0. Genes with an adjusted The core active ingredient target of drug-pairs of AM and CPV was matched with the disease target of GC to obtain the compound target. The Venn map was drawn by the Venn of R4.1.0. In order to study the functional relationship of DEGs, the PPI network of the target was obtained by using the String, with protein type “homosapiens” and the highest confidence level (0.700) as the setting standard of the drug-disease intersection gene.The “herbs-components-targets” network (H-C-T network) of AM and CPV in the treatment of GC was constructed by using Cytoscape3.7.2. According to the topological characteristics of the network, three most important parameters were selected by CytoNCA to screen the core targets of AM-LA drug-pairs in the treatment of GC: degree centrality (DC), closeness centrality (CC), and betweenness centrality (BC). They indicated the function and impact of the associated nodes in the overall network, and their relevance was positively connected with the network's output value. According to relevant literature, the target with the median value for DC was chosen , and theR based on KEGG data.The biological pathways associated with the DEGs were then examined using gene ontology (GO). Biological processes (BP), molecular functions (MF), and cellular components (CC) are controlled by differentially expressed PRGs. PRGs were further investigated using π, anion-π, π-π stacking, and hydrogen bonding). Finally, the Pymol 2.4 software was used to visualize the molecular docking.The protein configurations of the core targets were obtained from the Uniprot by using the minimum resolution (Resolution) and the source (Method), and the crystal structure of these protein configurations was obtained from the RCSB PDB database. 2D structures of 6 active components of core targets were available from PubChem database, and these 2D structures were minimized by chem3d software. The binding strength and activity of active components and targets were evaluated by SYBYL2.0 software, and the functional components of binding TotalScore greater than 3 were selected for subdocking. Then, crystal was imported into the Pymol 2.4 for dehydration, hydrogenation, and separation of ligands; it then imported AutoDockTools 1.5.6 to construct the docking grid box for each target. Docking was completed by Vina 1.1.2, and the molecules with the lowest binding energy in the docking conformation were selected to observe the binding effect by matching with the original ligands and intermolecular interactions | > 1 as screening conditions, the volcano and heat map were used to display 5918 DEGs (P < 0.05 and |log2(FC)| > 1 as screening conditions, a total of 176 DEGs were gathered and depicted using a volcano map and a heat map , cellular composition (CC), and biological process (BP). The MF mainly involves the channel activity (GO: 0015267), passive transmembrane transporter activity (GO: 0022803), and ion channel activity (GO: 0005216). The BP mainly involves ion channel complex (GO: 0034702), transmembrane transporter complex (GO: 1902495), and transporter complex (GO: 1990351). The CC mainly involves metal ion transport (GO: 0030001), calcium ion transport (GO: 0006816), and calcium ion homeostasis (GO: 0055074) , 4xdx , 2yk9 , 3bes , 1alu , 6y3v , 6esm , and1bf5  were downloaded. Evaluation of the binding strength and activity of active components and targets by SYBYL2.0 software showed that the TotalScore of formononetin was 2.6282 less than 3. So, molecular docking of formononetin was not done. The affinity energies of the molecules were CCL2: quercetin-3ifd were −6.1 kcal/mol, CXCL8: quercetin-4xdx were −7.5 kcal/mol, HSP90AA1: isoastragaloside ii-2yk9 were −8.0 kcal/mol; pinocembrin-2yk9 were −8.0 kcal/mol, IFNG: quercetin-3bes were −7.4 kcal/mol, IL6: quercetin-1alu were −7.0 kcal/mol; procurcumenol-1alu were −6.5 kcal/mol, and JUN: quercetin-6y3v were −6.4 kcal/mol. Kaempferol-6y3v were −6.3 kcal/mol, MMP9: quercetin-6esm were −10.7 kcal/mol, STAT1: kaempferol-1bf5 were −9.8 kcal; quercetin-1bf5 were −9.4 kcal/mol , belongs to top-grade medicine. It can promote qi and secure the exterior, diuresis, suppressing toxin, and purulent muscle and is typically used in the treatment of shortness of breath, palpitation, fatigue, etc. [Astragalus polysaccharides may independently induce apoptosis of GC cells and enhance the apoptosis-promoting effect of adriamycin by reducing the expression and inhibitory function of CD4+ and CD25+ [β-catenin and Sp-1, cysteine, and other protein apoptosis pathways [AM, a plant of the genus ue, etc. . Modern ue, etc. . Astragand CD25+ , which cnd CD25+ . CPV, a nd CD25+ . The maind CD25+  mainly tpathways  playing pathways .α signal and Akt-mTor signal to affect the level of inflammatory factors in tumor microenvironment and initiate the autophagy process of GC cells [α (ERR α) reverse agonists, which can inhibit oxidative stress and tumor glycolysis through PI3K-Akt signal pathway and ErbB signal pathway to promote tumor cell apoptosis [We found most of the targets are quercetin (61 targets) and kaempferol 25 targets), and the literature on the analysis of the mechanism of quercetin and kaempferol acting on GC also confirmed the feasibility of drug therapy taken into account this active ingredient . As we k targets,GC cells . It can GC cells . KaempfeGC cells . It has poptosis . Song Hapoptosis  showed tpoptosis . In addipoptosis . Inhibitpoptosis . It can STAT1, MMP9, IL6, HSP90AA1, JUN, CCL2, IFNG, CXCL8, and other genes screened are the principal effect genes. MMP9 and STAT1 were chosen as core genes by overlapping intersection and union. MMP9, a member of the matrix metalloproteinases (MMPs) family, is one of the keys protease involved in the decomposition of the extracellular matrix in physiology and disease . NumerouFollowing that, we analyzed the enrichment of core targets by GO and KEGG. GO and KEGG enrichment analysis showed that the core target added value in a biological process, cell composition, and molecular function. In MF, 172 items are enriched mainly involving channel activity, passive transmembrane transporter activity, ion channel activity, etc. In BP, 1363 items are enriched, mainly involving metal ion transport, calcium ion transport and homeostasis, divalent inorganic cation homeostasis, etc. In CC, 39 items are enriched, mainly involving neuronal cell body membrane, ion channel complex, transmembrane transporter complex, transporter complex, synaptic membrane, etc. In addition, we also observed 20 pathways related to GC and constructed a “target-pathway” network with immune regulation, classical inflammation, multiple cancer pathways, retroactive ligand-receptor interaction, cell proliferation, and apoptosis involved. Among the signal pathways screened in this study, the highest correlation with the effective target of AM-CPV drug-pairs is the AGE-RAGE signal pathway, followed by calcium signal pathway, PI3K-Akt signal pathway, immune-inflammatory regulation, and cancer-related signal pathway. The combination of AGEs and its cell surface receptor RAGE can exert its pathophysiological effects in age-related diseases, diabetic complications. and the course of cancer . The comIn addition to the above two most important pathways, the following pathways also play a part in AM-CPV in the treatment of GC. ① PI3K/AKT signal pathway is one of the most frequently activated signal transduction pathways in tumors, which are closely linked to the growth, proliferation, and apoptosis of GC cells . PI3K-AkκB pathway to amplify the inflammatory response [κB to enter the nucleus and promote the production and release of inflammatory factors such as TNF-α, IL-8, and IL-6 [Experiments have shown that MAPKl4 and MAPK3/1 pathways have the function of controlling the migration and proliferation of malignant tumors . ③ IL-17response . ④ P53 sresponse . P53 sigresponse . It has response . ⑤ TNF sand IL-6 . TNF infand IL-6 .Immuno-inflammatory signaling pathways such as HIF-1, toll-like receptor signaling pathway, and JAK-STAT signaling pathway are also involved in the treatment of GC with AM-CPV. In addition, chemical carcinogenesis-receptor activation, neuroactive ligand-receptor interaction, lipid and atherosclerosis, and other cancer-related signal pathways are rarely mentioned in the study of GC, which may become a new research direction.AM-CPV has the following characteristics on the regulation of differentially expressed target genes in GC: ① STAT1, MMP9, IL6, HSP90AA1, JUN, CCL2, IFNG, and CXCL8 are affected by multiple active components at the same time, indicating that the target genes are regulated by multiple active components. ② Active components such as kaempferol and quercetin are targeted and regulated with multiple target genes at the same time, which shows that the active components play different roles in regulating GC lesions through multitarget gene crossover. ③ Quercetin targets 59 target genes at the same time, of which 30 are downregulated and 29 are upregulated. It can be observed that some of the functional components in AM-CPV drug-pairs act on both upregulated and downregulated genes at the same time. Therefore, it can be recognized that the drug may be bidirectional in targeting regulation of GC lesions.To summarize, the AM-CPV medication combination governs the therapy of GC through a multitarget, multicomponent, and multisignal route, with a cooperative and bidirectional regulation mechanism. Its active ingredients may treat GC by regulating the expression of STAT1, MMP9, IL6, HSP90AA1, JUN, CCL2, IFNG, CXCL8, and other targets, activating or inhibiting immune-inflammatory and cancer signaling pathways such as the AGE-RAGE signal pathway, calcium signal pathway, and PI3K/AKT signal pathway, and regulating immune-inflammatory reaction, cell proliferation, differentiation, and apoptosis and antioxidant stress response, etc. Despite the fact that this work gives some theoretical foundation and research suggestions, it still has certain limitations. Improvements should be made in this direction: ① we might continue to conduct focused fundamental research on the key components of the AM-CPV drug-pairs, such as serum pharmacology and extraction of active components of the compound comprising pharmacology, based on the network pharmacology research results. ② More clinical trials are needed to see if the medication is suitable for long-term maintenance treatment in GC patients.


Toutongning capsule (TTNC) is an effective and safe traditional Chinese medicine used in the treatment of migraine. In this present study, a multiscale strategy was used to systematically investigate the mechanism of TTNC in treating migraine, which contained UPLC-UESI-Q Exactive Focus network pharmacology and experimental verification. First, 88 compounds were identified by the UPLC-UESI-Q Exactive Focus method for TTNC. Then, the target fishing for these compounds was performed by means of an efficient drug similarity search tool. Third, a series of network pharmacology experiments were performed to predict the key compounds, targets, and pathways. They were protein-protein interaction (PPI), KEGG pathway enrichment analysis, and herbs-compounds-targets-pathways (H-C-T-P) network construction. As a result, 18 potential key compounds, 20 potential key targets, and 6 potential signaling pathways were obtained for TTNC in treatment with migraine. Finally, molecular docking and experimental were carried out to verify the key targets. In short, the results showed that TTNC is able to treat migraine through multiple components, multiple targets, and multiple pathways. This work may provide a theoretical basis for further research on the molecular mechanism of TTNC in the treatment of migraine. Migraine is a common chronic and paroxysmal neurological disease. The most characteristic symptoms associated with migraine include nausea, emesis, photophobia, and phonophobia. In addition, patients tend to have a variety of other neurological symptoms, such as dizziness, tinnitus, and cognitive impairment , 2. EpidTraditional Chinese medicine (TCM) has been widely used in the treatment of migraine for more than 2000 years. Migraine belongs to the category of Shou Feng or Nao Feng in TCM, which has been recorded as early as in the Yellow Emperor's Medicine Classic (Chinese name in pinyin Huang Di Nei Jing), a classical literature from the Han Dynasty (AD 25–220). In TCM, “wind, heat, phlegm, and blood stasis” are considered to be the main pathogenic factor of migraine. In this regard, the critical points were extinguishing wind and resolving phlegm activate blood and relieving stasis in the treatment of migraine. Toutongning capsule (TTNC) functions calming wind, clearing phlegm, removing blood stasis, and relieving pain. Hence, Toutongning capsule is frequently used in the treatment of migraine in clinical and has a significant effect. The components of the formula include Smilacis Glabrae Rhizoma, Gastrodiae Rhizoma, Polygoni Multiflori Radix, Angelicae Sinensis Radix, Saposhnikoviae Radix, and Scorpio. Among these herbs, Gastrodiae Rhizoma is able to calm the liver to stop endogenous wind and combined with Smilacis Glabrae Rhizoma can pacify the liver to subdue yang. Polygoni Multiflori Radix has the effect of replenishing lean and nourishing blood. Angelicae Sinensis Radix can nourish blood and activate blood. Saposhnikoviae Radix is able to dispel wind and eliminate dampness. Scorpio can break blood, expel stasis, and free the collateral vessels to relieve pain. The results of evidence-based medicine research showed that the effective rate of TTNC was 72.5%, with a prevention rate of 86.5%. No adverse events took place for the treatment of 400 patients with primary migraine by a randomized, double-blind, placebo-controlled multicenter clinical trial . At presIn this present study, an integrated strategy was used to investigate the mechanism of TTNC in treating migraine, which contained UPLC-UESI-Q Exactive Focus and network pharmacology. The UPLC-UESI-Q Exactive Focus method was adopted to identify the chemical composition for TTNC. The technologies of network pharmacology , 15 wereAcetonitrile (HPLC-grade) and methyl alcohol (HPLC-grade) were obtained from Fisher Scientific Company. The MS-grade formic acid was obtained from Sigma Aldrich (Germany) Trading Co., Ltd. Toutongning capsule (TTNC) (batch no. 190312) was provided by Buchang Pharmaceuticals .μm). In addition, column temperature was maintained at 30°C. The mobile phase consisted of two solvents A (0.1% formic acid in water) and B  at a flow rate of 0.30 mL/min. A linear gradient elution of A and B was used according to the following program . The injection volume was set 3 μL. Liquid chromatography HESI-MS analysis was conducted by means of the positive and negative electrospray model. The mass spectral scan ranges from 80 Da to 1200 Da. The spray voltage was set at 3.5 kV. Besides, the other conditions are shown as follows: capillary temperature, 320°C; Aux gas heater temperature, 350°C; sheath gas flow rate, 40 arb; Aux gas flow rate, 10 arb; S-lens RF level, 50.0 V; resolution, full MS 70000, dd-MS2 35000; and scanning mode, full MS-dd-MS2.The instrument used for the chromatographic separation was a Thermo Scientific Q Exactive Focus system coupled with the Thermo Scientific Ultimate 3000 system , 17. SepThe potential targets of the chemical composition by UPLC-UESI-Q Exactive Focus for TTNC were predicted in an encyclopedia of traditional Chinese medicine (ETCM) platform using MedChem Studio (version 3.0), which is an efficient drug similarity search tool to identify the similarity between the known drugs in the DrugBank database with the tested compounds , 19. TheThe targets related to the migraine were collected from the GeneCards database. The targets with relevance score above the average were regarded as the migraine targets. Relevance score is the Novoseek score of the relevance of the disease to this gene based on their literature text-mining algorithms . All tarhttps://bioinformatics.psb.ugent.be/webtools/Venn/) was used to screen the overlap of disease-causing genes and component targets for TTNC.The nonhuman targets were deleted from all the component targets and disease-causing genes. After that, Venn online tool free software (https://string-db.org/) to obtain the interaction between proteins and predicted proteins [Homo sapiens,” and the minimum interaction threshold with the highest confidence was −0.900, and the targets with poor connection of the primary network were removed. After then, the PPI network was constructed using Cytoscape 3.7.2, and the network topological properties were analyzed for each node using the CytoNCA plugin for Cytoscape 3.7.2, such as degree centrality (DC), betweenness centrality (BC), and closeness centrality (CC) [The targets were introduced into the online system public database STRING  .The biological functions and participated pathways of the candidate drug targets have been investigated according to KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis by means of DAVID  , 25.The H-C-T-P network was constructed for TTNC in the treatment of migraine by Cytoscape 3.7.2. The network topological properties, including DC, BC, and CC, were analyzed by the NetworkAnalyzer plugin in the software to explore the key compounds and targets for TTNC in the treatment of migraine.https://pubchem.ncbi.nlm.nih.gov). The structures of the targets were obtained from the PDB database (https://www.pdb.org). The docking simulation was performed by means of AutoDock 4.2.6 software. During the docking calculations, hydrogen atoms and Gasteiger charges were added to the protein using the automated docking tool. The auxiliary program AutoGrid was used to set the docking boxes. These docking boxes were defined according to the crystal structures of complex of the proteins with known ligands. The Lamarckian genetic algorithm (LGA) was adopted for each docking progress.Molecular docking was performed to investigate the interactions between some key components and targets. The structures of the key components were obtained from the PubChem database (n = 24), 6 weeks, weighing 180–220 g, were purchased from Xi'an Jiaotong University; the certificate number was SCXK2017-003. The rats were fed with a standard pellet diet and tap water ad libitum, and they were housed and maintained in 12 h light/darkness with standard humidity and temperature in the laboratory. The principles of laboratory animal care guidelines, approved by the Animal Ethics Committee at the Shaanxi Academy of Traditional Chinese Medicine, were strictly followed.Sprague-Dawley (SD) rats . The TTNC group was given intragastric administration for 7 days, and the control group and model group were given equal volume normal saline. After the last administration of 30 min, the rats were subcutaneously injected with nitroglycerin injection 10 mg/kg  in the TTNC group and model group, and the control group was injected subcutaneously with equal volume of normal saline. The number of scratching and other behavioral manifestations of rats were recorded within 1 h after injection. When the rats showed irritability such as red ears, frequent head scratching, and cage climbing, it indicates that the modeling was successful. The rats were anesthetized with pentobarbital, blood was taken from the abdominal aorta, standing 2 h, centrifugation at 1000 rpm for 10 min, and then blood serum was obtained. At the same time, the brainstem was isolated and brain tissue was obtained and collected in the EP tube. Blood serum and brain tissue were stored at −80°C until used.Rat blood was taken from the abdominal aorta after anesthetized with pentobarbital, and serum was obtained. The brainstem was isolated, and the tissue was collected in EP tubes. Both serum and brain tissue were stored at −80°C until used. A certain amount of tissue was cut and weighed. Ten times the amount of normal saline was added for further grinding . After centrifuging twice at 4°C, the supernatant was collected, and the protein quantification was determined by the BCA protein concentration assay kit .β-EP in blood serum was detected by commercial enzyme-linked immunosorbent assay ELISA kits. The nitrate reductase method was used to detect the level of NO in serum, strictly following NO one-step detection instructions . The HTR1A and DRD2 levels in brain tissue were detected by ELISA according to the instructions of ELISA kits. All the ELISA kits were purchased from Elabscience Biotechnology Co., Ltd.According to the manufacturer's protocols, the content of t-test was used for pairwise comparison. The difference between the means was considered to be statistically significant at P < 0.05 and P < 0.01.All data were expressed as the mean ± standard deviation (SD). GraphPad Prism 5.01 software was used to ascertain statistically significant differences. The differences among multiple groups were evaluated using the one-way analysis of variance (ANOVA), and the https://www.chemspider.com), the mass spectrometry database (https://www.massbank.jp), and many related Chinese herbal medicine research chemical components [In terms of mapping the chemical profiles of the extracts of TTNC, data of mass fragmentation coupled with high-resolution spectrometry provided sufficient information. In addition, UPLC-UESI-Q Exactive Focus was used in this present study. The base peak chromatogram (BPC) of TTNC extracts in the mode of positive and negative ions is shown in mponents –38, the To obtain the targets of the 88 components for TTNC, target fishing was performed in accordance with the method described in The 102 targets of TTNC were introduced into the STRING 11.0 platform for searching the interaction between potential targets . The interaction results were introduced into the Cytoscape 3.7.2 software to draw the PPI network. Also, the nodes represented targets and the edges represented the interaction between different nodes. After removing the targets far away from the main network, there were 73 nodes and 206 edges . The larq-value value are shown in q-value . The number of enriched targets in the PI3K-Akt signaling pathway was 12. The log q-value is −8.52E + 00. In addition, NADH: ubiquinone oxidoreductase, mitochondria have the smallest log q-value , and may be one of the other most important pathways for TTNC.The 73 targets were mapped to a total of 124 KEGG pathways. The obviously unrelated KEGG pathways were removed, such as “pathways in cancer,” “prostate cancer,” “hepatitis C,” and so on. Finally, the pathways with the top 20 KEGG pathways of the log The H-C-T-P network was constructed, and analysis for TTNC in treatment with migraine was done by using Cytoscape 3.7.2 . The net1According to the results, we obtained 18 potential key compounds, 20 potential key targets, and 6 potential key pathways with higher than the average of DC. In the key compounds, gastrodin, the main effective constitute from Gastrodiae Rhizoma, has been indicated for migraine treatment and prophylaxis more than 30 years, with demonstrated safety . The resIn terms of the key targets, AKT1 is one of 3 closely related serine/threonine-protein kinases  called the AKT kinase, which regulates many processes including the metabolism, proliferation, cell survival, growth, and angiogenesis through serine and/or threonine phosphorylation of a range of downstream substrates –44. PrevAmounts of evidences supported the significance role of the PI3K-Akt signaling pathway in migraine , 56. A sThe molecular docking was carried out to further explore the interaction between some compounds and targets. We selected some key targets that have been reported to be closely related to migraine  and the corresponding compounds for molecular docking. The binding energy is given in P < 0.05). Compared with the model group, the incubation period was significantly prolonged, and the number of head scratching was significantly decreased in the TTNC group (P < 0.05). The results showed that TTNC can improve the behavioral changes of migraine rats.As shown in β-EP in blood serum. The content of NO in the animal serum model group increased significantly compared with the control group (P < 0.05), indicating that the model was successful. Compared with the model group, the content of NO in the TTNC group was significantly decreased (P < 0.05). It is well known that NO plays a key role in the mechanism of migraine and other vascular headache. NO can expand blood vessels, result in neurogenic inflammation, activate the sensitivity of injured sensory neurons, and mediate pain signal transmission of the body and lead to migraine. The experiment in the present study is a classic migraine model. After injection of nitroglycerin, the content of NO in animal blood increase rapidly, resulting in migraine. In the TTNC group, the content of NO decreased significantly, indicating that TTNC can downregulate the production of NO induced by nitroglycerin, so as to alleviate the attack of migraine. In addition, the content of β-EP in the model group decreased significantly compared with the control group (P < 0.05), while the content of β-EP in the TTNC group increased significantly compared with the model group (P < 0.05). β-EP is α-endogenous opioid peptide with a strong analgesic effect. In the case of disorders of the endorphin system in the body, β-EP release decreased immediately, the pain threshold of body also decreased, and migraine are more likely to occur. In this experiment, NO induced endogenous opioid peptide system disorder, and the content of β-EP decreased. After the intervention of TTNC, the content of β-EP increased, indicating that TTNC could upregulate the level of β-EP induced by nitroglycerin, so as to relieve migraine. These results showed that TTNC has a good effect on migraine induced by nitroglycerin.P < 0.05), and the level was significantly higher in the TTNC group compared with the model group (P < 0.05). Besides, the level of DRD2 in brain tissue of the model group was significantly lower than that of the control group (P < 0.01), and compared with the model group, the level of DRD2 treated with TTNC improved significantly (P < 0.01).The two key targets of HTR1A and DRD2 in brain tissue were verified in vivo by ELISA detection  for the In the present study, a systematical research strategy was adopted to investigate the mechanism of TTNC in treatment with migraine. It contained UPLC-UESI-Q Exactive Focus, network pharmacology, and in vivo experiment. Also, the target fishing, protein-protein interaction (PPI), KEGG pathway enrichment analysis, and herbs-compounds-targets-pathways (H-C-T-P) network construction were performed according to the network pharmacology to predict the potential pharmacodynamic components, targets, and pathways. After 18 potential key compounds, 20 potential key targets and 6 potential signaling pathways were obtained for TTNC in treatment with migraine. Finally, molecular docking and experimental were carried out to verify some key targets. More experimental verification and research still need to be carried out for TTNC in treatment with migraine. The results of this study may provide a theoretical basis for further research on the molecular mechanism of TTNC in the treatment of migraine.


Aim: To explore the diverse target distribution and variable mechanisms of different fangjis prescriptions when treating arrhythmias based on the systems pharmacology.Methods: The active ingredients and their corresponding targets were acquired from the three fangjis  and the arrhythmia-related genes were identified based on comprehensive database screening. Networks were constructed between the fangjis and arrhythmia and used to define arrhythmia modules. Common and differential gene targets were identified within the arrhythmia network modules and the cover rate (CR) matrix was applied to compare the contributions of the fangjis to the network and modules. Comparative pharmacogenetics analyses were then conducted to define the arrhythmia-related signaling pathways regulated by the fangjis prescriptions. Finally, the divergence and convergence points of the arrhythmia pathways were deciphered based on databases and the published literature.Results: A total of 187, 105, and 68 active ingredients and 1,139, 1,195, and 811 corresponding gene targets of the three fangjis were obtained and 102 arrhythmia-related genes were acquired. An arrhythmia network was constructed and subdivided into 4 modules. For the target distribution analysis, 65.4% of genes were regulated by the three fangjis within the arrhythmia network. ZT and GLT were more similar to each other, mainly regulated by module two, whereas HET was divided among all the modules. From the perspective of signal transduction, calcium-related pathways  and endocrine system-related pathways  were associated with all the three fangjis prescriptions. Nevertheless, heterogeneity existed between the biological processes and pathway distribution among the three prescriptions. GLT and HET were particularly inclined toward the conditions involving abnormal hormone secretion, whereas ZT tended toward renin-angiotensin-aldosterone system (RAAS) disorders. However, calcium signaling-related pathways prominently feature in the pharmacological activities of the decoctions. Experimental validation indicated that ZT, GLT, and HET significantly shortened the duration of ventricular arrhythmia (VA) and downregulated the expression of CALM2 and interleukin-6 (IL-6) messenger RNAs (mRNAs); GLT and HET downregulated the expression of CALM1 and NOS3 mRNAs; HET downregulated the expression of CRP mRNA.Conclusion: Comparing the various distributions of the three fangjis, pathways provide evidence with respect to precise applications toward individualized arrhythmia treatments. Cardiovascular disease (CVD), which includes arrhythmia, has long been recognized as the major cause of global death and morbidity. Epidemiological studies have shown that atrial fibrillation (AF) affects 1–2% of the population and may reach unprecedented levels as populations around the world age . IndirecNotably, fangjis prescriptions have a long history of use and their effectiveness has been previously verified in clinical settings –9. FangjThe crucial question is how to accurately identify the target distribution for each of the fangjis preparations and to match these precisely to the corresponding treatment population .Systems pharmacology builds a paradigm for drug–target interactions, which enables a better understanding of the mechanism of action of drugs at the molecular level , 16. Furhttp://lsp.nwu.edu.cn/tcmsp.php) and the TCM mesh database (http://mesh.tcm.microbioinformatics.org/). Arrhythmia-related genes were acquired from the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/) database, using the search terms “arrhythmia” with “Homo sapiens” set as the background.The constituent herbs of these three classical fangjis including ZT, GLT, and HET are given in https://string-db.org/) database using the “Homo sapiens” background to establish associations between the genes and then to acquire the arrhythmia-related network. The three decoctions of ZT, GLT, and HET were independently mapped to the arrhythmia-related network; the Molecular Complex Detection (MCODE) method was used to conduct cluster analysis in order to discover structural modules based on topology, and their similarities and differences were characterized both in the disease networks and modules.The arrhythmia-related genes were uploaded to the Search Tool for Recurring Instances of Neighboring Genes (STRING)  was defined as follows:CRfm was the coverage rate of formula f on module m in the arrhythmia-related network; Ym was the overall genes in module m; Xfm was the number of targets covered by formula f in module m. Letting f = {f1, f2, ……, fn} be a given set of formulas and m = {m1, m2, ……, mn} be a given set of modules, Yf × Ym was considered as a binary matrix of module-formula association.F represented any formula and p-value, once corrected with the Bonferroni correction, at <0.05.The online database such as the Database for Annotation, Visualization, and Integrated Discovery (DAVID) was used to perform the GO biological processes and the KEGG pathway analysis. The GO and the KEGG significance levels were both set at a https://www.ncbi.nlm.nih.gov/pubmed/), the common and unique mechanisms of the three classical prescriptions for the treatment of arrhythmia were compared from the extracted information.According to the results from the bioinformatics analysis and literature search resulted from PubMed .The experimental procedures used in this study were approved by the Animal Welfare Ethics Committee of Sino Animal (Beijing) Science and Technology Development Co. Ltd. and were in accordance with the U.S. National Institutes of Health (NIH) (Publications No. 8023). A total of 48 8-week-old SD male rats were obtained from SPF Biotechnology Corporation Ltd. . The rats were placed in polycarbonate cages under a 12-h light/dark cycle in an air-conditioned room under a constant temperature (25 ± 1°C) and humidity (50 ± 10%) in a specific pathogen-free environment. They were randomly divided into the following five groups: control group (deionized water), vehicle group (deionized water), ZT group , GLT group , and HET group . The rats were gavaged daily for 14 days and anesthetized 30 min after intragastric administration on the 14th day . Anesthetized rats were then linked to ECG and IV lead. ECGs were recorded using the PowerLab biological signal processing system. Once the ECG was stable, the control group was injected with saline  through the tail vein and the vehicle group, ZT, GLT, and HET groups were injected with calcium chloride solution  through the tail vein. The ECG was recorded for 15 min and the occurrence and duration time of ventricular arrhythmia (VA) were observed. The left cardiac myocardium (left ventricle) was frozen for analysis.t-test and multiple groups were compared using the one-way ANOVA. p < 0.05 were considered as statistically significant.Samples from five rats from each group were used for qRT-PCR. Total RNA was extracted from the left cardiac myocardium of each rat using TRIzol  and reverse transcribed into complementary DNA (cDNA) using the Reverse Transcription System Kit  in accordance with the manufacturer's protocols. qRT-PCR was performed on the ABI 7500 Real-Time PCR System  using the SYBR Green PCR Kit to determine messenger RNA (mRNA) expression levels. The relative expression of CALM1 (primers: GCTACATCAGTGCGGCAGA and ACCTGTCCGTCTCCATCAATA), CALM2 (primers: CGGGGATGGGACAATAACA and TACCGTCGGCATCTACTTCAT), CALM3 (primers: GGAATGG- CTACATCAGTGCTG and CCACTTCCTCATCAGTCAGCTT), CRP (primers: GGACAAATG- CAAGCATCATCT and GTGCCCGCCAGTTCAAA), interleukin-6 (IL-6) (primers: GATTGTATGAACAGCGATGATGC and AGAAACGGAACTCCAGAAGACC), and NOS3 (primers: GATCCTAACTTGCCTTGCATC and CTCAATGTCGTGTAATCGGTCT) was analyzed using the comparative CT method for relative quantitation and the 2-DDCt method by normalizing to β-actin expression and relative expression was presented as the percentage change compared to matched controls. All the quantitative data were obtained from at least three independent experiments and were presented as the mean ± SD. The SPSS statistical software version 27.0  was used for statistical analysis. The variances between the two groups were compared using Student's Based on our search strategy involving the constituents of the three prescriptions, ZT , GLT , and HET , we retrieved 187, 105, and 68 active ingredients and 1,139, 1,195, and 811 corresponding gene targets for ZT, GLT, and HET, respectively . There wSLC22A5 and ECHS1. Analysis of the fangjis-related molecules in the arrhythmia network showed 27, 31, and 25  genes in the ZT, GLT, and HET decoctions, respectively, overlapped with the arrhythmia network  were co-associated with ZT, GLT, HET, and arrhythmia; 5 genes  were co-associated with ZT, GLT, and arrhythmia; 3 genes  were co-associated with ZT, HET, and arrhythmia; and, lastly, 1 gene (HRAS) was co-associated with GLT, HET, and arrhythmia. There were 8, 4, and 2 unique genes for GLT, HET, and ZT, respectively, in the arrhythmia network .Representative ECGs of the control and vehicle groups are shown in p < 0.05). The ZT, GLT, and HET groups showed statistically significant differences compared with the vehicle group (p < 0.05).The results of PCR analysis showed that the expression of CALM2 and IL-6 mRNAs were consistently altered in individual samples from the control, vehicle, ZT, GLT, and HET groups. CALM1 and NOS3 mRNA expression was altered in individual samples from the control, vehicle, GLT, and HET groups . There wThis study aimed to identify the similarities and characteristic therapeutic mechanisms of the different fangjis decoctions against arrhythmia using network- and module-based approaches. ZT, GLT, and HET have been used in clinical settings for a considerable period of time, playing an important role in complementary and alternative therapies. This method gathered potential targets for ZT, GLT, and HET to investigate the biological basis of treating arrhythmias and to clarify the precise clinical applications of the various prescriptions.The etiology of complex diseases might be polygenic, perhaps even “omnigenic” –26. TherBy comparing the homogeneity and heterogeneity of the three prescriptions for arrhythmia, we found that: (1) although the molecules targeted by ZT, GLT, and HET are different, a majority of shared targets, biological processes, and pathways converge to denote certain primary characteristics; (2) the targeting rate of ZT, GLT, and HET in the network and modules was comparable, but the target distribution within the modules was different, for example, module two was the main target module for ZT and GLT, whereas module 1 was the main target for HET; (3) among the pathways enriched by the three prescriptions, most were related to the calcium signaling pathways; and (4) each fangjis were also uniquely associated with a particular target pathway.Regardless of whether the target distribution was similar or identical, shared molecular backgrounds may target the arrhythmia network to produce pharmacological effects . This siBased on the fangji-related target pathways network analysis, we found the commonly enriched pathways involving the three fangjis converged on signal transduction processed related to the cyclic adenosine 3′,5′-monophosphate (cAMP), cyclic guanosine 3′,5′-monophosphate (cGMP)-PKG, and calcium signaling pathways. Consistently, according to the Vaughan–Williams classification scheme, the effect on receptors and/or ion channels represents one of the primary mechanisms of antiarrhythmic drugs .f), thereby promoting an increase in heart rate  from the cardiac sympathetic nerves and epinephrine (Epi) from the adrenal medulla, which binds to β-adrenergic receptors (β-ARs) on cardiomyocytes. This triggers a signaling cascade leading to an increase in cAMP and consequent protein kinase A (PKA) activation and phosphorylation of a myriad of targets, thereby increasing heart rate and conduction velocity, increasing contraction strength, and the speed of relaxation . HoweverInterestingly, the estrogen (E2) signaling pathway was shared by GLT and HET, while the GnRH signaling pathway was only linked with GLT, although both the pathways belong to the same endocrine system category. Previous epidemiological studies suggested that women have a lower incidence of cardiovascular disease during their reproductive age, partly due to E2 . These fThe aldosterone synthesis and secretion pathway were only shown in conjunction with ZT, but is related to renin secretion and adrenergic signaling in cardiomyocytes. A randomized, double-blind, placebo-controlled study showed that excess aldosterone secretion resulted in proarrhythmic effects and moreover confirmed that drugs affecting the renin-angiotensin-aldosterone system (RAAS) were of benefit for arrhythmia and its comorbidities . A syste2+) can regulate a variety of biological functions including muscle contraction, cell exocytosis, neuronal activity, and also triggering of programmed cell death , catecholaminergic polymorphic ventricular tachycardia (CPVT), and atrial fibrillation (AF)  . As showion (AF) . Moreove to LQTS  and muta to LQTS  and Brug to LQTS . Besides to LQTS . Invasivernation , 66. IndJun-Chen-Zuo-Shi”  Science and Technology Development Co.Ltd.HL contributed to the conception or design of the study. XL and WX revised the manuscript. DL and YT are responsible for data collection. KY was responsible for statistics. PW drafted the manuscript. All authors read and approved the final version of the manuscript.This study was supported by Grants from the National Natural Science Foundation of China (No. 81273741) and China Postdoctoral Science Foundation (No. 2021M702311), and the National Administration of Traditional Chinese Medicine (No. 2019XZZX-XXG001).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 ofinterest.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.