The surfactants were prepared via one-pot reductiveamination, allowing the introduction of different alkyl chain lengthsand methyl modifications. Optimal reaction conditions were establishedto achieve high yields and easy purification. The synthesized surfactantsincluding the tertiary amines exhibited desirable properties, includingsolubility, foamability, and reduction of surface tension. Notably,the anionic surfactants derived from d-GalA demonstratedbetter solubility and foam performance compared to those derived from l-Ara. In addition, these surfactants exhibited surface tensionand critical micelle concentration (CMC) comparable to those of thecommercial surfactant sodium lauryl ether sulfate (SLES). Furthermore,the biodegradable surfactant GalA1.8 displayed excellentemulsifying properties and low skin irritation potential. On the l-Ara surfactant with a short chain, Ara1.6 haspotential as a hydrotrope. These findings suggest that biobased surfactantsderived from SBP monosaccharides have promising applications in variousindustries, including pharmaceuticals, cosmetics, detergents, andchemicals.Herein, we reporta method for the synthesis of biobased surfactantsderived from sugar beet pulp (SBP) monosaccharides,  l-Ara and d-GalA using a simple and environmentally benignprocess. The surfactant properties of the prepared derivatives offerversatile potential applications in cosmetics and detergents.Herein, we report the synthesis of biobasedsurfactantsderived from the sugar beet pulp-derived monosaccharides  Hence, this source of biomassdoes not interfere with food production and does not require additionalarable land.5 Agricultural side streamsare available at a large scale in the form of residues produced duringcultivation, harvesting, and processing of crops. Depending on thefeedstock, biomass is available in the form of leftover straw, roots,leaves, cobs, pulps, peels, and seeds.6The utilization ofbiomass has become a subject of great interestas a sustainable alternative for products derived from nonrenewableresources.7 Yearly, sugar beets are being processed into 36.4 millionmetric tons of sucrose worldwide. During this process, an equal amountof SBP is obtained, which is a cheap and abundant source of biomass.7 Moreover, SBP is particularly attractive because its utilizationdoes not affect food production to an appreciable extent. Currently,SBP is being sold as animal feed, has a low commercial value, andis rich in polysaccharides such as pectin and cellulose and hemicellulose.These polysaccharides can be enzymatically processed to provide variousmonosaccharides such as d-galacturonic acid and l-arabinose (l-Ara).10 Effortsdirected at the large-scale recovery of pure d-GalA and l-Ara may enable their production at large scale in a sustainablemanner.11 This makes them an interestingstarting material for the production of high-value commercial products.13Sugar beet pulp (SBP) is an interesting source of agriculturalwaste, which is obtained during the production of sucrose 1.7 Yearl14 Surfactants or surface active agents have the ability to reducethe surface tension and are therefore essential ingredients in thepharmaceutical, cosmetic, detergent, and chemical industries.15 Microbial biosurfactants such as rhamnolipids and sophorolipidscan be produced in various microorganisms and from different feedstocks.16 Carbohydrate-based surfactants can also be preparedvia chemical synthesis from monosaccharides isolated from biomass.The commercial production of carbohydrate-based surfactants startedin the 1980s and is mostly based on sucrose and glucose derivatives.21 Much less is known about surfactants derived from l-Araand d-GalA, monosaccharides that can be obtained from SBPin bulk quantities without direct competition with food production.23The hydrophilic nature of monosaccharides enablestheir applicationas the polar headgroup for the synthesis of biobased surfactants.d-GalA and l-Ara via one-pot reductive amination. Different chain lengths ofalkylamines were introduced using this method, leading to a varietyof secondary amine products. Using a second hydrogenation with formaldehyde,novel surfactants containing a tertiary amine were also prepared.In particular, we found that the tertiary amine surfactants are moresoluble in water and display favorable surfactant properties suchas foamability, stable foams, and emulsions, lowered surface tensionof solutions, and a variable critical micelle concentration.25 Hence, this class of molecules may represent a promising set ofbiobased surfactants derived from sugar beet pulp.Herein, we report a simple,green method to prepare biobased surfactantsderived from the SBP monosaccharides 27 This reactioncan be used to selectively modify monosaccharides by making use ofthe aldehyde present in the open form with secondary or tertiary amines.29 Several sugar monomers converting into their corresponding aminesvia reductive amination reactions have been reported.30 For example, a reaction with d-glucose, methylamine,and a reducing agent affords N-methylglucamine, aprecursor in the synthesis of Glucopure.32 In contrast to glucose-based surfactants,much less is known about the reductive amination reactions with theSBP monosaccharides l-Ara and d-GalA.19 Hence, we set out to investigate the synthesisand properties of biobased surfactants based on l-Ara and d-GalA formed by the reductive amination with various alkylamines.Reductive amination of carbonyl-containingcompounds is an effectivemethod to form C–N bonds.d-GalA via reductive aminationusing primary or secondary amines . We will refer to the compounds accordingto their monosaccharide abbreviations, l-arabinose (Ara)and d-galacturonic acid , followed by a number denotingthe optional presence of a methyl group (1) and the length of thealkyl chain (6–12). Reaction conditions were optimized to developan efficient and environmentally benign process  was obtainedusing precipitation as a purification method. Under the same conditions,a secondary amine was used to afford the corresponding tertiary amine GalA1.8. However, this product was obtained in a much loweryield (19%), which was likely the result of its higher solubilityand hence poor recovery using the precipitation protocol  of GalA1.8 . Moreover, two other modifications were introduced:a variety of alkyl chain lengths and R1 may or may notbe methylated. Due to the commercially available N-methylalkyl chains (R1 = Me) with 6, 8, and 12 carbonchains, these were used to react with the two sugars to obtain sixdifferent surfactants. Using the reactions conditions of Ara1.6Ara1.8, and Ara1.12 were synthesized in 85–95%yield with GalA1.6, GalA1.8, and GalA1.12 synthesized in 86–90%. The N-methyldecylchain was not commercially available; hence, the corresponding surfactantswere made via a one-pot two-step synthesis starting from the respectivecommercially available primary amine  and GalA1.10 (73%).To investigate the properties of the methyl modification on the surfactants(R1 = Me), previously described reference surfactants weresynthesized including an N-alkyl chain (R1 = H).22 Primary alkyl amines with 6,8, 10, and 12 carbon chain length were used to react with l-Ara and d-GalA. Because poor solubility purification wasdifficult, some of them were synthesized via the previously describedmethod (1 =H) were obtained in lower or similar yields than with methyl modification(R1 = Me).With the optimal reactionconditions established, we set out toexplore the synthesis of a panel of biobased surfactants using aterials 2. These Page S9 for experimental details). Various physicochemical properties suchas solubility, foamability, surface tension, and critical micelleconcentration (CMC) were determined  was determined to compare them with their methylated counterparts(R1 = Me). All methylated compounds (R1 = Me)showed an increased solubility compared to their corresponding nonmethylatedcounterparts (R1 = H). An interesting example is the solubilityof Ara1.6, which is 30% and much higher than that ofnon-N-methylated Ara6 (0.75%). We presumethat this effect is caused by the disruption of surfactant stackingby the addition of the methyl group, which in turn increases the solubilityof the surfactant.To establish the optimal applicationof the prepared surfactants,their properties were established (see termined 3.24 Sinc1 = Me) were determined. Foamwas created in a measuring cylinder with an Ultraturrax, and the foamvolume (in mL) was monitored over 30 min . Furthermore,a compound can be classified as a surfactant if it decreases the air–waterinterfacial force and lowers the surface tension of the system. Thesurface tension and corresponding critical micelle concentration (CMC)of the synthesized surfactants were measured using a tensiometer . The l-Ara-based compounds Ara1.8, Ara1.10, and Ara1.12 hada lower surface tension  than thatof SLES (33.8 mN/m). In addition, the CMC of these surfactantswas comparable to that of SLES, indicating that withlow concentrations between 0.01 and 0.25%, micelles are formed. Thesefindings demonstrate that these molecules display suitable propertiesto be used as surface-active agents. Likewise, the anionic surfactantsderived from d-GalA, GalA1.8, GalA1.10, and GalA1.12 reduced the surface tension to 32.1, 32.9, and 33.9mN/m at a CMC of 1.15, 0.06, and 0.1%, respectively, similar to thecommercial surfactant. Interestingly, anionic surfactants GalA1.12 and SLES, both with a 12-carbon chain, had very similarfoamability and surface tension performance. This would suggest thata potential application as a wetting agent could be interesting forthese compounds. Finally, we compared the performance of the SBP-derivedsurfactants to Glucopure, a commercially available biobased surfactantbased on glucose. Performance was very similar in terms of the solubility,foamability, surface tension, and CMC.Thefoamability and surface tension of the more soluble and interestingmethylated surfactants  for experimentaldetails). To this end, detergency and emulsion properties of the newsurface-active agents were determined using the TurbiscanLab protocols(Table S1 and Page S11). Emulsions consistingof 6% oil, 1% surfactant, and 93% H2O were prepared. Thecommercially available surfactant alkylpolyglucoside with a six-carbonchain (APG-6) is a well-known oil-in-water emulsifierand was used as a reference. The emulsion with APG-6 hada TSI score of 11.1. The sample of GalA1.8 showed morestability with a TSI of 1.6 . The Turbiscan was used to measure the transmittance andbackscattering of pulsed near-infrared light (880 nm) to monitor thestability of emulsions, which are then expressed as the TSI, witha low TSI score referring to a stable emulsion . Zein is a protein found in corn and isan accepted mimic of skin proteins. The more zein is solubilized bythe surfactant, the harsher the potential to irritate and dry thehuman skin. Notably, it is an indication of its harshness to the skinand allows a comparison of our surfactants to some commercially availablesurfactants, even though in vivo testing is always the most idealfor measuring mildness. SDS and SLES arecommercial surfactants and known to be harsh on the skin.34 Hence, SDS is set as a default of 100 to comparewith the potentials of other surfactants, which are all executed inDuplo (Table S2 and Page S12). Moreover,the surfactants were measured at different pH values, a pH of 4–5,7, and at its original pH as is. SLES showed a skin irritationpotential value of 49 at a pH of 7, thus half as irritating as SDS.In contrast, our biobased surfactants all have very low values between0 and 3 (Table S2). For example, GalA1.8 had a zein score of 1 at pH 7  or the differencein solubility of the two surfactants.To investigate if the prepared surfactant may becompatiblewitha personal care application, the skin irritation potential was investigatedusing zein solubilization tests . Biodegradation is important in order to prevent pollutionof the environment. The commercially available surfactant SLES has a primary degradation of 94% in 30 days,35 depending on the initial concentration, using the Organizationfor Economic Cooperation and Development (OECD) 301 procedure.36 This procedure was used to test GalA1.8 resulting in a faster biodegradability and was followed by CO2 production . Furthermore, the condition to evaluatethe toxicity of GalA1.8 on the inoculum revealed thatCO2 production from acetate was not inhibited by GalA1.8, suggesting that the compound was not significantlytoxic to the inoculum.The biodegradation ofthe novel surfactant Ara1.6 and GalA1.6 are not suitable for application as a surfactant becausethey do not foam and form stable oil-in-water emulsions. We reasonedthat due to their high solubility, they may act as a hydrotrope, compoundsthat enhance the solubility and reduce the viscosity of other organicsubstances in water.38 One well-known example is sodiumxylene sulfonate (SXS), a commercially available hydrotrope.39 To evaluate the hydrotropic behavior of Ara1.6 and GalA1.6 against SXS as a benchmark, we conducted experimentsusing a hydrophobic dye Disperse Red 13 (DR-13) dissolved in water.DR-13 is insoluble in water, and hence, the solution has a low absorptionat 525 nm (see Page S15 for details). Uponthe increased solubilization of DR-13 in water by the addition ofa hydrotrope, the solution absorption will also increase, which wasdetected using a ultraviolet–visible (UV–vis) spectrophotometerat a wavelength of 525 nm.39 The obtainedabsorbance (Abs) values for DR-13, in relation to the molar concentrationof Ara1.6, GalA1.6, SXS, and the cosolventacetone, are plotted in Figure S4 (PageS15). SXS exhibited an Abs of 1866 L mol–1 cm–1 at a concentration of 0.9 M (40 wt %), indicatingthat the dye could be dissolved with a low concentration of SXS. Acetone,as a cosolvent, showed the same solubilization potential (Abs at 1829),however, only at high concentrations. Our surfactant Ara1.6 demonstrated significant solubilization capabilities at a very lowconcentration of 0.25 M with an Abs at 1854. This result suggeststhat Ara1.6 can be effectively employed as a hydrotropein various applications such as improving the solubility of less solublesurfactants in cosmetic formulations. To exclude pH effects on thesolubilization of the DR-13 dye upon addition of the Ara1.6 compound, the experiment was also carried out in a buffer, whichled to a very similar performance to SXS. Finally, we utilized Ara1.6 to boost the solubility of Ara 1.10 . Addition of Ara1.6 effectively enhanced the solubility of a 1% solution of Ara1.10.The results mentioned above have revealedthe potential applicationsof the synthesized surfactants, particularly those with alkyl chainsof eight carbons or more, as effective cleaning, emulsifying, andfoaming agents. The shorter chain derivatives l-Ara and d-GalAwas developed. By employing one-pot reductive amination, a varietyof surfactants with different alkyl chain lengths and methyl modificationswas synthesized. We optimized the reaction conditions to achieve highyields and easy purification, eliminating the need for excess reagentsand using (bio)ethanol as the solvent. Furthermore, we conducted acomprehensive evaluation of various surfactant properties, includingsolubility, foamability, surface tension, emulsion stability, skinirritation, and biodegradability. Remarkably, the newly synthesizedmethylated biobased compounds from the Ara1.X and GalA1.X series exhibited increased solubility compared totheir nonmethylated counterparts (R1 = H). Additionally,surfactants containing octyl, decyl, and dodecyl alkyl chains, like GalA1.8, displayed foamability and surface tension comparableto those of SLES, a commonly used commercial surfactant. On the otherhand, the study demonstrated the effectiveness of the short six-alkylchain-containing surfactant Ara1.6 as a hydrotrope. Thesefindings demonstrate the promising potential of SBP-derived molecules,which not only exhibit nonirritating properties but also demonstratecomparable or improved performance compared to existing commerciallyavailable molecules. By employing a sustainable hydrogenation processto generate the key N-methylalkyl (R1 =Me) compounds, we have successfully synthesized biobased surfactantswith interesting properties for various ecofriendly applications.Further scale up and application testing are needed to identify theultimate application of the developed molecules.A simple method to prepare biobased surfactantsderived from sugarbeet pulp (SBP) monosaccharides


Hedysarum, a traditional Chinese herbal medicine and food with a long history of clinical application, is used to improve health conditions and treat various diseases. Hedysarum polysaccharides (HPS), flavonoids, saponins, and alkaloids, are the primary components of Hedysarum. HPS is the most important natural active ingredient of Hedysarum, which has many pharmacological effects. Currently, HPS exhibits significant promise in drug development for various ailments such as tumors, diabetes, cardiovascular diseases, Alzheimer’s disease, and fibrosis. This review paper discusses the extraction, separation, and content determination techniques of HPS, along with the investigation of its chemical constituents. More importantly, we reviewed the anti-inflammatory pharmacological effects of HPS, such as inhibition of inflammatory factors and NF-κB signaling pathway; antitumor activity through apoptosis induction in tumor cells and blocking tumor cell proliferation and metastasis; antioxidant effects; regulation of various cytokines and immune cells; regulation of blood sugar levels, such as in type I and type II diabetes and in diabetic complications; improvement in symptoms of Alzheimer disease; anti-aging and anti-fibrosis properties; and improvement in cerebral ischemia-reperfusion injury. This review paper establishes the theoretical foundation for future studies on the structure, mechanism, and clinical use of HPS. Hedysarum polybotrys Hand.-Mazz. is a plant that belongs to the family Leguminosae. It invigorates the qi and aids the ascending yang, stops sweating, induces diuresis and relieves swelling, promotes salivation and flow of nourishing blood, relieves arthralgia, and supports the expulsion of poison and pus , which is extracted from the dried root of Hedysarum and contains active polysaccharides  and have discovered that HPS is more effective than APS in terms of immunomodulation and antioxidation  from Hongqi using water extraction and 30% ethanol precipitation which was then purified to yield four uniform components. Similarly, Chen Tongqiang et al. (Hedysarum polysaccharide 3 (HPS3) to isolate four uniform components, namely HPS3-A, HPS3-B, HPS3-C, and HPS3-D. Thin layer chromatography and gas chromatography analysis revealed that HPS3-A primarily consisted of glucose, while HPS3-B, HPS3-C, and HPS3-D were composed mainly of arabinose and galactose. Furthermore, Hui He Ping . HPS is a heteropolysaccharide composed of various types and numbers of monosaccharides, resulting in different HPS structures. Presently, research on the separation of HPS is progressing.Currently, there are limited studies on the separation methods of HPS. Madan  employedg et al.  conducte He Ping  reportedHedysarum stilbene, with glucose being the reference substance. Ouyang Yihua et al. , Hedysarum polysaccharide 2 (HPS2), Hedysarum polysaccharide 3 (HPS3), and Hedysarum polysaccharide 4 (HPS4)  . The kno5, and its main chain consists of 1, 4-α-D-Glcp and 1, 4, 6-α-D-Glcp. HPS1-C contains 1, 6-α-D-Glcp, 1, 4, 6-α-D-Glcp, 1,2-α-L-Rhap, and 1, 2, 4-α-L-Rhap in the main chain. HPS1-D is a neutral heteropolysaccharide with a relative molecular weight of 4.593 × 104, andits main chain consists of 1, 4-α-D-Glcp and 1, 4, 6-α-D-Glcp. HPS1 has been separated and purified to yield HPS1-A, HPS1-B, HPS1-C, and HPS1-D . HPS1-A HPS2 is a heteropolysaccharide with predominantly β-glycosidic bonds, but determining precise structure requires further research. HPS3 comprises four components, namely HPS3-A, HPS3-B, HPS3-C, and HPS3-D. HPS3-A contains α-D-(1→4) glucopyranosyl group as the primary chain, while HPS3-B contains β-D-(1→4) galactose and β-D-(1→4) galacturonic acid in the main chain. HPS3-C is composed of β-D-(1→4) galactosyl and β-D-(1→4) galacturonic acid in the main chain, whereas HPS3-D consists of β-D-(1→4) galactopyranosyl and β-D-(1→4) galacturonic acid group as the main chain. The relative molecular masses of these components require further investigation. 4 to 6.68 × 105, and its main chain comprises 1,5 and 1, 3, 5 linked α-L-furanyl arabinose groups and 1, 6 and 1, 2, 6 linked α-D-galactopyranosyl groups. HPS4-1B has a relative molecular mass ranging from 20 to 5.023 × 104, with 1, 4-α-D-Glcp and 1, 6-α-D-Galp formingthe main chain. HPS4-2A is a thioylacetylaminoamino heteropolysaccharide with a relative molecular mass of 2.72105, and its main chain consists of 1, 3,4-β-D-2-acetyl aminogalactose, 1, 4, 6-α-D-glucose, 1, 4,6-β-D-galactose, and 1, 3, 6-β-D-galactose. HPS4 consists of three components: HPS4-1A, HPS4-1B, and HPS4-2A . HPS4-1AHPS has many pharmacological effects. In particular, nine of these effects have been thoroughly studied, including its anti-inflammatory, antitumor, antioxidation, anti-aging, and anti-fibrosis properties and its ability to lower blood sugar levels, regulate immune functions, improve Alzheimer’s disease (AD) symptoms, and improve cerebral ischemia-reperfusion injury .in vivo, closely related to the inhibition of IL-6, TNF-α, IL-1β, and other inflammatory cytokines.Wang Jing et al.  on studyNuclear factor-κB (NF-κB) is a transcription factor that is activated by various signals through the degradation of IκBs. Once activated, NF-κB enters the nucleus to bind with DNA . NF-κB ain vitro by MTT assay, microfluorescence, and flow cytometry. HPS-3 at 200 g/L and 400 g/L for 72 h could induce cell apoptosis and inhibit the MGC-803 tumor cell growth and proliferation, thus, achieving an antitumor effect. The mechanism and regulation of HPS have been found to be closely associated with the modulation of Bax and Bcl-2 proteins’ expression in the mitochondria. That is, HPS can inhibit the growth of oral cancer, lung adenocarcinoma, and human gastric adenocarcinoma, thus, possessing a wide range of effects. HPS can prevent the formation of tumors and help treat tumors by suppressing tumor growth, stimulating cell apoptosis, and supplementing the effects of chemotherapy drugs . Zeng et4 Da in molecular weight from the Hedysarum root. The scavenging experiments with 1,1-diphenyl-2-methylhydrazine, superoxide, and hydroxyl radical showed the good antioxidant activity of HPS3aS in vitro, implying that it is a potent antioxidant. HPS may also enhance Bcl-2 expression, suppress Bax expression, and effectively block the apoptosis of cells, thus, delaying the aging of brain cells  γ protein, and mRNA expression were upregulated, while that of NF-κB mRNA and protein was downregulated. HPS can potentially alleviate oxidative stress and inflammatory reaction and inhibit the progress of myocardial fibrosis caused by hyperglycemia . Wang etMajor vascular complications of diabetes include Diabetic nephropathy (DN), DCM, DR, nonalcoholic fatty liver disease (NAFLD), and diabetic neuropathy, with lipid-lowering and hypoglycemic therapy being the primary treatment methods used to prevent complications. HPS at different doses in DM rats can significantly improve blood glucose levels, decrease TC, TG, and LDL levels, and improve lipoprotein metabolism . The avaDiabetic neuropathy (DN) is a chronic complication of diabetes that not only contributes significantly to the disease but also leads to end-stage renal disease (ESRD). The pathogenesis of DN is mainly related to oxidative stress caused by hyperglycemia due to abnormal glucose and lipid metabolism . NumerouDCM, a cardiovascular complication of DM, has no specific treatment protocol. The primary mechanisms causing DCM, extracellular matrix remodeling, and myocardial fibrosis include oxidative stress and inflammatory responses caused by glucose and lipid metabolism disorder. HPS can help delay DCM onset and other cardiac protection . Recent DR is another important complication of diabetes, which can lead to blindness in patients with diabetes. HPS can potentially inhibit neovascularization and proliferation in DR by elevatingTSP-1 expression level and reducing the platelet-derived growth factor (PDGF)-B expression level in the retina of diabetic rats to protect the retina . HPS canNonalcoholic fatty liver disease (NAFLD) refers to a range of liver conditions caused by metabolic stress and strongly associated with insulin resistance, including nonalcoholic simple fatty liver, nonalcoholic fatty liver hepatitis, liver cirrhosis, and hepatocellular carcinoma . The incin vivo by upregulating Nrf2 and downregulating the expression of Keap1 protein and gene. The lifetime incidence of diabetic neuropathy is about 45% in T2D and 54%–59% in T1D . HyperglIn the late 18th century, Roberts , in the + T and CD4+ T cell counts. Subsequent studies have also shown that HPS can regulate immunity, enhance immunity, and increase the T lymphocyte subset levels  antagonists and acetylcholinesterase (AChE) inhibitors, but their effectiveness is not satisfactory. In China, because of the known curative effects of TCM, these have been applied to treat AD for hundreds of years. AD is a type of senile dementia, with its primary clinical manifestation being the gradual decline of cognitive and life functions. Commonly used therapeutic drugs are The aforementioned research findings indicate that HPS modulates various proteins, such as choline kinase A, LAM-TOR1, AP-2-δ, and ferritin, while also upregulating PRKCB expression. Moreover, the ERK1/2 signal-mediated PRKCB has a therapeutic effect on Alzheimer’s disease.Drosophila melanogaster, improve the nonspecific resistance of aged mice to harmful stimuli, reduce lipid peroxide levels in plasma, and reduce lipofuscin formation. It is further proved that HPS can increase the SOD content of red blood cells and serum cortisol and testosterone levels in aged rats. DNA methylation, changes in telomere and telomerase, abnormal free radicals, and abnormal regulation of nervous, endocrine, and immune systems are factors responsible for accelerating the aging process . HPS hasHedysarum. A rat model of pulmonary fibrosis was created by administering bleomycin through a laryngoscope, after which the rats were given different drugs for intervention. HPS, flavonoids, and saponins were found to inhibit the proliferation and deposition of collagen fibers and improve the pulmonary interstitial fibrosis and alveolar inflammatory reaction in the rats. HPS can significantly improve liver fibrosis caused by carbon tetrachloride in mice and also considerably improve bone loss caused by liver fibrosis . Wang etHedysarum, a traditional Chinese medicine possessing multi-component and multi-target characteristics. Its pharmacological activities are wide-ranging, playing an irreplaceable role in the treatment of modern diseases. Nine main pharmacological effects of HPS have been detailed in this study, namely, inhibition of inflammatory reaction, immunomodulation, antioxidation, anti-aging, antitumor, lowering blood sugar levels, preventing and treating AD, lowering blood lipid levels, anti-fibrosis, improving cerebral ischemia-reperfusion injury. HPS promotes the repair and regulation of the immune system. Among the several anti-aging effects of HPS, its antioxidation mechanism is the most important one; HPS is also effective in preventing and treating cancer. HPS has been shown to exhibit antitumor properties by suppressing the growth of tumor cells and promoting tumor cell apoptosis. In addition, HPS has potential therapeutic applications for managing diabetes and associated complications. HPS has been found to significantly decrease levels of blood glucose, TC, TG, LDL, NO, NOS, and lipid peroxide, while also improving lipoprotein metabolism. Moreover, HPS has been shown to increase serum leptin and fat leptin levels, fasting blood glucose, and insulin sensitivity index. HPS has also been found to enhance the spleen and thymus index, thereby providing a potential treatment option for diabetes complications. HPS, as a natural medicine, has almost no toxic or side effects and is suitable for long-term use by patients with chronic diseases. Therefore, HPS has great potential in treating diseases.HPS is the primary active ingredient of Hedysarum monomer or its compound, clinical application research is still lacking. (2) Except on HPS, studies on the pharmacological effects of isoflavones from Hedysarum stilbene are few. (3) At present, research on the effect of HPS has been conducted on only cells and animals, and its specific pharmacodynamic material basis remains unclear. (4) Only a few reports exist on the structural modification of HPS. Therefore, future studies should expand the research scope and depth of HPS, explore whether different HPS modifiers can enhance its immunomodulatory function, and further determine the pharmacological effects of Hedysarum active ingredients from the classic prescription. The biological activity of more structural modifiers of HPS and the efficacy comparison of different administration methods need to be explored. The mechanisms of its effects of reducing blood lipid levels, resisting fibrosis, and preventing radiation need to be clarified. Moreover, although HPS exerts various pharmacological effects, the precise control of its dosage needs further study.However, there are some shortcomings in the current research situation. (1) Whether it is HPS is a natural compound. Currently, the main challenge in HPS research is to identify its specific components and determine its precise targets. It is important to accurately detect the nine pharmacological targets of HPS and demonstrate the remarkable effect of multi-target integration in traditional Chinese medicine. This can help fill the gaps in the research of pharmacological effects from the perspective of HPS and provide significant theoretical support for the clinical management of illnesses. It can also provide direction for further research on new ways of treating diseases with traditional Chinese medicine.


Kidney diseases are worldwide public health problems affecting millions of people. However, there are still limited therapeutic options against kidney diseases. Semaphorin 3A (SEMA3A) is a secreted and membrane-associated protein, which regulates diverse functions, including immune regulation, cell survival, migration and angiogenesis, thus involving in the several pathogeneses of diseases, including eyes and neurons, as well as kidneys. SEMA3A is expressed in podocytes and tubular cells in the normal adult kidney, and recent evidence has revealed that excess SEMA3A expression and the subsequent signaling pathway aggravate kidney injury in a variety of kidney diseases, including nephrotic syndrome, diabetic nephropathy, acute kidney injury, and chronic kidney disease. In addition, several reports have demonstrated that the inhibition of SEMA3A ameliorated kidney injury via a reduction in cell apoptosis, fibrosis and inflammation; thus, SEMA3A may be a potential therapeutic target for kidney diseases. In this review article, we summarized the current knowledge regarding the role of SEMA3A in kidney pathophysiology and their potential use in kidney diseases. Semaphorins are guidance proteins regulating cellular morphology and functions, and, thus, have important roles in developments and diseases, including cancers and metabolic diseases . SemaphoKidney diseases, characterized as kidney dysfunction, are a growing public health burden, with a huge economic cost on healthcare systems worldwide . There aSEMA3A, as with other semaphorin family members, has a conserved N-terminal SEMA domain that contains 500 amino acids, and a diverged C-terminus. The central feature of the SEMA domain is a disulfide-rich seven-blade β-propeller fold. A small cysteine-rich plexin–semaphorin–integrin (PSI) domain is between the SEMA domain and the immunoglobulin (Ig)-like domain. The basic domain is located at the C-terminus of SEMA3A. The C-terminus determines its affinity to neuropilin-1 (NRP1)  3,38]. . 38]. SE165); thus, SEMA3A plays a role in suppressing angiogenesis by the competitive inhibition of VEGF signaling through the inhibition of binding to NRP1 [It has been reported that excess SEMA3A expression may accelerate kidney injury in a variety of kidney diseases via the JNK or Akt signaling pathway ,44,45,46 to NRP1 . It has  to NRP1  Figure 65; thus, to NRP1 . In addi to NRP1 . To spec to NRP1 .165 and decreased activity of Akt survival pathways. Conversely, the deletion of SEMA3A in mice enhances ureteric bud branching [−/− mice showed increased endothelial cells and defects in renal vascular patterning, whereas SEMA3A+/+ mice had normal wide-open capillary loops, observed using light microscopy. The morphometric analysis of transmission electron microscopy (TEM) revealed that SEMA3A−/− mice had effaced podocyte foot processes, which were associated with albuminuria. On the other hand, the overexpression of podocyte SEMA3A resulted in glomerular hypoplasia, undifferentiated podocytes and congenital proteinuria [SEMA3A has roles in axon pathfinding, and cardiovascular, lung and kidney patterning during organogenesis ,57. Duriranching . Thus, Steinuria . Taken tteinuria . In additeinuria ,49. Take+ nuclei counts were not altered, suggesting that SEMA3A caused decreased nephrin expression without podocyte loss. In addition, GST-binding assays revealed a direct interaction between plexin A and nephrin [Podocytes, which have crucial roles in the kidney filtration barrier, line out of the glomerular basement membrane (GBM), prevent urinary protein loss . Podocyt nephrin , indicat nephrin   caused Kimmelstiel-Wilson-like nodular glomerulosclerosis, massive proteinuria and kidney insufficiency [+ gain of function [Diabetic nephropathy (DN) is one of important complications of DM , and is ficiency . Increasficiency , as wellficiency . The genficiency . Importafunction . A recenfunction . miR-15bfunction . SEMA3A function  and miR-function . KCNQ1 ofunction . It has function . CollectAKI is recognized as a major public health problem, affecting millions of patients worldwide and leading to higher mortality , CKD proIn addition to biomarkers, SEMA3A signaling is also expected to be a therapeutic target. SEMA3A expression was increased after LPS-induced AKI in mouse tubular epithelial cells, as well as in an LPS-treated rat-kidney-proximal-tubular-epithelial-cell line in vitro via Rac1/nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) p65 and JNK pathways . In addiCKD is characterized by progressive damage and a loss of kidney function, in which parenchymal cell loss, chronic inflammation, fibrosis and the reduced regenerative capacity of the kidney are involved in its progression . It was SEMA3A also regulates immune systems, especially enhancing T-cell and B-cell regulatory properties , rheumatIt was reported that SEMA3A downregulated autoimmune responses by suppressing the over-activity of both B and T cells ,100. SEMCardiorenal syndrome (CRS) is a complex interaction between the heart and kidneys, where dysfunction in one organ causes or exacerbates dysfunction in the other . It was We have summarized the current knowledge regarding the role of SEMA3A in kidney pathophysiology and its potential use in kidney diseases. On the basis of these studies, SEMA3A plays an important role in kidney morphogenesis and kidney diseases. SEMA3A loss-of-function studies indicate that SEMA3A is required for the maintenance of structure and function in the glomerular filtration barrier. In contrast, excess SEMA3A causes the progression of a variety of kidney diseases, including DN, AKI and CKD, through an increase in albuminuria, kidney fibrosis, apoptosis and inflammation. Current speculations around SEMA3A and the pathophysiology of kidney diseases is summarized in


Diabetic retinopathy (DR) is the most frequent microvascular retinal complication of diabetic patients, contributing to loss of vision. Recently, retinal neuroinflammation and neurodegeneration have emerged as key players in DR progression, and therefore, this review examines the neuroinflammatory molecular basis of DR. We focus on four important aspects of retinal neuroinflammation: (i) the exacerbation of endoplasmic reticulum (ER) stress; (ii) the activation of the NLRP3 inflammasome; (iii) the role of galectins; and (iv) the activation of purinergic 2X7 receptor (P2X7R). Moreover, this review proposes the selective inhibition of galectins and the P2X7R as a potential pharmacological approach to prevent the progression of DR. Diabetes is known as a chronic disease with multiple complications . One of Historically, DR has been considered as a microangiopathy, and is characterized by increased retinal vascular permeability and endothelial damage ,6,7. In Accordingly, DR can be clinically classified as either non-proliferative diabetic retinopathy (NPDR) or proliferative diabetic retinopathy (PDR) , which aWhile the glycemic, lipidic and blood pressure controls are needed for DR primary prevention, the current surgical and pharmacological options for DR management include pan-retinal laser photocoagulation in severe NPDR and PDR patients, focal retinal laser photocoagulation in patients with diabetic DME, anti-VEGF intravitreal injections in PDR and DME patients, and steroid intravitreal injections for non-responder DR patients or naïve patients when appropriate . In partCurrently, intraretinal inflammation and neurodegeneration have emerged as key processes in DR progression ,28. IndeIn this review, we focus on the importance of endoplasmic reticulum stress (ERS) and the consequent activation of the nucleotide-binding oligomerization domain-, leucine-rich repeat- and pyrin domain-containing 3 (NLRP3) inflammasome during retinal neuroinflammation. In particular, ERS modulation by specific proteins binding beta-galactoside residues on glycated proteins known as galectins is detailed. The galectin family consists of 15 identified members, all of which contain conserved carbohydrate-recognition domains (CRDs) of about 130 amino acids . The proMoreover, the importance of purinergic 2X7 receptor (P2X7R) in NLRP3 activation underlying neuroinflammation is analyzed, along with the options for their selective inhibition in several clinical settings. Specifically, the state of the art on galectins and P2X7R inhibitors in DR is described, along with their potential use as pharmacological tools to prevent DR progression.α) and monocyte chemoattractant protein-1 (MCP-1), have been found in serum and in ocular samples (vitreous and aqueous humor) of DR patients. The accumulation of these cytokines is mainly mediated by cells from the macroglia and microglia [The “retinal neurovascular unit” includes specific retinal glia elements, such as microglial cells and macroglia (Müller cells and astrocytes) ,33,34. Ticroglia .α and interferon gamma (IFN-γ), known inducers of inflammation, apoptosis and neurotoxicity [Microglial cells represent the first defense line in neural retina ,42,43. Stoxicity ,56,57. Mtoxicity ,58.Retinal microglia polarization directly influences Müller cells and macroglia cells, spanning the entire retinal width and connecting neurons and vascular components ,60. In hLastly, astrocytes are the most abundant central nervous system (CNS) macroglial cell type, forming the inner retinal BRB . These aIn the diabetic retina, hyperglycemia leads to elevated ROS production by various mechanisms, including the reduction of antioxidant enzymes  and the During DR, the hyperglycemia-induced ER stress (ERS) affects all the components of the “neurovascular unit” and leads to the apoptosis of both vascular and neuronal cells ,73. In pERS-induced microglial activation occurs during the different DR stages ,47. IndeConversely, ERS-induced Müller cell activation seems to precede the DR vascular alterations , with NPROS-induced ER stress leads to the activation of the NLRP3 inflammasome, a multiproteic complex found as monomer when inactive ,89,90,91The NLRP3 complex consists of a sensor component (NLRP3 protein), an effector component (caspase-1) and, in some cases, an adapter protein (known as apoptosis-associated speck-like protein—ASC) linking the sensor and the effector ,95,96. TThe pyrene domain connects to the NLRP3 pyrene domain through an oligomerization process; in addition, the adaptor protein ASC brings pro-caspase 1 monomers near to each other through the CARD domain by inducing a proximity-mediated caspase-1 autoactivation . Active Similarly, NLRP3 is pivotal in DR progression ,106,107.NLRP3 activation is shown by retinal epithelial ,110,111 Overall, galectin dysregulation has been linked to different pathological conditions, such as fibrosis, heart disease, cancer, and diabetes . In thisGalectins, induced by AGEs and considered as receptors for AGEs (RAGE), have emerged as ERS regulators. For example, the elevation of Galectin 9  has been linked to inflammatory processes in both type 1 and type 2 diabetes ,128. HowIn addition, Gal-1 and Gal-3 have also emerged as ERS regulators . SpecifiGal-3 is localized at the ER-mitochondria interface and regulates the UPR . It is iThis could be firstly due to the high affinity showed by galectins for β-galactosides ,143, whiP2X7R is a trimeric ion channel that belongs to the P2X family of ionotropic receptors preferably permeable to sodium, potassium and calcium, which are exclusively triggered by extracellular adenosine triphosphate (ATP) . P2X recP2X7R presents five domains: one extracellular domain; two transmembrane domains (classified as transmembrane 1 and 2); and two intracellular domains (N- and C-terminus), which form homotrimeric receptors after their activation ,157. ItsP2X7R is highly expressed in different cell types and tissues, such as retinal neural cells and the retinal vasculature , but alsP2X7R has a key role in linking inflammation with purinergic signaling: this receptor and the NLRP3 inflammasome interact at distinct cytoplasmic sites, where changes in P2X7R-dependent ion concentrations  occur ,161,162,Indeed, P2X7R activation in glial cells overall results in the release of the pro-inflammatory cytokines, thereby triggering or potentiating neuroinflammation . This coSeveral studies evidenced that P2X7R expression is upregulated in the activated microglia of AD patients and is concentrated in amyloid plaques ,171. SimIt is well known that galectins and P2X7R are two key mediators in DR pathology and progression ,188,189.In this regard, different compounds inhibiting galectins have been evaluated in clinical trials. In particular, although Gal-1 inhibitor OTX0008 has been tested in patients with advanced solid tumors , Gal-3 iGal-3 inhibition by GB0139 and TD139 compounds is under evaluation in idiopathic pulmonary fibrosis (IPF) ,198,199,Regarding P2X7R, different compounds have been tested on healthy volunteers to assess their safety and tolerability, bioavailability, pharmacokinetics, and pharmacodynamics as P2X7R antagonists. These include GSK1482160 , AZD9056To date, none of these inhibitors has been considered for DR patients. However, several pre-clinical studies evidenced the potential role of galectin and P2X7R inhibition strategies in modulating diabetic retinal damage ,183,184.Gal-1 and Gal-3 have been recently associated with the insurgence and progression of DR pathology .In particular, Gal-1 levels were increased in the vitreous fluid and epiretinal fibrovascular membrane of PDR patients compared with non-diabetic controls . This waGal-1 also seems to be involved in the regulation of the RPE that forms the BRB. Indeed, ARPE-19 cells exposed to high glucose exhibited high Gal-1 levels, which was associated with epithelial fibrosis and epithelial-mesenchymal transition . Both prAlthough Gal-1 has been associated with neuronal alterations in a mouse OIR model, and particularly with neuroglial injuries at post-natal day 26 , retinalRecent evidence suggests a role of the P2X7 receptor in controlling the BRB function and integrity ,211. OncA specific role for the P2X7R-NLRP3 inflammasome pathway in retinal endothelial inflammation and pericyte loss during the NPDR stage has been also described ,183,184 Therefore, a P2X7R inhibition strategy could represent a useful therapeutic tool to manage the early phase of DR. In this regard, the P2X7R selective inhibitor A74003 reduced the apoptosis of mice retinal endothelial cells (mRECs) stimulated with high glucose and lipopolysaccharide (LPS) . FurtherDue to the role of galectins in ERS modulation and the role of P2X7R in NLRP3 inflammasome activation, both these mediators could represent two new potential targets, whose specific inhibition could help counteract the inflammatory process underlying DR progression, although evidence for a specific molecular relation and interlink between them is not available yet .


By influencing transcription, transportation and translation, m5C modification of mRNA, tRNA, rRNA, lncRNA and other RNAs has been proven to affect gene expression and metabolism and is associated with a wide range of diseases, including malignant cancers. RNA m5C modifications also substantially impact the tumor microenvironment (TME) by targeting different groups of immune cells, including B cells, T cells, macrophages, granulocytes, NK cells, dendritic cells and mast cells. Alterations in immune cell expression, infiltration and activation are highly linked to tumor malignancy and patient prognosis. This review provides a novel and holistic examination of m5C-mediated cancer development by examining the exact mechanisms underlying the oncogenicity of m5C RNA modification and summarizing the biological effects of m5C RNA modification on tumor cells as well as immune cells. Understanding methylation-related tumorigenesis can provide useful insights for the diagnosis as well as the treatment of cancer.RNA modification plays an important role in epigenetics at the posttranscriptional level, and 5-methylcytosine (m Commonlrylation –5. The drylation –11. For mination , m7G modbacteria , and m6Anescence .6A modification is the most widely and comprehensively investigated because of its abundance in eukaryotic cells  cells  cells . Howeverogressed , showingan cells , 21. RNAquencing . Immunopquencing . Other dquencing .5C in the modulation of gene expression, metabolism and diseases , generally influences tRNA methylation . The m5CAVal-AAC , 40, andAVal-AAC . StudiesAVal-AAC . In addiAVal-AAC .5C levels  , catalyzes rRNA m5C modifications, thus affecting biological processes including ribosome biogenesis  of SOX9 (SRY-box transcription factor 9) mRNA is necessary for adaptation to higher temperatures  , 34, 51,cer (GC) , 53, hepcer (GC) , 54, hypcer (GC) , prostatcer (GC) , cervicacer (GC) , nasophacer (GC)  and uveacer (GC) . NSUN2 anslation . In addin of p21 . Note thby NSUN2 . Other mogenesis , cell progenesis , healthsogenesis  and HIV-ogenesis . NSUN3, ogenesis , 63, 64,ogenesis  and multogenesis . Mechanidon loop , 117. Cuial mRNA . NSUN3-aial mRNA  and M2 mial mRNA . NSUN4 fial mRNA . In addiial mRNA  and mRNAial mRNA  methyltreratures  and choneratures . NSUN4 aeratures , 72 and eratures . NSUN5 p28S rRNA , 74. Ove28S rRNA  and colo28S rRNA , while N28S rRNA . In pati28S rRNA  and Will28S rRNA , NSUN5 i tRNAThr , 78, but tRNAThr , 121. mR tRNAThr . The rol tRNAThr , pancrea tRNAThr , testis  tRNAThr , thyroid tRNAThr  and ovar tRNAThr  but is a tRNAThr , 81. One tRNAThr , but in  tRNAThr . In addi tRNAThr , 80, and tRNAThr . NSUN7 m tRNAThr .5C sites, include ALYREF and YBX1. ALYREF is reported to be an important oncogenic factor and is associated with poor prognosis in patients with various types of cancer, including HCC (5C modification of YAP (Yes-Associated Protein) mRNA in the 3’-UTR, thus increasing the stability of YAP mRNA and causing enhanced exosome secretion, tumor malignancy and drug resistance  and alpha-ketoglutarate-dependent m5C dioxygenases that convert 5-methylcytosine (m5C) to 5-hydroxymethylcytosine (hm5C)  . These ee (hm5C) . OverallA damage . TET2 man in RNA , 109, bun in RNA  but downn in RNA , 111, ovn in RNA  and prosn in RNA  patientsn in RNA . TET3-merognosis .5C RNA modifications to either hm5C or f5C (5-formylcytidine) RNA modifications  oncoprotein, a potential enhancer of MYC accumulation  rate  (Kip2 (a type of cyclin-dependent kinase (CDK) inhibitor) -2/3, which directly interact with NSUN2 to stabilize and mediate its nuclear transport, promote the development of GC  and OS (The role of mOS) rate . ModificOS) rate , 34, 51 OS) rate , 139 havhibitor) . After m) and OS .5C-mediated cell proliferation is considered one of causes of bladder cell malignancy. Overexpressed ALYREF in bladder cancer cells interacts with the 3’-UTR of PKM2 (pyruvate kinase M2) mRNA, causing its stabilization and enhanced PKM2-associated glycolysis  (mycolysis . ALYREF ycolysis . Anotherotein 1) . Moreoveotein 1) .5C modulators (5C-related lncRNAs (5C modification of androgen receptor (AR) mRNA by NSUN2 is recognized by YBX1, increasing AR mRNA stability and translation -insensitive chromatin structure during leukemia, which causes drug resistance , a conserved RNA-binding protein that interacts with the lineage-determining transcription factors GATA binding protein 1 (GATA1), recombinant spleen focus forming virus proviral integration 1/purine rich box-1 (SPI1/PU.1) and cyclin-dependent kinase 9 (CDK9)/positive transcription elongation factor b (P-TEFb) to recruit RNA-polymerase-II to RNA precursors, forming chromatin structures that are sensitive to 5-AZA  mRNA, which upregulates its expression level , which is recognized and stabilized by YBX1, is associated with advanced TNM stage and poor prognosis  , B cells and plasma cells, NK cells, NKT cells, macrophages, granulocytes and mast cells, manifest alterations in cell expression, infiltration and recruitment rate, which is concluded by several prognostic models constructed based on m lncRNAs , m5C-reged genes , 148 or s (DEGs) , especias (DEGs) Figure 4Subgroups of T lymphocytes (mainly CD4+ and CD8+ T cells) are distinguished in T cells by surface markers using flow cytometry . Upon le5C RNA modification of T cells in the context of cancer. For instance, the abundance of CD4+ T cells is higher in patients with soft tissue sarcoma (STS) (Recent studies have mainly focused on the mma (STS) , ccRCC (ma (STS)  and HCC ma (STS)  but the ma (STS) . Tregs ama (STS)  and HCC ma (STS)  patientsma (STS)  and bladma (STS)  patientsma (STS)  and lungma (STS)  patientsma (STS)  patients5C-related regulators include NSUN1  patients, while the number of m5C-containing RNAs increased. In addition, m5C sites were mainly distributed in mRNA translation initiation sites, and hypermethylated m5C and/or upregulated genes in SLE were enriched immune-related and inflammatory pathways, including immune system signaling pathway, cytokine signaling pathway, and interferon signaling pathway -dependent manner.For CD4+ T cells, associated mde NSUN1 , NSUN2  in the bone marrow. Naïve B cells, once properly activated, mature into plasma cells, the antibody-secreting form of B cells, following an intrinsic developmental process . Recent rognosis . Activatrognosis  and lungrognosis  patients the TME , suggestand TET3 , 38, 80.and TET3 .5C RNA modifications in macrophages are in the context of cancer development, especially macrophage infiltration in the TME. The risk scores based on m5C-related genes of patients with four types of cancer  were positively correlated with infiltration of resting macrophages (M0), M1 or M2 macrophages  cells constitute a first line of innate immunity against tumors due to their capabilities of killing aberrant cells . Regardiin HNSCC , glioma in HNSCC  and lungin HNSCC  patients(5C gene .5C in DCs (6A/m5C/m1A-associated lncRNAs showed that the high-risk group has a significantly higher concentration of activated DCs within the TME (5C writer DNMT3A was found to be involved in regulating DCs (Dendritic cells (DCs) are generally regarded as the most potent antigen-presenting cells, thus modulating both immunity and tolerance . In lungC in DCs . In cont the TME , showingting DCs .6A/m1A/m5C-regulated genes in HCC patients indicated the links between poor prognosis and high infiltration of resting mast cells (Mast cells are tissue-resident cells that function in inflammatory responses and tissue homeostasis . These cst cells .5C RNA modification of immune cells in the context of tumorigenesis largely remain to be further clarified. A better understanding of the mechanisms by which alterations in cell expression, infiltration and activation are regulated by methylation can be extremely helpful for the development of novel methods for tumor diagnosis as well as treatment.In summary, the roles of m5C RNA modifications in cancer by discussing m5C RNA-related genes and alterations in gene expression and immune cell infiltration. The modifications involve mainly mRNAs but also other noncoding RNAs such as tRNA, rRNA, and lncRNA, and are regulated by RNA m5C writers, readers and erasers, leading to changes in RNA processes, including transcription, transportation, translation and metabolism. Risk models made for prognosis prediction are based on m5C regulators as well as genes with m5C modification, which indicates the relationship between prognosis and alterations in immune cell infiltration in the TME.In this review, we provide a detailed review concerning the roles of m5C RNA modifications remain to be elucidated. For example, the mechanisms by which m5C writers, readers and erasers function have only been investigated in a limited manner. What are the target genes of RNA m5C modifications? How are these genes linked to cell signal transduction and tumor malignancy? Additionally, we now know that RNA m5C modifications are related to immune cell infiltration within the TME, but scarce and controversial study results offer no comprehensive and fully convincing conclusions. What alterations do RNA m5C modifications cause indifferent groups of immune cells? How are these alterations linked to the progression of cancer? Is immune cell infiltration protective or destructive for patients with malignant tumors? These questions might provide deeper insights into the diagnosis and treatment of different cancer types.However, numerous questions regarding oncogenic m5C modifications in cancer patients have massively progressed within the last five years, providing new analytical results from clinical samples. It should be pointed out that most of the studies based on clinical samples were only limited at the laboratory level, and no m5C-related clinical trials against cancer has been performed so far. However, as detection methods for RNA m5C sites continue to improve, cancerous RNA m5C modifications will most likely remain a popular scientific topic in the years to come and, hopefully, instill new hope for millions of patients fighting cancer.Nevertheless, studies on RNA mXG and XM have equal contributions to this study. XG and HZ designed the whole study. XG and XM drafted the manuscript. CC, JG, JW, and SW made the relevant edits to the manuscript. XG and XM revised the manuscript. All authors contributed to the article and approved the submitted version.


CASZ1/Castor (Castor zinc finger 1), initially identified in Drosophila, plays a critical role in neural, cardiac, and cardiovascular development, exerting a complex, multifaceted influence on cell fate and tissue morphogenesis. During neurogenesis, CASZ1 exhibits dynamic expression from early embryonic development to the perinatal period, constituting a key regulator in this process. Additionally, CASZ1 controls the transition between neurogenesis and gliomagenesis. During human cardiovascular system development, CASZ1 is essential for cardiomyocyte differentiation, cardiac morphogenesis, and vascular morphology homeostasis and formation. The deletion or inactivation of CASZ1 mutations can lead to human developmental diseases or tumors, including congenital heart disease, cardiovascular disease, and neuroblastoma. CASZ1 can be used as a biomarker for disease prevention and diagnosis as well as a prognostic indicator for cancer. This review explores the unique functions of CASZ1 in tissue morphogenesis and associated diseases, offering new insights for elucidating the molecular mechanisms underlying diseases and identifying potential therapeutic targets for disease prevention and treatment.The zinc finger transcription factor gene,  CASZ1/Castor (Castor zinc finger 1) gene, initially discovered in Drosophila, encodes a zinc finger transcription factor (TF) that regulates neural fate [CASZ1 gene across species from Drosophila to humans [Drosophila, Casz1 functions as a neuronal fate-determining gene, controlling nervous system development [Xenopus laevis and humans, CASZ1 plays a critical role in heart development, cardiomyocyte differentiation, vascular assembly, and lumen morphogenesis [The ral fate . Highly o humans –5 are sto humans . In Droselopment , 8, and elopment –9. In Xeogenesis –12.CASZ1 gene in tumor samples from patients with a poor prognosis of neuroblastoma (NB) [CASZ1 gene locus in a patient with cervical cancer, which disrupted CASZ1 gene expression [In addition to its crucial role in both neural and cardiac development, the involvement of CASZ1 in various pathological conditions, including cancer, is being increasingly recognized . In cancoma (NB) . A clinipression . Furtherpression . Similarpression –19. Howepression , 21. Thepression –25, and pression . Additiopression .This review provides a comprehensive summary of the role of CASZ1 in both development and disease, offering novel insights into the molecular mechanisms underlying various diseases. Moreover, it highlights the potential of CASZ1 as a prognostic indicator and therapeutic target for diagnosing and treating these diseases, thereby contributing to the advancement of medical research.Castor, which is required for the development of the central nervous system in Drosophila embryos [In the 1990s, an enhancer detection screen identified a new gene,  embryos –31.Drosophila at a developmental stage comparable to later stages of human embryonic neurogenesis [I 8.4), containing five TFIIIA class C2H2 ZnF motifs. In addition, the hCasz5 isoform has two nuclear localization signals (NLS) located at 23–29 and 232–248, a nuclear export signal (NES) located at 168–186 [The expression of Castor in ogenesis , led to ogenesis . The CAS 168–186 , a nucle 168–186 , a serinI 6.64). In addition to the information contained in CASZ1b, CASZ1a has six additional TFIIIA class C2H2 ZnF motifs. Furthermore, CASZ1a has one extra NLS located in 1401–1418, one Glu Asp-rich region located in 1672–1729, and two Ala-rich regions located in 1635–1670 and 1726–1756 [The cDNA sequence of CASZ1a is 7946 bp in length and codes for a protein with 1759 amino acids , castor, and grainyhead, during development. These temporal recognition factor cascades act as a timing mechanism, coordinating the output of numerous neuroblast lineages within the CNS of Drosophila [Neural progenitor cells undergo identity transitions, generating diverse neurons and glial cells in a precise manner during development. Throughout neurogenesis, the majority of neural stem and progenitor cells alter their output, initially producing neurons followed by glial cells . The occosophila .Casz1 is expressed in the dorsal root ganglia and spinal cord of mice during neurogenesis , 42, 43,Drosophila neuroblasts, Casz1 regulates temporal progression through a conserved transcriptional cascade. Additionally, Casz1 plays a crucial role in regulating progenitor cell potential and controlling the generation of mid/late-born neurons in the mouse retina. Predominantly observed in middle and late RPCs, conditional Casz1 loss enhances early retinal neuron generation at the expense of late fates [In mouse retinal progenitors, similar to te fates . Furtherte fates . In addite fates , relyingte fates .Early heart development is characterized by hyperplastic growth, wherein cardiac cells undergo mitogen-dependent activation during the G1 phase of the cell cycle . During Xenopus embryos, CASZ1 deficiency leads to the failure of ventral midline progenitors to differentiate into cardiomyocytes, resulting in abnormal cardiac morphogenesis and death [TNNI2, TNNT1, and CKM), contractile fiber genes (ACTA1), and genes encoding arrhythmia-associated ion channels (ABCC9 and CACNA1D) occurs in Casz1-null mouse embryonic hearts, leading to myocardial hypoplasia and congenital ventricular septal defects (VSDs) [In humans, CASZ1 exhibits high expression levels in various organs, including the heart, lungs, skeletal muscle, pancreas, testes, small intestine, and stomach. Notably, the highest relative expression level of CASZ1 is observed in the heart  where itnd death . Furthers (VSDs) . Additios (VSDs) , 53.During embryonic development, endothelial cells (ECs) serve as the foundation for functional vasculature formation. The initial phases involve vasculogenesis, where mesodermal cells differentiate into EC progenitors, which subsequently proliferate and migrate, assembling into vascular cords at specific embryo sites. Then, the umbilical cords undergo tubulogenesis or lumen formation and mature through angiogenesis, which involves vessel budding, branching, and remodeling. Finally, pericytes and smooth muscle cells provide structural support by surrounding and stabilizing the vessels , 55.Casz1 locus and hypertension [EGFL7) gene, activated by Castor (CASZ1), is crucial for vessel assembly and luminal morphogenesis [Xenopus and human epithelial cells, CASZ1 directly regulates Egfl7, thereby promoting RhoA-mediated vascular development in vertebrates. CASZ1 deficiency not only impedes the branching and luminal vasculature development of Xenopus embryos, but also induces marked changes in human EC adhesion, morphology, and sprouting [Two genome-wide association studies (GWASs) have revealed a genetic association between the human rtension , 57, sugogenesis , 58, 59.prouting , 59. TheNB is a prevalent solid tumor that affects the pediatric population, predominantly targeting the brain and accounting for ~ 15% of all pediatric tumor-related deaths, and has a poor prognosis . The CASThe loss of CASZ1 in NB tumors correlates with poor prognosis. An examination of CASZ1 protein expression in a primary NB tissue microarray revealed that tumors from patients with NB and favorable prognoses exhibited higher levels of nuclear CASZ1 protein expression. Conversely, tumors from patients with unfavorable prognosis exhibited cytoplasmic-restricted CASZ1 staining or low nuclear CASZ1 staining . FurtherCASZ1, CLU, NGFR, and RUNX3, are direct targets of EZH2- and H3K27me3-mediated gene silencing [Histone 3 trimethylation at lysine 27 (H3K27me3), a histone modification associated with gene silencing, is catalyzed by the methyltransferase EZH2, an enzymatically active component of the PRC2 . EZH2 isilencing . Knockinilencing .CASZ1 gene, determined via bisulfite sequencing, appears insufficient to explain the low CASZ1 expression level [Although demethylating agents can induce CASZ1 expression, the methylation status of the 5ʹ and 3ʹ CpG-rich regions of the on level , 63. HDAon level . This suCyclin D1 and Chk1, leading to an undifferentiated NB phenotype. The increased expression of cyclin D1 and enhanced cyclin D-dependent kinase activity contribute to pRb hyperphosphorylation and E2F release, thereby activating E2F-dependent gene transcription and promoting cell cycle progression in the G1-S phase [The dysregulation of the cell cycle mechanism marks cancer progression , 68. Pre-S phase , 71. CAS-S phase . Further-S phase .CASZ1 exhibits distinct expression patterns across various solid tumors, manifesting dual biological functions contingent on tumor type. For instance, Wang et al. reported reduced CASZ1 expression in hepatocellular carcinoma tissues, which hindered abnormal tumor cell proliferation by modulating the MAPK/ERK signaling pathway alongside MMP2 and MMP9 expression in vitro . Similarp75NTR [Conversely, contrary to the that reported by several previous studies, high CASZ1 expression has been observed in glioma tissues, where it functions as an oncogene by regulating the transcription of its target gene p75NTR . Additiop75NTR . Furtherp75NTR , 77. Recp75NTR . The comCASZ1 gene is located on chromosome 1p36, and 1p36 deletion is the most prevalent telomere deletion. This deletion is causally related to congenital cardiovascular malformations and cardiomyopathy, which are the most common phenotypes of 1p36 deletion syndrome [CASZ1 gene, c.2443_2459delGTGGGCACCCCCAGCCT , was identified in a patient with DCM and left ventricular noncompaction cardiomyopathy (LVNC), highlighting the role of CASZ1 as a pathogenic gene for human LVNC [CASZ1 gene [CASZ1 loss-of-function mutations with human cardiac disease susceptibility holds potential implications for the personalized prevention and treatment of cardiac diseases.In humans, the syndrome . Specifisyndrome  and dilasyndrome , respectSZ1 gene . Associa+ channel-α and serum/glucocorticoid-regulated kinase 1 expression, which ultimately lowers BP levels [An epigenome-wide association study, leveraging genome-wide transcriptome data, has revealed that CASZ1 methylation may serve as a regulatory element linked to mortality in patients with cardiovascular disease . GWASs hP levels . NeverthP levels . In a reP levels . These fP levels , 84, 85,P levels  and rs12P levels , are ass+ T cells lacking Casz1 to experimental autoimmune encephalomyelitis. Furthermore, the loss of Casz1 results in the severe impairment of Th17 and Treg responses during mucosal Candida infection, rendering mice deficient in Casz1 less capable of clearing secondary infections [Osteoarthritis is an age-related condition characterized by articular cartilage degeneration and joint inflammation that has garnered research attention in recent years. Notably, CircANKRD36 has emerged a key player in preventing chondrocyte apoptosis and counteracting inflammatory responses induced by IL-1β treatment. This protective effect is attributed to the ability CircANKRD36 to target miR-599, leading to the upregulation of Casz1 expression . Casz1, fections .Genome-wide DNA methylation analysis of whole blood from monozygotic twins has revealed that CASZ1 DNA methylation variants are negatively associated with fasting plasma glucose (FPG) levels . AbnormaSince its discovery in the early 1990s, the TF CASZ1 has been found to play a crucial role in neural, cardiac, and cardiovascular development. Its utility extends to elucidating the cellular and molecular mechanisms underlying the diversification and subsequent differentiation of neural stem/progenitor cells and cardiomyocytes. In diseases marked by cardiac developmental anomalies, such as DCM, VSD, and LVNC, the loss of CASZ1 expression or genetic mutations can contribute to aberrant cardiac morphogenesis. Notably, the methylation of CASZ1 serves as a regulatory element associated with mortality in patients with cardiovascular disease, and it is reported hypomethylated in Chinese patients with hypertensive cerebral infarction. Furthermore, CASZ1 holds clinical importance in the context of cartilage degeneration and autoimmune inflammation.CASZ1 plays dual roles in cancer, functioning both as a tumor suppressor and promoter, thereby influencing cancer cell metastasis. The loss of CASZ1 expression or its mutational inactivation is associated with diseases or cancers linked to developmental defects. Moreover, the epigenetic silencing of CASZ1 is linked to poor prognosis in NB. Conversely, CASZ1 is downregulated in hepatocellular carcinoma, colorectal cancer, esophageal cancer, lung adenocarcinoma, and clear cell renal cell carcinoma, and its decreased expression is linked to patient prognosis, with this diminished expression serving as a potential prognostic indicator. Conversely, CASZ1 is highly expressed in glioma and EOC, suggesting a tissue- and tumor-specific expression pattern. The exploration of the potential of CASZ1 as a cancer biomarker for diagnosis and prognosis involves the evaluation of its prognostic value in tumor cells.In conclusion, as a tumor suppressor gene of neuroblastoma, CASZ1 is involved in the progression of NB through cell cycle regulation and is associated with poor prognosis. Low expression of CASZ1 in hepatocellular carcinoma, colorectal cancer, esophageal cancer, lung adenocarcinoma and clear cell renal cell carcinoma is associated with poor prognosis, while high expression of CASZ1 in glioma, EOC and lung cancer promotes cancer progression. These studies suggest that CASZ1 can be used as a diagnostic and prognostic indicator or as a therapeutic target for cancer. In addition, CASZ1 loss-of-function mutations or mutations at SNPs loci are associated with susceptibility to human diseases, which is of great significance for personalized prevention and treatment of diseases. Therefore, it will be intriguing to investigate the regulatory mechanisms of CASZ1 itself and to identify additional transcriptional targets that could drive development of innovative therapeutic strategies for disease.


Diabetic Kidney Disease (DKD) is a common microvascular complication of diabetes mellitus. Multi-center, randomized controlled trials have shown that Qidan Dihuang Granule (QDDHG) reduces the levels of albuminuria of DKD. However, the specific mechanisms of QDDHG on DKD are not clarified. Thus, this study utilized network pharmacology, UHPLC-MS/MS (Ultra-High Performance Liquid Chromatography - Mass Spectrometry) and animal experiments to reveal the mechanisms of QDDHG on DKD.In vivo experiment with db/db mice were used to verify the targets and pathway predicted by network pharmacology.Screening and retrieving active ingredients and corresponding targets of QDDHG on DKD through the TCMSP, ETCM, Disgenet, GeneCards, Omim and DrugBank databases. The PPI were performed with BioGrid, STRING, OmniPath, InWeb-IM. AutoDock Vina molecular docking module to estimate the validation from the compounds and target proteins. Free energy to estimate the binding affinity for identified compounds and target proteins. The ingredients of QDDHG were analyzed utilizing UHPLC-MS/MS. In vivo experiment verified the effects of QDDHG on protecting the renal function mainly through inhibited the expression of EGFR, STAT3 and pERK in the db/db mice.The results demonstrated that QDDHG has 18 active compounds and 13 target proteins of QDDHG exerted a crucial role in treatment of DKD. QDDHG affect the multiple biological processes included cellular response to lipid, response to oxidative stress, and various pathways, such as AGE-RAGE, PI3K-Akt, MAPK, TNF, EGFR, STAT3. The results of UHPLC-MS/MS showed that six ingredients predicted by network pharmacology were also verified in experiment.  •This work is the first to reveal the mechanism underlying the effect of QDDHG on DKD based on RCT results.•QDDHG was studied using network analysis, UHPLC-MS/MS, and animal experiments.•The mechanism of QDDHG involved inhibition of the expression of EGFR, STAT3 and pERK in DKD mice.•This study demonstrates that TCM exerts its the effects through multiple components and pathways. AGEadvanced glycosylation endAlbUrine albuminBCbetweenness centralityBUNblood urea nitrogenCCcloseness centralityCOX-2c oxidase IImitochondrially encoded cytochrome CTGFconnective tissue growth factorDKDdiabetic kidney diseaseDLdrug-likenessDNDiabetic NephropathyELISAEnzyme-linked immunosorbent assayERK1/2Extracellular signal-regulated protein kinase 1/2ESRDend-stage renal diseaseETCMThe Encyclopedia of Traditional Chinese MedicineGOGene OntologyHEhematoxylin-eosinHIF-1Hypoxia-inducible factor 1HPLCHigh-performance liquid chromatographyIKBaNFKB inhibitor alphaIL-17Interleukin 17IL-1βInterleukin 1 betaIL22Interleukin 22IL-6Interleukin 6KEGGKyoto Encyclopedia of Genes and GenomesMAPKMitogen-activated protein kinaseMassonMasson's trichromeMSMass SpectrometryNCNormal ControlNF-kappa BNuclear factor-kappa BOBOral bioavailabilityPASPeriodic acid-SchiffPDBProtein Data BankpERK1/2Phospho-extracellular signal-regulated protein kinase 1/2PI3KPhosphoinositide 3-kinasesPPARPeroxisome proliferator-activated receptorPPIprotein-protein interactionQDDHGQidan Dihuang GranulesRAGEadvanced glycosylation end-product specific receptorRap1Ras-related protein 1ROSreactive oxygen speciesScrSerum creatinineSDStandard DeviationSIRT1sirtuin 1STRINGSearch Tool for the Retrieval of Interacting Genes/ProteinsTCMtraditional Chinese medicineTCMNPASTCM Network Pharmacology Analysis SystemTCMSPTraditional Chinese Medicine Systems Pharmacology Database and Analysis PlatformTEMTransmission Electron MicroscopeTGF-β1transforming growth factor beta 1TNFTumor necrosis factorUHPLC-MS/MSUltra-High Performance Liquid Chromatography - Mass SpectrometryVEGFvascular endothelial growth factor1Astragalus mongholicus Bunge), Danshen , Dihuang (Rehmannia glutinosa (Gaertn.) DC.), Shanyao (Dioscorea oppositifolia L.), and Gancao (Glycyrrhiza echinata L.), were proposed to treat DKD. Multi-center, randomized controlled trials (RCT) have demonstrated that QDDHG combined with Angiotensin Receptor Blockers administration can significantly lower the levels of albuminuria, improve the TCM syndrome score, and delay the disease process [Diabetic Kidney Disease (DKD) is one of common microvascular complication of diabetes mellitus  and has  process .in vivo and in vitro studies. Therefore, this study utilized network pharmacology methods to retrieve active compounds and corresponding genes of QDDHG, and targets of DKD, and reveal the potential therapeutic mechanisms. Furthermore, molecular docking was applied to verify putative targets. Finally, identification of the chemical components of QDDHG was performed using UHPLC-MS/MS and animal experiments were used to verify the targets and pathway predicted by network pharmacology methods.However, the specific mechanisms of QDDHG on DKD are not clarified. Unlike synthesized compounds or single herbal extracts, QDDHG has multiple components, and maybe performs systemic function via multiple pathways in treatment of DKD. Traditional studies’ method concentrating on specific mechanisms may have shortcoming for the assessment of mechanisms owing to QDDHG with multiple components and targets. As a systemic research method, network pharmacology could identify the potential mechanisms and target proteins of TCM for subsequent verification 22.12.1.1https://tcmsp-e.com/) and ETCM (http://www.tcmip.cn/ETCM/) database were accessed, and the active ingredients of QDDHG were retrieved. Oral bioavailability (OB) ≥ 30 % and Drug-Likeness (DL) ≥ 0.18 or Drug-likeness Grading of “good” were set as the thresholds for identifying active ingredients [https://www.uniprot.org/), with the screening conditions “homo sapiens” and “reviewed”. For ingredients not included in the TCMSP and ETCM, the Swiss Target Prediction database (http://swisstargetprediction.ch/) was searched to identify the corresponding genes.The TCMSP , Omim (http://www.omim.org/), GeneCards (https://www.genecards.org/), and DrugBank (https://go.drugbank.com/) databases with the terms “DN” (diabetic nephropathy) or “DKD” (diabetic kidney disease).Screening targets of DKD was carried out by searching the Disgenet (2.1.3https://metascape.org/gp/). Then, Cytoscape 3.6.1 was used to construct a PPI network diagram, and topology analysis was carried out with the “Network Analyzer” plug-in network structure. “CC”, “BC”, and “Degree” were calculated to discover core targets [A Venny diagram was constructed using the common genes of QDDHG and DKD. The PPI enrichment of intersecting targets were performed with the Metascape databases .GO and KEGG analyses were constructed using Metascape. The Cutoff values were set as follows: 2.1.5The experimental data, including the herb, active compounds, targets, and KEGG pathways were input to Cytoscape 3.6.1 to establish an herb-compounds-targets-pathways network. Network Analyzer was used to filter the core chemical components with BC ≥ median, CC ≥ median, and degree≥ 2 times the median degree.2.2https://www.rcsb.org/), and the default Vina compound ligand preparation program was used [The built-in AutoDock Vina version 1.1.2 docking module of the TCMNPAS online analysis platform was used for molecular docking verification . The smawas used . The pro2.32.3.1The QDDHG formula used in this study was of Chinese granule herbal extracts. The grains were purchased from Tianjiang Pharmaceutical Co., Ltd. . The herbal composition is shown in 2.3.2db (db/db) mice and their non-diabetic Lepr db/m(db/m) male mice were purchased from the Changzhou Cavens Laboratory Animal Co. Ltd. . All mice were raised in SPF environment, after an acclimation period of 14 days, 8-week-old mice randomly assigned to four groups. The mice of the QDDHG1 group were fed a chow diet containing 1 % QDDHG (containing 10 g of QDDHG per 1000 g of the chow diet). The mice of the QDDHG2 group were fed a chow diet containing 2 % QDDHG (containing 20 g of QDDHG per 1000 g of the chow diet). The NC  group consisted of the db/m mice, and the DKD group consisted of the db/db mice, which were fed a drug-free chow diet. Fasting body weight (FBW) and caudal vein fasting blood glucose (FBG) were measured at regular intervals over a 12-h fasting period. After 16 weeks of treatment, the mice were sacrificed, and the blood samples and renal tissue were collected.The use and care of animals and experimental protocols was processed in accordance with European Community guidelines of experimental animals, and were approved by the Ethics Committee of the University of Chinese Academy of Science-Shenzhen Hospital (Ethics number: LL-KT-2021231). Male Lepr 2.3.3Blood was centrifuged at 5000 rotation per minute for 10 min, and serum samples isolated. Serum creatinine (Scr) and blood urea nitrogen (BUN) were tested using a Hitachi-7600 Automatic Biochemical Analyzer  using the recommended procedures. 24-hour urine albumin (Alb) levels were determined using a commercially available ELISA kit .2.3.43 and transferred into an EP tube containing fresh TEM fixative. The tissue was then fixed at 4 °C for preservation and sent to Wuhan Servicebio Technology Co. Ltd. (China).Renal tissue was embedded in paraffin after fixation in 4 % (w/v) paraformaldehyde. Tissue sections were stained with hematoxylin-Eosin (HE), Periodic Acid-Schiff (PAS) and Masson's trichrome (Masson). Glycogen intensity and collagen fiber intensity in the glomerular mesangial region were calculated on PAS-stained and Masson-stained tissue, respectively, using NIS-Elements Viewer (NIKON). The PAS-positive material was identified by its purple-red color, while the nucleus was stained blue. A NIS-Elements Viewer was utilized to measure the area of the PAS-positive material and the total glomerular area. The volume ratio of glomerular glycogen was calculated as the PAS-positive material area/glomerular area. For Masson trichrome staining, the blue collagen deposition was taken as positive material, and the ratio of collagen accumulation was calculated as the Masson -positive material area/glomerular area. The ultrastructure of glomeruli, renal tissue was examined under a Tecnai Transmission Electron Microscope (TEM). The cortical portion of the renal tissue was sliced into small sections of 1 mm2.3.5Sections of kidney tissue were deparaffinized, rehydrated, and submitted to antigen retrieval. Then, the tissue sections were covered with 3 % BSA to block non-specific binding for 30 min. The slides were incubated with primary antibody  overnight at 4 °C; incubation was performed in a wet box containing a small amount of water. Next, the slides were treated with Alexa Fluor 488-conjugated goat anti-rabbit IgG  as the secondary antibody. They were then incubated with DAPI solution at room temperature for 10 min, in the dark. Next, spontaneous fluorescence quenching reagent was added, and the slides were incubated for 5 min. The slides were then cover-slipped with anti-fade mounting medium. Finally, microscopy detection was performed, and images were collected with a fluorescent microscope . The nucleus was stained blue with DAPI and positive cells were labeled green.2.3.6Renal tissue was lysed in RIPA buffer supplemented , then, the protein quantification with the Bradford assay . Subsequently, 50 ng protein solution was electrophoresed via 7.5%–10 % SDS-PAGE , transferred to PVDF membrane , blocked with 5 % BSA and incubated with antibodies  overnight at 4 °C. Next, membranes were washed and incubated with goat anti-rabbit IgG  at 1:5000 dilution for 1 h. Blots were rinsed in PBS containing 0.2 % Tween 20, three times for 5 min each at room temperature, and an enhanced chemiluminescence reagent to visualize the protein bands . The images were captured with Tanon-5200  and quantified with the image analysis software Image J .2.42. Significant differences were considered when the P-values <0.05.Statistical tests were performed using SPSS 19.0. Statistical analysis was carried out using ANOVA, followed by Tukey's post hoc test or Tamhane's T33.1There were 190 active compounds that meet the screening criterion, of which, Huangqi had 17 active compounds, Danshen had 59 active compounds, Dihuang had 8 active compounds, Shanyao had 15 active compounds, and Gancao had 91 active compounds. The 190 compounds corresponded to 921 genes, of which, the active compounds of Huangqi corresponded to 204 genes, Danshen corresponded to 132 genes, Dihuang corresponded to 256 genes, Shanyao corresponded to 68 genes, and Gancao corresponded to 261 genes . After rThe number of DKD genes in each database was 3319 (GeneCards), 43 (DrugBank), 293 (Omim), and 1189 (Disgenet). After removing the duplicates, 3872 genes were retained .3.2In total, 275 putative targets were identified by intersecting the genes corresponding to the active compounds and DKD . The put3.3The GO analysis returned a total of three thousand and two items, including two thousand five hundred and eighty-four items for biological processes (BP), primarily relating to response to hormone, response to oxidative stress, cellular response to lipid, regulation of kinase activity, regulation of MAPK cascade, response to growth factor. One hundred and fifty-five items for cellular component (CC) were identified, primarily relating to membrane raft, dendrite, membrane microdomain, vesicle lumen, side of membrane. Two hundred and sixty-three items were identified for molecular function (MF), primarily related to protein kinase activity, protein serine or threonine or tyrosine kinase activity, phosphotransferase activity .Fig. 2GOKEGG pathway enrichment of two hundred and twenty-two pathways were found, primarily relating to the AGE-RAGE in diabetic complications, PI3K-Akt, MAPK, TNF, EGFR tyrosine kinase inhibitor resistance, HIF-1, VEGF, Rap1, NF-kappa B, and JAK-STAT .3.4The compounds-targets-pathways network contained 480 nodes and 2772 edges. In figure, HQ, DS, SDH, SY, and GC refer to Huangqi, Danshen, Dihuang, Shanyao, and Gancao, respectively. The connection between the nodes indicates the targeting relationship between the active compounds, target, and pathway. The greater the number of connections, the larger the font of the node, the more important it is in the network .Fig. 3CoThe network was analyzed by Network Analyzer with the following filter conditions: BC ≥ 0.00068, CC ≥ 0.3731, and degree greater than or equal to 20. In total, eighteen key chemical compounds in the network were screened . These h3.5−1, it indicates that the binding of the active component and protein is feasible. The results indicated that 235 docking effects were feasible.Molecular docking was performed between 18 active compounds and 13 proteins. The protein, PDB-ID, and Unique Ligands data are shown in 3.6According to the peak integration, retention time, peak alignment, peak extraction and peak area, there have 16 components of QDDHG was detected, namely, 6-Methyl-3-pyridinol, 7-isoquinolinol, calycosin, naringenin, pinocembrin, baicalin, formononetin, licochalcone B, 3′,7-dihydroxy-4′,6-dimethoxyisoflavone (odoratin),10-deoxyeucommiol, cryptotanshinone, salvianolic acid B, ononin, bifendate, sugiol and 18-β-glycyrrhetinicacid .Fig. 5Th3.7During the experiment, fasting blood glucose declined in the QDDHG1 and QDDHG2 groups  A. The Q3.8Glomerular lesions, including mesangial matrix proliferation, decreased numbers of endothelial cells and mesangial cells, and nodular sclerosis were observed in HE-stained tissue from db/db mice. QDDHG1 and QDDHG2 alleviated pathological alterations of the glomerulus in db/db mice, as revealed by histological examination B. In gloTEM revealed foot processes with a tight arrangement in NC group, these were tightly apposed with each other, and filtration slits were narrow. In contrast, slits between foot processes were wide and foot processes with clearance leakage were observed in DKD group, compared to QDDHG1 and QDDHG2 group. The thickness of the GBM in db/db mice was displayed an increase compared with QDDHG1 and QDDHG2 group D.3.9In addition to the pathological lesions, fewer glomerular podocytes were also observed in the DKD group; these are known to contribute to the development of DKD. Nephrin, a podocyte-specific marker, was used to verify whether podocyte loss occurred in DKD mice A. As sho3.10In order to further verify the mechanism predicted by network pharmacology, the protein expression of EGFR, STAT3, ERK and pERK1/2 were adopted. The Western blot analysis revealed significant upregulation in the expression of EGFR, STAT3 and pERK1/2 proteins in DKD group, compared with NC group, using β-actin expression as the inner control. Treatment with QDDHG1 and QDDHG2 induced a decrease in protein expression of EGFR, STAT3 and pERK1/2 in db/db mice, compared with DKD group C and D.4In the previous study, QDDHG was proved to be a promising drug in the aspect of reducing albuminuria and protecting the renal function . However4.1Potential targets of QDDHG on DKD were identified by network pharmacology. Studies show that intervention of EGFR tyrosine kinase inhibitors in model animals can postpone the progression of DKD . When th4.2O-methylisomucronulatol, licochalcone A, dihydrotanshinlactone and jaranol.In this study, 18 key ingredients were predicted as the main components in QDDHG for DKD, including quercetin, kaempferol, 7-isoquinolinol, ferulic acid methyl ester, formononetin, isorhamnetin, luteolin, rehmapicrogenin, calycosin, naringenin, tanshinone IIA, 3′,7-dihydroxy-4’,6-dimethoxy isoflavone, pinocembrin, 7-methoxy-2-methyl isoflavone, 7-In vivo and in vitro studies have revealed that calycosin inhibits diabetes-induced kidney inflammation, mainly by inhibiting NF-κB p65 phosphorylation [O-methylisomucronulatol, 3′,7-dihydroxy-4′,6-dimethoxyisoflavone, pinocembrin, dihydrotanshinlactone and jaranol in DKD have not been reported in the literature. Therefore, future research is needed on these compounds and their role in treating DKD.Quercetin can decrease the levels of albumin in rat urine, increase the excretion rate of urinary creatinine and urea nitrogen, and improves renal function . Kaempferylation . Naringerylation . Licocharylation . In addiFurthermore, the molecular docking was employed to verify active compounds and potential proteins of QDDHG to treat the DKD. The binding energy with a more negative represents a more stable complexes of the bound ligand-protein conformation . The bin4.3Go enrichment shows that the top 5 most significant terms of biological process included response to hormone (GO:0009725), cellular response to nitrogen compound (GO:1901699), response to oxidative stress (GO:0006979), cellular response to lipid (GO:0071396), regulation of kinase activity (GO:0043549). In the biological process, STAT3 was involved of response to hormone and regulation of kinase activity, EGFR, STAT3, MAPK1 and MAPK3 protein were involved in the process of cellular response to nitrogen compound, EGFR, MAPK1 and MAPK3 protein were participated in the biological process of oxidative stress and cellular response to lipid. From KEGG enrichment analysis, the AGE-RAGE, PI3K-Akt, IL-17, MAPK, TNF, EGFR, HIF-1, Apoptosis, Toll-like receptor, Insulin resistance, VEGF, Rap1, NF-kappa B, JAK-STAT, PPAR and Wnt pathway were displayed as the critical signaling participated in the role of QDDHG on DKD. Especially, STAT3, EGFR, MAPK1 and MAPK3 were extensively involved in the above multiple biological processes and pathways.4.4Identification of the chemical components of QDDHG was performed by UHPLC-MS/MS. The results showed that 13 ingredients predicted by network pharmacology were also confirmed by UHPLC-MS/MS. Moreover, six of the key ingredients predicted by network pharmacology were also verified by UHPLC-MS/MS, namely, calycosin, 7-isoquinolinol, naringenin, pinocembrin, formononetin, 3′,7-Dihydroxy-4′,6-dimethoxyisoflavone (odoratin). In view of the role of these components, we evaluate the overall effect of QDDHG on DKD treatment via animal experiments.Traditionally, urinary albumin, Scr and BUN levels have been used to monitor the progression of DKD . In thisSubsequently, we utilized animal experiments to verify the mechanism of QDDHG predicted by the GO and KEGG enrichment. The protein expressions of EGFR, STAT3, ERK1/2 in the QDDHG1-and QDDHG2-treated db/db mice demonstrated an obvious reduction, compared with that in the untreated db/db mice. Study from the literature also shown that regulating the STAT3 pathway improves DKD . The del5in vivo and in vitro experiments. Because of TCM action based on synergy and interaction of multiple components, experimental research performed on a single drug or components not necessarily increase the evidence of QDDHG on DKD. In addition, there are other potential active compounds and putative pathway from the KEGG enrichment, more in vivo experiments should be conducted to elucidate the action and mechanisms.However, there are still some limitations in this study. we did perform UHPLC-MS/MS analysis on the active compounds of the QDDHG, but did not verify the single drug or components The mechanism of QDDHG on reducing the levels of albuminuria and protecting the renal function mainly through inhibited the expression of EGFR, STAT3 and pERK in DKD.LX, XSC and XSZ designed the study. YLL, YX and PPJ performed the data analysis. LX and XSC drafted the manuscript. XFJ, HLY and DS revised the manuscript. All authors gave approval for this study to be published.10.13039/501100001809National Natural Science Foundation of China (81804030), the Fellowship of 10.13039/501100002858China Postdoctoral Science Foundation , the 10.13039/501100003453Natural Science Foundation of Guangdong Province, China , the 10.13039/501100010883Traditional Chinese Medicine Bureau of Guangdong Province, China (20222203) and the Major project of University of Chinese Academy of Science-Shenzhen Hospital , China (HRF-2021003).This research was supported by the The data used to support the findings of this study are included within the manuscript and supplementary information files.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.