Current Biotechnology

Structure and Clinical Application of mRNA Drugs
Dongfeng CHANG, Zhaopeng SUN
2024, 14(1): 1-16. DOI: 10.19586/j.2095-2341.2023.0139
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Because of the characters of effective， safe and easy to be produced on a large scale， mRNA drugs have showed great potential on preventing viral infections. mRNA represents an emerging field of precision medicine， several mRNA drugs targeted therapies for infectious diseases and cancer have shown beneficial results both in vivo and in vitro. mRNA is stable， immunogenic， and not limited by the receptor's major histocompatibility complex （MHC） type. In theory， mRNA can achieve in vivo expression of different target proteins， which makes the development of mRNA drugs more flexible and can be used to prevent and treat many refractory or hereditary diseases. This paper introduced the primary structure and higher structure of mRNA， and summarized the progress of mRNA drugs in clinical application， which was expected to help understand the drug function of mRNA， and the clinical application， and provide the direction for the development of mRNA drugs.

Overview of Norovirus Vaccine Research
Hongyan JIAO, Guochao LI, Liang CHANG, Yanyi LI, Lili ZHAI
2024, 14(1): 17-25. DOI: 10.19586/j.2095-2341.2023.0124
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Norovirus （NoV） is one of the main pathogens causing acute gastroenteritis. NoV is prone to mutating and producing multiple strains that pose a significant threat to human health. Subsequent evaluation of anti-NoV drugs and vaccines is limited by the lack of successful animal models， and no vaccines are currently on the market for NoV prevention. In this paper， the research progress of NoV vaccine was reviewed， focusing on the research status and development prospects of virus-like particle （VLP） vaccine， viral vector vaccine and P-particle based vaccine， in order to provide new ideas for NoV vaccine research and development.

Advances on Prevention and Treatment of Human Respiratory Syncytial Virus
Na LYU, Yanyi LI, Zhuqing MA
2024, 14(1): 26-34. DOI: 10.19586/j.2095-2341.2023.0140
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Respiratory syncytial virus （RSV） is a leading cause of respiratory and lung infections， especially in young children and the elderly， which are prone to serious infections. Despite the global impact of RSV infection on healthcare， the current therapy is still mostly supportive. Over the past few decades， our understanding of the pathogenesis and immunopathology of RSV had evolved， leading to significant advances in RSV prevention strategies. More than 50 years have passed since the RSV vaccine was first trialed in 1966， and two RSV vaccines have been approved. In this paper， current treatment options for RSV infection （including RSV-specific and non-specific） and the development progress of different RSV prevention vaccines were reviewed， which was expected to provide reference for the treatment and vaccines research and development of RSV.

Application of IgY in the Field Antiviral Medical
Yonghong ZHANG, Yanyi LI, Ronglin LIU, Na LYU, Weiting ZHANG
2024, 14(1): 35-41. DOI: 10.19586/j.2095-2341.2023.0107
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Immunoglobulin Y （IgY）， as a potential therapeutic antibody， is present in many species， from lower amphibians and reptiles to higher animal birds. The development of vaccines has become a competitive arena for global scientists to compete against viruses. Although the vaccine can effectively prevent the infection of the virus， reduce the incidence and mortality of the disease， but the development of the vaccine is time-consuming and costly. IgY has the advantages of easy production， low cost， take effect quickly， and antibody therapy strategies basedon IgY have been widely recognized in recent years. This review focused on the application of IgY in the treatment of viral diseases by reporting the latest research on the characteristics of IgY and the progress of production technology， so as to provide some enlightenment for the application of IgY in the treatment of viral infectious diseases.

Therapeutic Effect of Recombinant Collagen Gynecological Gel on Cervicitis in Rats
Xin LIU, Hua LI, Lei QI, Lixia YANG, Yuexin YU, Lanju XU
2024, 14(1): 42-47. DOI: 10.19586/j.2095-2341.2023.0129
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Cervicitis is a kind of frequent gynecological disease， which brings trouble to more and more women. Antibiotics are often used in clinical treatment of cervicitis， which has great side effects. In this study， recombinant collagen was developed into a topical gynecological gel and applied to the treatment of cervicitis in rats. 25% phenol glue was used to induce cervicitis animal model in SD rats， and then recombinant collagen gynecological gel was injected into vagina by local administration for treatment. The changes of vulva swelling and purulent secretion in cervicitis model rats were observed. Hematoxylin-eosin（HE） and masson staining were used to examine the histopathologic changes and collagen deposition in rats. The expression levels of IL-6 protein and Ki-67 protein in the tissues were detected by immunohistochemical experiments. The regulation of inflammatory response and promoting cell proliferation were evaluated in the vaginal and cervical tissues of each group. Results showed that， compared with normal group， the body weight of rats in model group was significantly reduced （P<0.05）. Vaginal opening was red， swollen， and purulent secretions flowed out （P<0.05）. Cervical tissues showed cell degeneration， necrosis and exfoliation， and a large number of inflammatory cells infiltrated. The expression of IL-6 protein was significantly up-regulated （P<0.05）， and the expression of Ki-67 was decreased （P<0.05）. Compared with model group， the body weight of rats in positive control group， GG01 group and GG02 group was significantly increased after treatment（P<0.05）. The swelling and secretions of the vaginal opening were significantly reduced （P<0.05）， the inflammatory cell infiltration was significantly reduced， there was no obvious edema and congestion， collagen deposition and orderly arrangement were shown in the tissues， and the treatment effect of GG02 group was more significant. The expression levels of IL-6 and Ki-67 in GG02 group and positive control group were close to those in normal group， the inflammatory response was reduced， and the proliferation level of damaged tissue cells was restored. The recombinant collagen gynecological gel has a good therapeutic effect on acute cervicitis in rats and provides data support for its clinical application.

Study on the Repairing Effect of Recombinant Human Type Ⅲ Collagen on Guinea Pig Skin Damaged by Ultraviolet Radiation
Liqin DIAO, Hua LI, Lin SHI, Yuexin YU, Yuan XIE, Yaru WANG, Lanju XU
2024, 14(1): 48-54. DOI: 10.19586/j.2095-2341.2023.0130
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Due to the gradual destruction of the ozone layer， the amount of ultraviolet radiation is greatly increased， which would lead to chemical reactions in the skin of nucleic acids， proteins and other large molecules， resulting in skin erythema， pigmentation， sunburn， photoaging and even cancer. Taking recombinant human type Ⅲ collagen （rhCol Ⅲ） as the research object， the cytotoxicity of recombinant human type Ⅲ collagen on fibroblasts （L929） and its repairing effect on skin damaged by ultraviolet radiation in guinea pigs were investigated. The results showed that rhCol Ⅲ had no potential cytotoxicity to L929 cells in vitro. In vivo animal experiments have confirmed that rhCol Ⅲ had obvious repairing effect on the skin damaged by ultraviolet radiation phototoxicity in guinea pigs， which could reduce the skin wrinkles after skin injury， improved the epidermal hyperplasia of the injured skin tissue and the inflammation of the dermis. The expression of Vimentin and human melanoma monoclonal antibody 45 （HMB45） in injured skin were decreased. The above results indicated that rhCol Ⅲ had good safety in the repair treatment of the skin damaged by ultraviolet radiation in guinea pigs， rhCol Ⅲ could significantly improve the degree of epidermal layer thickening and keratinization of the skin of guinea pigs exposed to ultraviolet radiation， reduced the inflammatory response in the dermis and effectively regulate the degree of fibroblast fibrosisand the risk of melanoma. Results showed that， rhCol Ⅲ raw material has obvious therapeutic effect on skin damage caused by ultraviolet radiation.

A New Perspective to Evaluate the Treatment of Rheumatoid Arthritis with Biological Products
Xiaohan MA, Lijing HUANG, Fei WANG, Chenhui LI
2024, 14(1): 55-59. DOI: 10.19586/j.2095-2341.2023.0135
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Rheumatoid arthritis （RA） is a widespread systemic autoimmune disease characterized by joint immune dysfunction with severe synovitis and joint erosion， which can further lead to progressive disability. At present， the main therapeutic drugs for RA in clinical practice are disease modifying anti rheumatoid drugs （DMARDs） that alleviate the condition， including traditional synthetic DMARDs （csDMARDs）， biological DMARDs （bDMARDs）， and targeted synthetic DMARDs （tsDMARDs）. In addition， non steroidal anti-inflammatory drugs （NSAIDs） and glucocorticoids （GCs） are also included. Among them， bDMARDs and tsDMARDs have become the absolute mainstay of the RA market， but due to their long usage cycle and some safety issues， it is impossible to achieve effective and sustained relief， and the phenomenon of relapse often occurs after drug withdrawal， which further limits the clinical application. The article reviewed the research progress on the recurrence of major RA treatment drugs after discontinuation in clinical practice， in order to provide an effective theoretical basis and new ideas for the clinical drug cycle and method.

Preservation Effect of Tissue Protectant on Viral Nucleic Acid and RNA of Guinea Pig Skin Tissue Samples
Hua LI, Lanju XU, Xueliang HUANG, Yaru WANG, Liqin DIAO, Yuexin YU
2024, 14(1): 60-65. DOI: 10.19586/j.2095-2341.2023.0123
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Viral nucleic acid and RNA of biological tissue samples are easy to be affected by external environment and easily degrade， resulting in serious deviations in experimental results. Taking viral nucleic acid and guinea pig skin tissue sample RNA as research objects， the protective effect of tissue protectant on viral load and RNA in guinea pig skin tissue sample after preservation of them at different temperatures and different times was explored. The results showed that after storage at 4 ℃ for 30 days （d）， 25 ℃ for 7 d or 37 ℃ for 24 hours （h）， the viral load stored in tissue protectants did not change significantly compared with the initial viral nucleic acid dose （P>0.05）. After storage at 4 ℃ for 30 d， 25 ℃ for 7 d or 37 ℃ for 24 h， compared with liquid nitrogen storage group， both tissue protectant group and commercially available RNA later group could maintain the extraction amount and purity of sample RNA in guinea pig skin tissue， with no statistical significance （P>0.05）. The results showed that the tissue protectant could be used as a storage solution for scientific research or clinical tissue samples. It was placed at 4 ℃， 25 ℃ and 37 ℃ for a certain time， without affecting the viral load of viral nucleic acid and the amount and purity of RNA extraction from guinea pig skin tissue samples， which made the sample preservation more convenient and efficient.

Progress on Integration Site Analysis Technology
Yonghong ZHANG, Yanyi LI, Weiting ZHANG
2024, 14(1): 66-71. DOI: 10.19586/j.2095-2341.2023.0104
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Lentiviral vectors have been widely used to transfer exogenous DNA into human cells to treat various genetic diseases. Lentiviral vectors can integrate into the host genome， but their integration sites are often unpredictable， which may increase the uncertainty of their therapeutic efficacy. With the wide application of gene and cell therapy， regulators have also issued a series of technical guidance documents to ensure the continuous safety of products. Integration site analysis （ISA） is a key tool for evaluating the biological safety of gene therapy vectors by characterizing their integration profiles， as well as for tracking transgenic cells. This review mainly described the technological evolution of integration sites for retroviruses， and the advantages and development trends of analysis methods. At the same time， strategies to reduce the random integration of viruses into the genome were also reviewed， in order to provide reference for the analysis and detection of lentiviral vector integration as a point of view and the safety evaluation of new drug clinical trials in cell therapy products.

The Application Progress of Freeze-drying Technology in Vaccine Research and Devolopment
Ronglin LIU, Ning WANG, Yanyi LI, Weiting ZHANG, Chu LUO, Weibing NIU
2024, 14(1): 72-84. DOI: 10.19586/j.2095-2341.2023.0111
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As the rapid development of biotechnology， the role of vaccines in the prevention and control of infectious diseases becomes increasingly significant， and widespread immunization is crucial for global health. However， there are considerable challenges in improving vaccine coverage in low- and middle-income countries. Due to the inherent instability and poor thermal stability of liquid vaccines， the high cost of cold chain distribution restricts the dissemination and administration of vaccines in these regions， leading to ineffective control of certain infectious diseases in remote areas. To ensure the stability and effectiveness of vaccines， freeze-drying （FD） technology has become one of the key technologies in vaccine preparation and preservation. This paper provided a comprehensive and systematic review of the principles of freeze-drying technology， its influencing factors， the application of FD technology in different types of vaccines， and its future development trends， which aimed to provide useful information and insights for further research and industrialization of vaccines.

Determination of Sulfonate Genotoxic Impurity Residues in Celecoxib Based on Gas Chromatography-mass Spectrometry Method
Chenyang ZHAO, Xiaomeng LIU, Liangbin XING, Yali LI, Lixin ZHAO, Lianfeng AI, Jing HA
2024, 14(1): 85-93. DOI: 10.19586/j.2095-2341.2023.0138
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A gas chromatography-mass spectrometry （GC-MS） method was developed for the determination of methyl celecoxib sulfonate and ethyl celecoxib sulfonate residues in celecoxib APIs and their formulations. The two impurities were derivatised into iodomethane and iodoethane by sodium iodide derivatisation with headspace injection， and separated on a DB-624 capillary column （60 m×0.25 mm， 1.4 μm）， with helium as the carrier gas and detected by mass spectrometry detector. Both methyl celecoxib sulfonate and ethyl celecoxib sulfonate showed good linearity in the concentration range of 10~500 ng·mL^-1； the recoveries were in the range of 80.87%~106.52% with the RSDs less than 10%； and the limits of quantification （LOQs） were both 10 ng·mL^-1. Methyl celecoxib sulfonate and ethyl celecoxib sulfonate impurities were not detected in any of the celecoxib samples. The method is simple and accurate， which can be used for the detection of genotoxic impurities of two sulfonate esters， methyl celecoxib sulfonate and ethyl celecoxib sulfonate， in celecoxib.

Research Progress of Biological Detection Reagent Preservation Technology
Yingying LIU, Kexin ZHAO, Yifei WANG, Kaixuan LU, Kongxin HU
2024, 14(1): 94-101. DOI: 10.19586/j.2095-2341.2023.0110
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The amplification performance of biological detection reagents， especially the biological activity of enzyme reagents， plays a decisive role in the detection results of real-time quantitative PCR. It is crucial to adopt different preservation technologies to maintain the biological activity of detection reagents， achieve long-term stable preservation and non-cold chain transportation of biological detection reagents at room temperature， and ensure the stability， accuracy， specificity and sensitivity of detection results. This paper reviewed the research progress of four different preservation technologies， including vacuum freeze-drying technology， spray drying technology， vitrification freezing technology， and summarized the advantages and disadvantages in application， in order to provide a certain scientific basis for the follow-up related work.

Application Progress of Hydrogen in Medical Field
Xingyu LIU, Guoli ZHAO, Xuejing LI
2024, 14(1): 102-110. DOI: 10.19586/j.2095-2341.2023.0097
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Hydrogen has attracted a lot of attention in energy field， at the same time， it is also a clean， efficient and economical technology in medical field. The field of hydrogen medicine mainly includes basic and clinical research on the effects of hydrogen on diseases， such as the usage methods， dosage， health promoting effects， therapeutic effects on diseases， and mechanisms of action of hydrogen. Hydrogen can remove hydroxyl radicals and peroxynitrites，which have significant therapeutic effect for oxidative stress and inflammation related diseases. At the same time， as an endogenous gas， it is non-toxic and harmless， and will not cause adverse reactions to human body. Targeted treatment of brain and nervous system diseases， cardiovascular diseases， diabetes， cancer and other diseases can be achieved through direct intake and controlled release. The article introduced the different ways of releasing hydrogen gas and its applications in the medical field， and looked forward to the scientific and practical issues of hydrogen medicine， in order to provide reference for the application research of hydrogen in the biomedical field.

Research Progress on Silver Ion Colorimetric Detection
Min YANG, Houjuan CHU, Longjiao ZHU, Qinghua HU, Xiuli XU, Feng ZHANG, Yushi XIE, Wentao XU, Songlin YANG
2024, 14(1): 111-119. DOI: 10.19586/j.2095-2341.2023.0127
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Due to the threat of silver ions （Ag⁺） to human health and ecosystems balance， the detection of Ag⁺ has attracted much attention in the environmental and food fields. The colorimetric method is one of the most commonly used methods for the detection of Ag⁺， which has advantages of simplicity， convenience of use， and quick on-site detection. The sensing mechanism and color development materials is an important research direction in the field of colorimetric method. In this paper， we reviewed the colorimetric principles and sensing strategies of Ag⁺colorimetric sensor. We first classified the colorimetric principles into three categories， including enzyme catalysis， localized surface plasmon resonance and chemical coloration， and the corresponding color development materials were also outlined. Then the sensing mechanism， sensing performance and practical sample applications for Ag⁺ detection were discussed. Finally， the current challenges and prospects of Ag⁺ colorimetric detection were also summarized. The purpose of this work was to promote the development of rapid detection technology for heavy metal ions and also assisting readers in better comprehending the principle of Ag⁺ colorimetric method.

Analysis of Method Validation of Transgenic Testing Standards
Yin ZHANG, Junwei JIA, Lan BAI, Yifan CHEN, Nguyen CONGTHANH, Beibei LYU, Aihu PAN
2024, 14(1): 120-124. DOI: 10.19586/j.2095-2341.2023.0131
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Genetically modified organism （GMO） testing standards provide important technical support for effective labeling， scientific supervision， promotion and application of genetically modified foods （GMFs）. Method verification means that the laboratory verifies the above transgenic testing standards meet satisfactory results under the conditions of existing laboratory personnel， equipment and site environment through certain scientific methods. Method verification has a direct impact on the implementation and application of the standard， but most of the existing verification methods of transgenic testing standards only provide the definition of method verification and lack of specific operational plans. This paper discussed the key elements of the verification work， such as instruments and equipment， reagents and standard substances， technical verification parameters， and environmental requirements， and put forward corresponding verification suggestions， aiming at providing detailed technical guidance for agricultural transgenic laboratories to carry out method verification， and providing reference for the application and promotion of transgenic standard system in China.

Development of Sheep-derived Genomic DNA Reference Material
Yi JI, Kaili WANG, Huiru YU, Xin ZHAO, Lin DING, Cheng PENG, Junfeng XU, Xiaoyun CHEN
2024, 14(1): 125-132. DOI: 10.19586/j.2095-2341.2023.0137
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In recent years， food safety incidents such as adulteration of mutton and its meat products have been occurring， and in order to improve the legal basis for market enforcement and inspection， a standard substance for sheep genomic DNA has been developed by using digital PCR to quantify the sheep HELZ gene. Since the standard substance can be used to measure the accuracy of detection methods， it can determine the adulteration of mutton in food and related products. In this research， the standard material of sheep genome DNA was developed as the positive control for the detection of mutton in food and related products. The uniformity and stability of the reference materials were evaluated by digital PCR. The results showed that the reference materials had good uniformity and could be stably stored at 4 ℃ and 25 ℃ for 14 days， and at -20 ℃ for 6 months. The standard values of sheep-derived genomic DNA reference material （high concentration） and sheep-derived DNA reference material （low concentration） and their extended uncertainties were （5.44±0.45） ×10³ copies·μL^-1 and （5.68±0.54） ×10² copies·μL^-1， respectively， which were jointly determined by nine different laboratories. The development of sheep-derived genomic DNA reference materials provides a technical basis for the development of animal-derived reference materials， and will establish a technical platform for the preparation， quantitative detection， quality control and quantitative value traceability of sheep-derived genomic DNA reference materials.

Study on Optimization of Culture Method and Improvement of Sleep Function of Limosilactobacillus reuteri HCS02-001
Jia SONG, Xuelong LI, Xinmei LIN, Ping YU, Sicheng YANG, Yongzhen PENG, Yanping JIAO, Xuejiao CHEN
2024, 14(1): 133-140. DOI: 10.19586/j.2095-2341.2023.0103
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In order to optimize the cultivation method of Limosilactobacillus reuteri HCS02001 and explore its sleep promoting function， the optimal cultivation was screened through single factor experiment and response surface experiment based on the γ-aminobutyric acid （GABA） yield，and its sleep promoting function was evaluated through mouse experiment. Under the condition of that culture temperature was 37 ℃， the culture time was 24 h and the initial pH was 6.5， the GABA yield of L. reuteri HCS02-001 was 5.58 g·L^-1. No direct sleep phenomenon occurred in mice in the direct sleep experiment. The experimental results of prolonging the sleep time of mice hypnotized by pentobarbital sodium showed that the mice in the high-dose group effectively prolonged the sleep time， and the results of pentobarbital sodium subthreshold dose hypnosis experiment showed that the sleep rate of mice in the high-dose group was as high as 40%. Barbiturate sleep latency experiments showed that mice in the high-dose group had reduced sleep latency and could act synergistically with barbiturate sodium. The results showed that L. reuteri HCS02-001 had the function of sleep aid.

Bioinformatics Analysis on the Regulation Mechanism of NHP2 on Hepatocellular Carcinoma Senescence
Shi HUANG, Yinyin MO, Lyujing LUO, Huiting LIU, Zhengyu CHEN, Genliang LI
2024, 14(1): 141-148. DOI: 10.19586/j.2095-2341.2023.0108
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To investigate the expression of ribonucleo protein NHP2 in hepatocellular carcinoma （HCC） and to explore the relationship with disease progression and prognosis. Differentially expressed genes of HCC were screened by Quick Go and GEPIIA 2 online databases， and NHP2 was identified as object of study. Furthermore， bioinformatics methods such as STRING， TIMER 2.0， UALCAN database were used to analyze the differential expression of NHP2 in pancreatic cancer and its correlation with pathological progress in HCC and other pancreatic cancers， and to predict the relationship between prognosis and survival. The target miRNAs and lncRNAs were analysed with the miRNet database， the possible regulatory network of CeRNAs was mapped by Cytescape v3.8.0. Results showed that， 113 genes related to cell aging and 2 206 differentially expressed genes（DEGs） in HCC were found in the online database. 19 DEGs were involved in the biological process of cell aging in HCC. Among them， NHP2 is significantly highly expressed in a variety of cancers including HCC， and the prognosis of HCC with high NHP2 expression is poor in statistical difference. There are 10 interacting proteins encoded by the NHP2 gene， it is mainly involved in 1 signalling pathway （KEGG）， 6 molecular functions （MF）， 11 cellular components （CC） and 7 biological processes （BP）. Two NHP2-targeting miRNAs and 57 NHP2-targeting lncRNAs had been predicted. The results indicated that， NHP2 is highly expressed in a variety of tumors， including HCC， and is significantly different in age and stage of patients with a variety of tumors. During the proliferation and senescence of HCC， it may be regulated by the regulatory axis of lncRNAs/miR-1-3p/NHP2 or lncRNAs/miR-124-3p/NHP2 and be a prognostic predictor and a potential therapeutic target of HCC.

Prognosis Prediction Model and Drug Sensitivity Analysis of Triple-negative Breast Cancer Based on m5C Related Genes
Yuemaierabola ANWAIER, Yeerkenbieke BUERLAN, Lili SUN, Fuzhong LIU, Yeerxiati DILINAER, Wenjia GUO
2024, 14(1): 149-159. DOI: 10.19586/j.2095-2341.2023.0134
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In order to explore the potential value of 5-methylcytosine （m5C）-related genes in the prognosis of patients with triple negative breast cancer （TNBC） and in the treatment of TNBC， a prognostic prediction model based on m5C-related genes was constructed for assessing the prognosis and survival of TNBC patients. TNBC gene expression profiles and corresponding clinical data were downloaded from the gene expression omnibus （GEO） database and the cancer genome atlas （TCGA） database.99 m5C related genes were identified through pearson correlation analysis and one-way cox analysis was used to identify m5C-related genes， from which five prognosis-related m5C-related genes （SLC6A14， BCL11A， UGT8， LMO4， PSAT1） were identified and a risk score prediction model was constructed， and the patients were classified into high-risk and low-risk groups according to the median value of the risk score. The predictive efficacy of the model was assessed using Kaplan-Meier （K-M） survival analyses， receiver operating characteristic （ROC） curves， multivariate Cox regression analyses， construction of column line plots and calibration curves. The K-M survival curve， area under the curve （AUC） analysis of the training and validation sets verified the good predictive ability of the model. Multivariate Cox regression showed risk score could be used as an independent prognostic biomarker. Analysis of differences in half maximal inhibitory concentration （IC₅₀） values of chemotherapeutic agents using ssGSEA， immune score analysis and chemotherapeutic agents in patients in the high and low risk groups showed significant differences in the expression of immune cells and immune checkpoint genes， as well as the IC₅₀ values of most chemotherapeutic agents， between the different risk groups. In this study， we constructed a risk score prognostic prediction model based on five m5C-related genes， which would help to elucidate the mechanism of action of m5C-related genes in TNBC， and then provided more valuable biomarkers for prognosis and diagnosis and potential therapeutic targets， and provided theoretical guidance for the clinical individualisation of treatment for TNBC patients.

The Mechanism Research of Tangshenbao Compound in Treating Diabetic Nephropathy Based on Network Pharmacology and Experimental Verification
Fanglin SU, Xin LUO, Sainan TANG, Leyun HUANG, Wei HU, Shilong LU, Longjiao RAN, Shaowei XIANG
2024, 14(1): 160-171. DOI: 10.19586/j.2095-2341.2023.0142
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The research investigated the mechanism of action of Tangshenbao compound （TSB） in treating diabetic nephropathy （DN） by combining network pharmacology methods with molecular biology experiments. The active ingredients and targets related to TSB were obtained using TCMSP， Pubchem， and Swiss Target Prediction databases， along with relevant literature. Candidate targets for TSB in treating DN were obtained through GeneCards， DisGeNET， and OMIM databases. A Venn diagram of the "TSB-DN" candidate targets was drawn using online plotting software， followed by GO and KEGG enrichment analysis using Cytoscape 3.7.2 software. Subsequently， molecular docking of active ingredient ligands with target protein crystal structures was completed using PyMOL， AutoDock Tools， and AutoDock Vina software. Cell experiments were then conducted using Mcs mesangial cells cultured in vitro to validate the findings. Real-time fluorescence quantitative PCR was used to detect the mRNA expression levels of STAT3， PIK3CD， PIK3R1， MAPK8， and their associated targets in the cells of each group， while cell viability after 24 hours was assessed using the CCK-8 method. Results showed that a total of 835 TSB target genes were obtained after processing the Swiss Target Prediction database. When combined with GeneCards， DisGeNET， OMIM， and other disease databases， there were 443 intersecting target genes for “TSB-DN”. Protein-protein interaction （PPI） network analysis identified the top 10 degree targets as SRC， PIK3R1， STAT3 and PIK3CA. GO and KEGG enrichment analysis results indicated that TSB treatment for DN may be related to functions such as protein phosphorylation， cell signal transduction， and positive regulation of gene expression. Finally， cell experiments in vitro demonstrated that TSB could effectively reduce the mRNA expression levels of PIK3CD， PIK3R1， and ETS-1 in SV40-MES-13 cells， resulting in an “inhibitory， sedative” effect following drug action on the cells. The results indicated that the TSB compound can inhibit the mRNA expression of target genes such as PIK3R1， PIK3CD， MAPK8， STAT3， and ETS-1， thereby regulating the PI3K-Akt and AGE-RAGE signaling pathways and exerting a therapeutic effect on DN.

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