Angelica sinensis (Oliv.) Diels are well known for their efficacy in promoting blood circulation. Although many studies have indicated that phthalides are the main chemical components responsible for the pharmacological properties of A. sinensis, the phthalide biosynthetic pathway and enzymes that transform different phthalides are still poorly understood. We identified 108 potential candidate isoforms for phthalide accumulation using transcriptome and metabolite profile analyses. Then, six enzymes, including phospho-2-dehydro-3-deoxyheptonate aldolase 2, shikimate dehydrogenase, primary amine oxidase, polyphenol oxidase, tyrosine decarboxylase, and shikimate O-hydroxycinnamoyl transferase, were identified and proven to be involved in phthalide accumulation by heterologously expressing these proteins in Escherichia coli. We proposed a possible mechanism underlying phthalide transformation and biosynthetic pathways in A. sinensis based on our findings. The results of our study can provide valuable information for understanding the mechanisms underlying phthalide accumulation and transformation and enable further development of quality control during the cultivation of A. sinensis.The roots of  Angelica sinensis (Oliv.) Diels is a high-altitude plant found in the marginal region of the Qinghai-Tibet Plateau. The roots of A. sinensis have a long history of being widely used in Traditional Chinese Medicine for treating various gynecological conditions  and prokaryotic expression. The results from our study expand the understanding of the changes in the phthalide content between early flowering and normal flowering in A. sinensis. This provides insights for developing a new plant variety with a high phthalide level or a characteristic phthalide content.In this study, the ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was utilized to identify six phthalides, including ligustilide, butylphthalide, butylidenephalide, senkyunolide H, senkyunolide I and senkyunolide A, in the roots of normal flowering and early flowering A. sinensis plants of the plant strain “Mingui No. 1,” were selected as the experimental materials. Samples were collected on August 22, 2018, from Tanchang, Gansu Province . Dr. Hui Yan from the Nanjing University of Chinese Medicine authenticated the roots. Fresh A. sinensis roots were collected, flash-frozen in liquid nitrogen, and transported in dry ice. The samples were stored at −80°C at the Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization. The high-throughput sequencing analysis was performed at Entrusted Frasergen Bioinformatics Co., Ltd. .In this study, root samples from the normal flowering (ZC-1 to ZC-6) and early flowering (ZT-1 to ZT-6) fresh A. sinensis “Mingui No. 1” were grown in a culture room at 23 ± 1°C under a 14-h photoperiod and 2000 lx. Methyl jasmonate  based on Illumina® manufacturer’s protocols, and index codes were added to attribute sequences to each sample.Two randomly selected plant roots were combined for transcriptome analysis, and each group (ZT and ZC groups) contained three samples for analysis. Libraries were constructed from root mRNA and sequenced using the PacBio Sequel and Illumina HiSeq X Ten PE150 platforms  by the Frasergen Biotechnology Company . Sequencing libraries were generated using the NEB NextA. sinensis, the full-length transcript sets from the roots of normal and early flowering A. sinensis plants by PacBio SMRT three-generation high-throughput sequencing technology were used as the reference isoforms for both subsequent bioinformatics analysis and comparative transcriptomics analysis. Sequencing reads were aligned to the reference isoforms using Tophat2 (v2.1.1) and Bowtie2 (v2.2.2) with default parameters  after MeJA treatment. The RNA prep pure plant kit  was used for RNA extraction with on-column DNA digestion according to the manufacturer’s protocol. Total RNA (1.5 μg) was reverse transcribed using random primers and conditions described in the EasyScript All in-one First-Strand cDNA Synthesis SuperMix for qPCR . RNA and cDNA concentrations and purities were estimated using a DS-11 spectrophotometer . qRT-PCR was performed on an ABI 7500 real-time PCR system  cells to express recombinant proteins. The positive clones were incubated in Luria-Bertani medium in the presence of kanamycin. E. coli BL21 (DE3) were grown in 250 ml Erlenmeyer baffle flasks containing 100 ml of Luria-Bertani medium in a rotary shaker at 160 rpm and 37°C. When the optical density of the cultures at 600 nm reached 0.5–0.6, recombinant proteins were expressed in E. coli cells following induction by the addition of 0.5 mM isopropyl-β-D-thiogalactoside and incubation overnight at 16°C. After centrifugation at 32000 × g for 10 min, the cells were resuspended in 1 ml phosphate buffer saline solution (pH 7.2–7.4). The cells were lysed using an ultrasonic disrupter at 15% power for 3 min, centrifuged at 12000 × g for 10 min, and then purified using Ni-NTA affinity chromatography under nature conditions, following the manufacturer’s instructions . After purification with Ni-NTA chromatography, the protein sample was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (The full-length cDNA of the seven candidate isoforms was cloned into the pET-28a (+) vector. The recombinant plasmids were transformed into phoresis . The pro2O MgCl2), 10 μl of 50 mM DL-dithiothreitol, and 20 μl of various phthalide (5 mg/ml)-methanol extracts, were incubated for 1 h at 30°C in the dark. Thereafter, the mixtures were centrifuged at 12000 × g for 10 min prior to UHPLC–MS/MS analysis  containing 250 μl reaction mixtures, which included 100 μl of crude enzyme solution, 110 μl of 25 mM Tris–HCl (pH 7.5), 10 μl of 100 mM magnesium chloride hexahydrate . However, the levels of senkyunolide I and butylidenephalide were not significantly different between the ZC  and ZT samples . The ratio of ligustilide was significantly higher in the ZT samples (98.33%) than that in the ZC samples . We then selected three characteristic phthalides with large differences for the diagnostic ratio analysis  and ligustilide/senkyunolide A  also revealed a steady-state imbalance when plants are flowering early. Therefore, cultivating early flowering-resistant plants has become more popular, resulting in more effective plant ingredients available for medicinal use.the same . Becausethe same , medicalthe same , and idethe same . It is aunctions . A largeunctions . Thus, iA. sinensis to reduce waste, improve the market supply of medicinal raw materials, and alleviate the economic losses of farmers. Studies on the mechanism underlying phthalide biosynthesis have been reported previously, but the enzymes related to the accumulation of phthalides, especially the enzymes involved in different phthalide transformations, have not yet been identified, requiring further investigation (O-hydroxycinnamoyl transferase (43), have shown potential for the regulation of phthalide accumulation.This study aimed to determine the key enzymes in phthalide accumulation and the molecular mechanism underlying the synthesis and accumulation of phthalide components to provide a feasible strategy for increasing the phthalide levels in the flowering tigation . In thiso-quinones (O-hydroxycinnamoyl transferase (43) catalyzes the synthesis of shikimate and quinate esters. It appears to control the biosynthesis and turnover of major plant phenolic compounds, such as lignin and chlorogenic acid  and shikimate dehydrogenase (21) catalyze the first and fourth committed steps of the shikimate pathway, respectively, both of which are required for the synthesis of aromatic amino acids and other aromatic metabolites in bacteria, microbial eukaryotes, and plants . Polyphequinones . Polyphequinones . Tyrosinquinones . In the quinones . Shikimanic acid . AlthougA. sinensis, ligustilide is a volatile and unstable compound with an α, β-unsaturated lactone in its structure. Senkyunolide I and 6, 7-epoxyligustilide were the major degradation products when ligustilide was stored at room temperature under direct sunlight. Ligustilide is likely to degrade into 6, 7-epoxyligustilide through oxidation and then transform into senkyunolide I by further hydrolysis (in vivo metabolites of ligustilide (+ generate shikimate and NADP+ under the action of shikimate dehydrogenase (21). In the above reaction, polyphenol oxidase (36) and shikimate dehydrogenase (21) mainly act on the conversion of the compound from a carbonyl to a hydroxyl group. The proposed reaction pathways from ligustilide to senkyunolide I are shown in Through the analysis of these enzyme reactions, it is speculated that they may regulate the conversion of different phthalide components through various pathways, such as oxidation, isomerization, and hydroxylation. As the most abundant phthalide in drolysis . In addiustilide . These sA. sinensis. These findings may also provide insights into the genes that can make for potential genetic engineering targets for enriching phthalides in A. sinensis.Overall, our study explored candidate enzymes corresponding to different phthalide compounds that attempt to build phthalide biosynthetic and transformed pathways. Seven candidate isoforms involved in phthalide accumulation and transformation have been identified. Due to the lack of relevant research, we could not fully clarify the mechanism of action of each enzyme. Only the proposed reaction pathway from ligustilide to senkyunolide I is shown. Further investigations into the enzymatic properties and their regulatory mechanisms are required to completely understand phthalide accumulation and transformation in https://www.ncbi.nlm.nih.gov/, PRJNA749925.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: W-MF, PL, HY, and J-AD conceived and designed the experiments. W-MF performed the experiments. W-MF, PL, E-XS, and SZ analyzed the data. J-AD, D-WQ, SZ, GY, HY, and SJ contributed reagents, materials, and analysis tools. W-MF and PL wrote the manuscript. All authors have read and approved the final manuscript.This research was supported financially by Innovation Team and Talents Cultivation Program of National Administration of Traditional Chinese Medicine (ZYYCXTD-D-202005). China Agriculture Research System of MOF and MARA (No. CARS-21), Ministry of Finance Central Level of the Special (No. 2060302), National Natural Science Foundation of China (81773848). This work was also partly sponsored by Six talents peaks project in Jiangsu Province (JNHB-066), Jiangsu Province 333 High-level Talents Training Project, Qing Lan Project, the Major Projects of Natural Science Foundation of Universities in Jiangsu Province (19KJA320002) and Natural Science Foundation of Jiangsu Province, China (BK20201403).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cordyceps militaris (CM) is a popular medicinal fungus; however, few studies have focused on its impact on the male reproductive system. We evaluated the effects of CM fermentation products on the reproductive development of juvenile male (JM) mice. Mice were divided into four experimental groups, each fed 5% CM products (weight per weight (w/w) in normal diet): extracellular polysaccharides (EPS), fermentation broth (FB), mycelia (MY), and whole fermentation products  for 28 days, while mice in the control group (CT) were fed a normal diet. Basic body parameters, testicular structure, sperm parameters, and sex hormones concentrations were analyzed. Compared to the CT group, mice in the EPS, MY, and FBMY groups showed a significantly increased mean seminiferous tubule area (p < 0.05), mice in the FB and MY groups had significantly higher sperm concentrations (p < 0.05), and mice in the EPS, FB, and FBMY groups showed significantly increased ratios of motile sperm (p < 0.05). Meanwhile, EPS significantly promoted the ability of JM mice to synthesize testosterone (p < 0.05). Furthermore, all CM products significantly increased the food intake of JM mice (p < 0.05) but did not significantly change their water intake and body weight gain (p > 0.05). In conclusion, CM products, especially EPS, exhibit strong androgen-like activities that can promote male reproductive development. Obesity2, environmental pollution3, psychological stress4, and even COVID-195 impact testicular function and spermatogenesis. Exposure to these risk factors early in life may have long-term effects on male fertility6. A meta-analysis reported that sperm concentration has decreased globally by approximately 50% over the past 50 years, and male infertility has become a major problem for many couples7. The World Health Organization (WHO) predicts that infertility will become the third major health threat after cancer and cardiovascular diseases in the twenty-first century8. Currently, some drugs have been developed for treating male infertility9. For example, sildenafil citrate (Viagra) is used clinically to treat erectile dysfunction. However, many adverse events have been observed, including flushing, headaches, and dyspepsia10. Therefore, it is necessary to identify natural products that enhance male reproductive development and function without significant side effects.Testis development during juvenile stages is essential for male reproductive functionOphiocordyceps sinensis (O. sinensis), referred to as the “Himalayan Viagra,” is a scarce and expensive medicinal fungus11. Cordyceps militaris (C. militaris), also known as Northern O. sinensis, is a species of the genus Cordyceps12. Studies have shown that there are the levels of some bioactive ingredients, such as amino acids, unsaturated fatty acids, and adenosine, are higher in C. militaris than in O. sinensis13. Recently, C. militaris has been widely used as a substitute for O. sinensis in traditional Chinese medicine and health supplement12.C. militaris using a liquid fermentation process can shorten the production cycle and increase yield14. This process yields large amounts of extracellular polysaccharides (EPS) and mycelia (MY) in a short period. EPS and MY have been proven to possess antioxidant, antimicrobial, and anti-inflammatory activities16. However, due to the large volume of fermentation broth (FB) and low content of active ingredients, it is often discarded as waste. In this study, we investigated the effects of C. militaris fermentation products on the reproductive development of juvenile male (JM) mice. Basic body parameters, testicular structure, sperm parameters, and serum concentrations of sex hormones were analyzed.Specifically, the culture of p < 0.05). There were significant differences in the contents of cordycepin and adenosine in the four C. militaris fermentation products (p < 0.05), and all had the highest content in the FB. Except between the FB and EPS, protein concentrations in different fermentation products were all significant differences (p < 0.05). The protein concentrations in the MY was the highest, followed by in the FBMY, in the FB, and in the EPS was the lowest. In this study, EPS were obtained from FB by ethanol precipitation. When the EPS in FB were precipitated with ethanol, cordycepin could not be simultaneously precipitated because of its good solubility in water and ethanol. Thus, the cordycepin content in the EPS was below detectable levels.Table C. militaris fermentation products  was significantly higher than that of the CT group that were fed a normal diet . A possible explanation for this phenomenon is that the sweetness of EPS increases the palatability of the pellets. Moreover, there were no significant differences in water intake . Although mice in the EPS group ate more than mice in the other groups, their body weight gains were only significantly increased when compared with the FB group (p < 0.05). These results imply that body weight gain in JM mice is controlled or regulated by EPS addition, thereby preventing obesity.Food and water intake and body weight gain in mice of all groups are presented in Fig. 22. Since intestinal bacteria can not only increase the absorption of intestinal monosaccharides and short-chain fatty acids and promote the fat accumulation in the liver tissue23, but the lipopolysaccharide of gram-negative bacteria can also bind to receptors on the surface of immune cells, causing an inflammatory response thereby aggravating obesity24. Previous studies have demonstrated that O. sinensis can reduce the amounts of Enterococcus cecorum and modulate a bile acid receptor, thereby attenuating lipid metabolism disorders and their associated inflammation in high-fat diet (HFD) mice25. C. militaris polysaccharides could increase gut bacteria negatively associated with obesity traits, decrease the population of gram-negative bacteria, effectively improve intestinal flora dysbiosis in HFD-induced mice, and regulate the levels of metabolites, thereby reducing body weight, fat accumulation, and pro-inflammatory cytokine levels in mice27. Selenium-enriched C. militaris could reduce serum triglyceride and low-density lipoprotein cholesterol levels, inhibit serum lipopolysaccharide-binding proteins, adiponectin levels, and pro-inflammatory gene expression in HFD mice, and improve the expression of related anti-inflammatory genes28. Although the mechanisms remain to be elucidated, C. militaris fermentation products, especially EPS, may effectively prevent obesity during the development of JM mice by modulating the intestinal flora.Recently, the regulatory role of some medicinal fungi on obesity has been gradually clarified through 16S rRNA gene sequencing and metabolomic studiesp > 0.05; Fig. p > 0.05; Fig. p = 0.004 vs. CT; p = 0.013 vs. FB; p = 0.002 vs. MY; p = 0.005 vs. FBMY).Testicular volumes and weights of JM mice in each group are shown in Fig. 29 and significantly reduces epididymal adipocyte sizes in a dose-dependent manner30. In addition, C. militaris polysaccharides can decrease epididymal fat index by modulating the expression of key genes and proteins in epididymal fat31, including reducing sterol regulatory element binding protein-1c level by approximately 49%, inhibiting the expression of peroxisome proliferator-activated receptor γ protein by approximately 67%32, thereby significantly reducing epididymal adipogenesis. This suggests that the decreased testicular weight of JM mice in the EPS group may be related to the fact that EPS can reduce lipid and adipocyte differentiation in testis and surrounding tissue. Since the testes play crucial roles in producing sperms and synthesizing androgens, the testicular structure, sperm parameters, and sex hormone levels in sera were further evaluated.It has been reported that cordycepin reduces fat accumulation around the epididymis of HFD ratsC. militaris fermentation products promote ST development in the testes of JM mice. Therefore, we evaluated the effect of C. militaris fermentation products on the morphological indicators of STs.The hematoxylin and eosin (H&E) staining images of testicular sections of JM mice from each group are shown in Fig. p < 0.05; Fig. p < 0.001 vs. CT, p < 0.001 vs. FB, p = 0.761 vs. MY, p = 0.052 vs. FBMY). Moreover, the mean area of STs in all experimental groups, except for the FB group, was significantly larger than that in the CT group . The larger the mean area of the STs, the more mature the STs.The number and mean area of the STs of JM mice in each group are presented in Fig. 33. In this study, except that EPS significantly reduced testicular weight, C. militaris fermentation products did not significantly affect testicular volumes or weights of JM mice, but significantly increased the size and maturation of STs. These results suggest that C. militaris enhances testicular development, thereby promoting spermatogenesis in JM mice.The STs of the testis are the site of spermatogenesis and are composed of spermatogenic cells and Sertoli cells, the former producing sperm and the latter supporting and nourishing spermatogenic cellsp < 0.05). Mice in the MY group had the highest sperm concentrations. Furthermore, the ratio of motile sperm was also higher in all experimental groups than in the CT group. Except for the MY group, the motile sperm ratios in all experimental groups were significantly higher than in the CT group . Ratios of motile sperm were the highest in the FBMY group.To further assess male reproductive development, sperm concentrations and ratios of motile sperm in the epididymis were analyzed Table . Sperm cC. militaris fermentation products promoted the development and maturation of STs, enhanced spermatogenesis in the testes, and increased mature sperm concentration and motile sperm ratio in the epididymis. These results are consistent with reports showing increased sperm concentrations and motilities in boars34 and Sprague–Dawley rats35 following MY supplementation.Based on the results shown in Figs. 36. C. militaris is a natural antioxidant with excellent ability to scavenge reactive oxygen radicals37. Among them, cordycepin can regulate the activities of downstream antioxidant enzymes through the SIRT1/Foxo3a signaling pathway38, as well as regulate NF-κB activation and MAPKs signaling39, effectively improving sexual dysfunction in aged rats. Moreover, another study showed that the EPS extracted from the C. militaris FB could promote the proliferation of spleen T and B lymphocytes through MAPK signaling40; thus, EPS may also be able to resist testicular oxidation through this pathway. Therefore, the antioxidant activities of C. militaris may explain why these fermented products can promote male reproductive development in JM mice.Oxidative stress is an important cause of testicular damage, which can lead to testicular dysplasia, spermatogenic cell necrosis and apoptosis, resulting in male reproductive dysfunctionC. militaris fermentation products on male reproductive development, the serum sex hormone levels of JM mice were analyzed , but not in the CT group.To investigate the effects of supplementation with zed Fig. . Althougp > 0.05; Fig. p < 0.05). In males, E2 is mostly converted through the aromatization of T41. The results shown in Figs. Since estradiol (E2) levels may antagonize the effects of T, serum E2 levels were also analyzed. Although mice in both the FB and MY groups showed higher E2 concentrations in sera than those in the CT group, there were no significant differences in E2 concentrations between each experimental group and the CT group . The synthesis and secretion of sex hormones are regulated mainly by the hypothalamus-pituitary-gonadal axis. Thus, increases in both T and E2 levels in the FB group may have a negative effect on the LH levels secreted by the pituitary gland. This explains why mice in the FB group had the lowest LH levels.Since luteinizing hormone (LH) is secreted by the anterior pituitary gland, which stimulates ovulation in females and androgen synthesis in males, serum LH levels were analyzed. Except for the FB group, there were no significant differences in LH concentrations among the other groups  were calculated to directly assess the ability of testes to synthesize T. Results showed that T/T ratios were higher in all experimental groups, except the FBMY group, than those in the CT group . Moreover, the T/T ratios of the FBMY group were the lowest and showed a significant decreases when compared with those of the EPS and FB groups ( p < 0.05), but there was no significant difference with the CT group.Since T concentrations in testes are at least 25–100 times higher than those in seraC. militaris fermentation products, especially EPS, promote the development of testes and elevate serum levels of T and T/T ratios in JM mice. Our findings agree with one previous study that demonstrated a C. militaris-induced increase in T productions in Sprague-Dawley rats 43. Another study showed that O. sinensis stimulates T productions in vivo and in vitro 44. Polysaccharides and glycoproteins of O. sinensis share structural similarities with LH and can be recognized by LH receptors on Leydig cells to stimulate T productions. Although the specific mechanism remains unknown, C. militaris fermentation products, especially EPS, showed androgen-like activity, which can promote male reproductive development in JM mice.The results shown in Fig. C. militaris fermentation products, especially EPS, promote the development of testes, elevate serum levels of T and T/T ratios, and then increase sperm concentrations and ratios of motile sperms in the epididymis of JM mice. Since all mice in this study were fed ad libitum, it is possible that the EPS-containing feed increased the mice's appetite due to its sweetness. From our results, increased EPS intake may better promote reproductive development in male JM mice. In addition, C. militaris extract has been reported to prevent obesity and fat accumulation in ovariectomized rats, lower triglyceride levels, and enhance estrogen receptor agonistic activity and phosphorylation, effectively regulating menopause-induced obesity45. Therefore, these findings suggest that C. militaris fermentation products can promote the reproductive system health and prevent metabolic syndromes including obesity.The results shown in Figs. C. militaris fermentation products on male reproductive development in JM mice. EPS showed the strongest androgen-like activity, which promoted male reproductive development. EPS supplementation can increase the mean area and maturation of STs in the testes, sperm concentrations and ratios of motile sperms in the epididymis, and serum T levels and T/T ratios. Therefore, EPS supplementation can effectively improve male reproductive development in JM mice, and may prevent or assist in treating male reproductive dysfunction or infertility. Furthermore, it is important to note that C. militaris fermentation products can effectively control or regulate body weight gain in JM mice during the experimental period of 28 days, thus it has the potential to assist in obesity treatment. However, it must be noted that clinical studies are required to determine if these beneficial effects of C. militaris can apply to humans.In this study, we evaluated the effects of four different C. militaris fermentation products including EPS, FB, and MY, were gifts from Dalong Biotechnology Co. Ltd. . EPS were obtained from ethanol precipitation of the FB, centrifugation, and freeze-drying. The yields of polysaccharides, cordycepin, adenosine, and proteins of the EPS, FB, MY and whole fermentation product (FBMY) were detected by the phenol-sulfuric acid method 46 high-performance liquid chromatography (HPLC) method 47, and Bio-Rad Protein Assay  according to the manufacturer's instructions, respectively , 25 in number, were purchased from the National Laboratory Animal Center . During the experimental period, they were kept in an air-conditioned room at 24 ± 2 °C and 50–60% humidity with a 12 h light/dark cycle. They were randomly divided into five groups (n = 5 each). Mice in the control (CT) group were fed Laboratory Rodent Diet 5001 only, while mice in the experimental groups were fed pellets containing 5% (w/w) EPS, FB, MY, and FBMY for 28 days. Mice were sacrificed using the cervical dislocation method after anesthesia with isoflurane  at the end of the experiment. Blood samples were obtained by cardiac puncture. In addition, testes and epididymides were removed for measurement and analysis.Each right testis was fixed in a 10% neutral formalin solution  for more than 16 h. After fixation, testes were cut in half at the widest point and embedded in paraffin. Five-micrometer histological sections were deparaffinized, rehydrated, and stained with H&E according to the manufacturer’s instructions . Morphological images were obtained using a TissueFAXS system  at the Instrument Center of Chung Shan Medical University. Seminiferous tubule (ST) numbers were recorded using Scope Photo 3.0 .6 cells/mL), and sperm images were captured at 5 s intervals using a Dino-Lite digital microscope  and DinoCapture 2.0. Active sperms were defined as sperm in different positions on the two images. Ratios of motile sperm (%) = /number of total sperm × 100%.The right epididymis was cut into three pieces and incubated in 1 mL of Ren’s solution at 37 °C for 30 min. After centrifugation at 100 g for 5 min, sperm concentrations were determined using a hemocytometer and Motic BA300 Binocular Compound Microscope . For each mouse, sperm numbers were obtained for five grids and then divided by five. Sperm concentrations were expressed in millions per milliliter , estradiol (E2), and luteinizing hormone (LH) levels were determined according to the manufacturer’s protocols .p < 0.05. All figures were created using Graph Pad Prism version 6.0.0 for Windows .Data are expressed as mean ± standard deviation (SD). The differences between multiple groups were evaluated using one-way analysis of variance (ANOVA) and post-hoc least significant difference (LSD) tests. Statistical significance was set at Experimental protocols were approved by the Animal Care and Use Committee of Da-Yeh University , all animals were treated according to the guidelines for animal experimentation of Da-Yeh University in Changhua, Taiwan. The animal experiments were also performed in accordance with the ARRIVE  guidelines.


Sijunzi decoction (SJZD), a classic Chinese formula, has been clinically used for the treatment of gastrointestinal disorders. However, few studies have uncovered its antitumor effects and its potential mechanisms against gastric cancer (GC). Therefore, this work aimed to identify the active compounds and putative targets of the SJZD and to further explore the potential mechanisms involved in the treatment of GC.  The active compounds and potential targets of the SJZD and related genes for GC treatment were collected from a public database. Traditional Chinese medicine (TCM)-compound-target-disease networks, Venn diagrams, protein–protein interactions (PPIs), gene ontology (GO), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to obtain the bioactive compounds, key targets, and potential pathways. Next, the human gastric adenocarcinoma cell line NUGC-4 was inoculated subcutaneously into the right flank of NCG mice to build a tumor-bearing mouse model to further verify the findings.  There were 117 compounds in the SJZD in total. The SJZD and GC had 161 and 3288 potential targets, respectively, among which 123 targets overlapped. The network analysis showed that quercetin, kaempferol formononetin, ginsenoside, atractylenolide III, etc., were bioactive molecules. The tumor necrosis factor (TNF), interleukin-6 (IL-6), cellular tumor antigen p53 (TP53), transcription factor AP-1 (JUN), and vascular endothelial growth factor A (VEGFA) were potential targets. A KEGG pathway enrichment analysis revealed 110 pathways involved in the pathways for cancer, including the PI3K-AKT signaling pathway. Validation experiments showed that the SJZD inhibited tumor growth and induced apoptosis in tumor cells. In addition, the SJZD downregulated expressions of VEGFA, iNOS, COX-2, and Bax/Bcl2 and inhibited the expressions of p-PI3K and p-AKT.  The SJZD treats GC by inhibiting blood vessel hyperplasia and inducing cell apoptosis by regulating the PI3K/AKT pathway. Helicobacter pylori infections, etc., can still significantly increase the risk of developing GC [With its heterogenous nature, gastric cancer (GC) is a highly aggressive malignancy, and it is the fourth most common cancer in the world, with more than 70% of the cases occurring in the developing world , 2. Due oping GC , 6. GC poping GC . Howeveroping GC . Traditioping GC , 12.Glycyrrhiza uralensis) [The Sijunzi decoction (SJZD) is a classic Chinese formula for “reinforcing asthenia qi” and nourishing the spleen; it consists of Renshen (Codonopsis pilosula), Baizhu , Fulin (Poria cocos), and Gancao  . In clinalensis) . Zhao Aialensis) . HoweverNetwork pharmacology is an integrated discipline that combines systems biology, multidirectional pharmacology, computational biology, and network analysis to investigate the relationships between drugs and diseases to find drug targets . ChineseGlycyrrhiza uralensis Fisch of legumes) were selected, and the herbs' compounds and targets were retrieved from the Traditional Chinese Medicine System Pharmacology Database . The compounds were screened according to the pharmacokinetic parameters “oral bioavailability (OB) ≥ 30%” and “drug likeness (DL) ≥ 0.18”. Then, the targets of GC were obtained from the GeneCards (https://www.genecards.org/), Online Mendelian Inheritance in Man , and Therapeutic Target Database  databases with “Gastric Cancer” as the keyword. Next, the corresponding gene names were found in the UniProt Knowledgebase (UniProtKB) (https://www.UniProt.org/) by searching for “Homo sapiens” [The keywords “Renshen” (Renshen is the root of the acanthaceae plant), “Baizhu” , “Fulin” (Fulin is the dried sclerotia of Poria cocos (Schw.)Wolf), and “Gancao”  with the following limiting condition: species (Homo sapiens). Then, the PPI network was analyzed by using Cytoscape 3.6.1, which obtained “Nodes” representing the target protein molecules and “Edges” representing the mutual relationships.The intersection targets of the SJZD and GC were uploaded to the STRING database (https://david.ncifcrf.gov/home.jsp) by inputting the above targets. Then, the top 10 terms were selected to make graphical structures in GraphPad Prism 8.0. The results of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were obtained from the DAVID database, and a bubble chart was made on imageGP (http://www.ehbio.com) to explore the correlated functional pathways of the selected genes.A gene ontology (GO) analysis including biological process (BP), cellular component (CC), and molecular function (MF) was performed in the DAVID database . The study was approved by the Medical Research Ethics Review Committee of the Beijing Shijitan Hospital of the Capital Medical University . When the tumor size reached 100 mm3, the tumor-bearing mice were randomly divided into 4 groups: model group (n = 6), SJZD-L group , SJZD-H group , and Urelumab group . Mice in the SJZD groups were administered SZJD once a day, and mice in the Model group were given an equal volume of distilled water. The mice in the Urelumab group were injected with Urelumab twice a week. The tumor size was measured every week. After the administration of the drugs for 21 days, the mice were sacrificed, and the tumors were isolated, photographed, and weighed. The tumor growth inhibition weight value (TGIW) and tumor growth inhibition volume value (TGIV) were statistically analyzed. TGIW% = (1-Treatment group weight/Model group weight) × 100%; TGIV% =  (1-Treatment group RTV/Model group RTV) × 100%; Relative tumor volume (RTV) = V21 day/V0 day; Tumor volume = 0.5 × major axis × minor axis2.The human gastric adenocarcinoma cell line NUGC-4 was maintained in a RPMI-1640 medium supplemented with 10% fetal bovine serum at 37°C in a humidified atmosphere containing 5% COL of PBS . The celμm sections using a microtome. After dewaxing and rehydration, the sections were stained with hematoxylin & eosin solution . Tumors were identified on sections. These sections were sealed using neutral gum and photographed under a microscope  at  × 200.Tissues were fixed with 10% neutral buffered formalin overnight, embedded in paraffin, and cut into 4.5 β-actin  was used as an internal control.Tumors were lysed using a radioimmunoprecipitation assay (RIPA) buffer to obtain the proteins. The protein concentrations were detected by a BCA protein assay kit. The proteins were separated by SDS/PAGE and blotted onto polyvinylidene fluoride (PVDF) membranes. Next, the membranes were blocked in skim milk for 2 hours and then incubated with primary antibodies against Bax , Bcl2 , p-PI3K , p-Akt , and VEGFA  at 4°C overnight, followed by HRP-conjugated secondary antibodies. Protein bands were detected using an ECL chemiluminescence system . After dehydration, hydration, and antigen recovery, tumor biopsies were incubated with the primary antibodies at the following dilution ratios: iNOS  and COX-2 . Subsequently, the sections were incubated with a secondary antibody for 2 hours at room temperature. Finally, these sections were sealed using neutral gum and photographed under a microscope at a magnification  × 200.t-test. P < 0.05 was considered to indicate a statistically significant difference.Data are presented as the mean ± standard deviation. Differences between the two groups were investigated using Student's As is shown in the Supplement To perform a comprehensive analysis, a TCM-compound-target-disease network was constructed , including pathways in cancer and the PI3K-AKT signaling pathway . The H&E results showed that the tumor cells in the model group were closely arranged and grew well, with hyperchromatic nuclei and normal morphologies. After treatment with a high dose of the SJZD, the cells showed necrosis and exhibited diffuse and ambiguous distributions . The results suggested that the SJZD regulated the PI3K/AKT/VEGF pathway to treat GC.According to the results of the PPI network and KEGG pathway enrichment analyses, specific targets, including VEGFA, Bax, Bcl2, and the PI3K/AKT pathway, were involved in the SJZD treatment of GC. Therefore, we used WB to measure the expression of these proteins. As shown in Figures P < 0.01). These results demonstrated that SJZD treatment downregulated the iNOS and COX-2 levels in the gastric tumor models.Furthermore, the expression levels of iNOS and COX-2 were evaluated by immunohistochemistry. Figures In this study, network pharmacology and in vivo experiments were performed to reveal the bioactivity and mechanism of SJZD action for treating GC. The TCM-compound-target-disease network showed connections between multiple compounds and targets, which is consistent with the holism of TCM . PPI resThe SJZD, a traditional Chinese prescription, has been used for the clinical treatment of gastrointestinal disorders for over 2000 years . Due to The PPI network and KEGG results indicated that VEGFA was the main target in the treatment with SZJD against GC. VEGF is a well-known angiogenic factor and plays an important role in the proliferation, migration, and invasion of vascular endothelial cells . The angTo further explore the mechanisms of SJZD action for the treatment of GC, KEGG enrichment analysis was performed, which showed that the PI3K-AKT signaling pathway was the most enriched gene involved in the SJZD treatment of GC. The PI3K-AKT signaling pathway participates in many cellular activities, including cell growth, differentiation, apoptosis, and cytoskeletal rearrangement . PI3K isIn summary, our study combined network pharmacology-based predictions and in vivo experiments to verify the antineoplastic efficacy and the potential mechanisms of SJZD action for the treatment of GC. We found that the SJZD exerted its anti-GC effects by downregulating the expression of VEGFA, iNOS, and COX-2 to inhibit blood vessel hyperplasia and by upregulating the Bax/Bcl2 ratio to induce cell apoptosis by regulating the PI3K/AKT pathway. However, there were still deficiencies in this study. The potential compounds involved in the SJZD treatment of GC need to be identified and validated in vivo or in vitro. In addition, the related targets should be further explored to provide a theory for the SJZD treatment of GC.


To analyze the visual prognosis of macular buckling in patients with high myopia foveoschisis (FS) and to identify factors that predict the final visual outcome.  We retrospectively included 155 eyes of 155 patients who underwent foveoschisis-related macular buckling. Best-corrected visual acuity (BCVA) and coexisting macular pathologies were assessed as a measure of surgical outcome, and multivariate linear regression was performed to identify factors affecting final visual prognosis. P < 0.001). Anatomical success was achieved in 151/155 eyes (97.42%) after the first surgery and in 155/155 eyes (100%) at the 2-year follow-up visit. Both preoperative and postoperative BCVA were better in eyes without macular hole (MH) than in eyes with MH. In patients with MH, the postoperative BCVA was significantly better than that before surgery when the MH was closed. However, the difference was not significant in patients with unclosed MH. Univariate analysis identified that baseline BCVA, age, MH, atrophic myopic maculopathy category, and postoperative intraretinal cyst were significantly related to BCVA at the postoperative 2-year follow-up. Multivariate analysis revealed that preoperative BCVA and age were significant factors.  The mean preoperative BCVA was 1.19 ± 0.55 logMAR (20/308), while the mean postoperative BCVA was 0.82 ± 0.51 logMAR (20/133) ( Better preoperative BCVA and younger age are predictors of better prognosis. Prompt surgery is advised for patients with myopic foveoschisis to improve their visual prognosis. Myopic foveoschisis (MF) is a severe complication of pathological myopia that causes visual impairment. A combination of posterior scleral elongation and counteracting anterior vitreomacular traction of the retinal arterioles causes MF , 2. PrevAdvancing foveal detachment (FD) or MH formation can cause visual impairment in patients with MF. Hence, identifying factors affecting postsurgery visual outcomes is important to achieve favorable results. Some studies have identified prognostic factors significantly related to final visual results –17. HoweThe aim of this study was to investigate the visual outcomes after MB surgery for MF and to identify prognostic factors related to BCVA at the postoperative 2-year follow-up.ClinicalTrials.gov with ID NCT03433547. Informed consent was obtained from all patients. The following inclusion criteria were adopted: (1) patients with the diagnosis of MF; (2) patients who had undergone MB for MF treatment; and (3) a refractive error with a spherical equivalent (SE) ≤ -6.0 diopters and/or axial length (AL) ≥ 26.5 mm. The exclusion criteria were as follows: (1) patients with any intraocular surgery history; (2) patients with dense cataract, advanced glaucoma, or amblyopia; (3) patients with a history of myopic choroidal neovascularization (mCNV); and (4) macular detachment extending to the peripheral retina. Only data corresponding to the surgical eye of each patient were used for the statistical analyses.This was a single-center investigation of 155 eyes from 155 patients who underwent MB for MF between January 2017 and February 2019. All surgeries were performed by a single surgeon (LL). MB was performed using a silicone sponge-titanium explant as had been previously described , 18. ThiAll patients underwent the following examinations: (1) BCVA assessment with refraction; (2) measurement of AL with the Intra Ocular Lens Master ; (3) fundus photography ; and (4) spectral-domain optical coherence tomography (SD-OCT) of the macula  before and after the surgery. Examination results obtained at 6 months, 1 year, and 2 years postoperatively were analyzed. Anatomical success was defined as the complete reattachment of the foveoschisis and any related FD. MH closure was defined as the recovery of normal anatomy without any remaining detachment of the inner retinal layers at the foveal region found on any optical coherence tomography (OCT) scan . The ATNt-tests. Univariate and multivariate linear regression analyses were both performed to evaluate the possible predictive factors associated with postoperative BCVA. Statistical significance was indicated by a P value of less than 0.05.Statistical analysis was performed using SPSS statistical software . All values are expressed as mean ± SD or proportions as appropriate. The VA was compared between the two groups using independent sample A total of 173 consecutive highly myopic patients (173 eyes) were initially selected for the study; however, 18 eyes were excluded due to missing data. A total of 155 eyes from 155 patients were finally included in the study, and their demographics and clinical characteristics are shown in Anatomical success was attained in 151/155 eyes (97.42%) after the first surgery and in 155/155 eyes (100%) at the 2-year follow-up, with 2 patients underwent a second PPV surgery due to retinal detachment, while the buckle of two patients had to be readjusted due to improper positioning. In those without an MH, initial success was achieved with one-time surgery in 102/103 eyes (99.03%) and in 103/103 eyes (100%) at the 2-year follow-up. In eyes with an MH, success after the first surgery was achieved in 49/52 eyes (94.23%), and the MH closure rate was 71.15% (37/52 eyes).P < 0.01). Both the pre- and postsurgery BCVA results were better in eyes without MH than in eyes with MH (P < 0.05). Although the improvement in BCVA was not significantly different between the two groups (P=0.762), the BCVA at the 2-year follow-up was significantly better in eyes without MH than in eyes with MH (P=0.004)  .P=0.003), but not in eyes without MH closure (P=0.091). However, neither the change from pre- to postsurgery BCVA nor the BCVA improvement between MH closure and nonclosure were significant (P > 0.05) (A significant improvement from preoperative to postoperative BCVA was noted in eyes with MH closure ( > 0.05) .P < 0.001). Those eyes with BCVA ≥ logMAR 1.0 at baseline showed a statistically significant difference between pre- and postoperative BCVA . However, those eyes with BCVA < logMAR 1.0 at baseline showed no statistically significant difference between pre- and postoperative BCVA . Additionally, we found that the group with better baseline BCVA indeed obtained a better postoperative BCVA with a statistical significance   .P < 0.001, P < 0.001, P=0.004, P < 0.001, and P=0.039, respectively). However, there was no change in the AMM category of each patient during the two years of observation after the surgery. To evaluate the association between these significant parameters and postoperative BCVA, a multivariate linear regression analysis was performed. Only age and preoperative BCVA remained significantly related to postoperative BCVA   .After macular buckling, patients tended to have a variable elevation of intraocular pressure within the first month, but all recovered to normal levels within three months using pressure-lowering agents or glucocorticoids. Three patients with a postoperative vitreous hemorrhage and five eyes with epiretinal hemorrhage were observed although all the hemorrhages were self-limiting and completely absorbed within three months without any treatment. No postoperative infections occurred. Almost all the patients exhibited some degree of surgically induced eye movement disorder, strabismus, metamorphopsia, and binocular diplopia. However, the symptoms were gradually reduced or fully resolved on their own without medication within two years. Postoperative intraretinal cyst was seen in 17 eyes, but no improvement was observed over time.In this study, we aimed to identify factors that predict the prognosis of visual function after MB in highly myopic foveoschisis. Better preoperative BCVA and younger age were significantly correlated with better visual outcomes.MB is an effective and safe intervention for the treatment of MF in highly myopic eyes , 14, 18.Our study revealed that better preoperative BCVA and younger age are indicative of good postsurgery outcomes in visual function. Several studies have identified prognostic factors for PPV in highly myopic foveoschisis , 20, 21.There was no independent correlation between final BCVA and other presurgical factors, including AL, refractive error, and FD. Previous studies , 23 haveIn our study, postoperative intraretinal cyst was seen in 17 eyes (10.97%) but in none after PPV surgery . 47.06% There were some limitations in our study. First, this was a retrospective study. Second, selection bias and allocation bias cannot be discarded, and the surgeon's preferences, experience, and skills might have affected the choices made during the surgical procedure and therefore the outcome. Third, variability would be expected at the level of indentation for each patient in terms of relative height and shape, as these performances were under the subjective judgement of the surgeon through indirect ophthalmoscopy. Fourth, two different types of OCT were used in our study, which might have an influence on the judgement due to the difference in their procedures though they have the same diagnostic capability.In conclusion, the main factors affecting postsurgery BCVA were preoperative BCVA and age. Current maneuvers in MB surgery are highly effective for foveal reattachment.


Methotrexate (MTX) is a first-line treatment for rheumatoid arthritis (RA), but its clinical use is greatly limited by the adverse effects and poor patient compliance caused by traditional oral administration or injection. In recent years, some transdermal drug delivery systems have received considerable attention due to overcoming these shortcomings. In this study, we developed dissolving microneedle patch (DMNP) for transdermal delivery of MTX to treat RA safely and effectively. The morphology, mechanical strength, skin insertion, drug content, in vitro transdermal delivery, and other properties of DMNP were characterized. Meanwhile, the adjuvant-induced arthritis model of rats was established to investigate the therapeutic effect of MTX-loaded DMNP in vivo. The results showed that the microneedles had excellent morphology with neat array and complete needles, good puncture performance and mechanical strength, and rapid intradermal dissolution rate. In vitro transdermal delivery results indicated that microneedles could significantly increase drug transdermal permeation compared with the cream group. The pharmacological study showed that MTX-loaded DMNP significantly alleviated paw swelling, inhibit inflammatory response via downregulating the levels of TNF-α and IL-1β, relieved synovium destruction with less cartilage erosion, and slowed the progression of RA in AIA rats. Besides, DMNP presented better therapeutic performance than cream or intragastric administration at the same dosage of MTX. In conclusion, the MTX-loaded dissolving microneedle patch has advantages of safety, convenience, and high efficacy over conventional administrations, laying a foundation for the transdermal drug delivery system treatment of rheumatoid arthritis. The DMNP was systematically characterized, including drug content, skin insertion, and in vivo dissolution. In addition, the therapeutic efficacy of DMNP on rheumatoid arthritis was observed in adjuvant-induced arthritis (AIA) rats through evaluating paw swelling, radiological change of ankle joint, histological analyses, and cytokines level in serum. The MTX–HA dissloving microneedle patch introduced in this article, a safe, effective, and convenient administration strategy of MTX, has great clinical application prospect in the treatment of RA .2.2.1.PVP K90 was purchased from BASF (Germany). Methotrexate  (purity >98%) was obtained from Heowns OPDE Biochemical Technology Co. Ltd. . HA was purchased from Huaxi Biotechnology Co. Ltd. . Dextran (Dex) was provided by Thermo Fisher Scientific (USA). Bacillus Calmette-Guerin was purchased from the Chengdu Institute of Biological Products Co., Ltd . Trypan Blue, stearic acid, and stearyl alcohol were provided by Sinopharm Chemical Reagent Co., Ltd (China). The reagents of acetonitrile (chromatographic grade), anhydrous sodium hydrogen phosphate, liquid paraffin, Span 80, polysorbate 80, glycerinum, and sorbic acid were provided by Macklin . Citric acid was from Biotechnology Co. Ltd. .2.2.Sprague-Dawley rats (SD rats)  were provided by Bengbu Medical College Laboratory Animal Centre. All animal experimental procedures were strictly in accordance with the guidelines for animal experiments of Bengbu Medical College and approved by the Animal Care Committee of Bengbu Medical College.2, and 90% relative humidity.Human fibroblast synovial cells were obtained from National Collection of Authenticated Cell Cultures. The cells were cultured in Dulbecco’s modified eagle medium in an incubator at 37 °C, 5% CO2.3.The brass master mold and polydimethylsiloxane (PDMS) mold of DMNP were fabricated according to our previous study  was applied to verify the structures of MTX, sodium hydroxide , HA , the physical mixture of the three components (PM), and the microneedle powder (MNP). In addition, the chemical compositions of these components were studied by Fourier-transformed infrared spectroscopy (FTIR).The morphology of MTX-loaded DMNPs was examined and the photos were taken by scanning electron microscopy (SEM) and an electron microscope .The mechanical properties of MTX-loaded DMNPs and blank DMNPs were evaluated using a texture analyzer according to previous studies  were prepared as in a previous method. After thawing and removing residual saline, the stratum corneum of skin was placed side up. Subsequently, trypan blue-loaded DMNPs were penetrated into the skin by thumb for 5 min. The insertion performance was obtained by a microscope and the penetration ratio was calculated simultaneously.2.6.To investigate the in vivo insertion performance of DMNPs and skin recovery, SD rats were anesthetized with 10% chloral hydrate and shaved with an electric razor. Drug-loaded DMNPs were inserted into the skin of thigh. The rats were sacrificed at different times, and the inserte site was removed and fixed in 4% paraformaldehyde solution. Then, it was embedded in paraffin, sliced into sections, and stained by hematoxylin and eosin (H&E). The insertion depth and skin recovery were observed under the microscope.2.7.Y = I/I0, I0 and I mean the initial and remaining length of the microneedles, respectively) of DMNPs at different time (X).MTX-loaded DMNPs were inserted into the hairless dorsal skin and removed after different predetermined times . The dissolution degree of microneedles was observed by a microscope. The dissolution curve was drawn by the remaining height ratio , stearyl alcohol (0.6 g), white valentine (0.6 g), liquid paraffin (0.9 g), and Sorbitan Oleate (0.16 g) were mixed and melted at 80 °C in a water bath to form the oil phase. The aqueous phase consisted of polysorbate 80 (0.44 g), glycerinum (1 g), and sorbic acid (0.02 g) and was heated to the same temperature. MTX (0.1 g) was dissolved in dilute alkali solution and added to the aqueous phase. Then, the aqueous phase was added to the oil phase dropwise and the mixed solution was stirred continuously until it cooled to room temperature. The final total weight should be complemented to 10 g with distilled water. Thus, the MTX cream (1%) was successfully prepared.2.9.The loading content of MTX in MTX-loaded DMNPs was measured by HPLC, which has gone through method validation. Drug-loaded DMNPs were separated into needles and base layer according to our previous study  and replaced with an equal volume of fresh PBS buffer. After filtrated by membrane, the MTX concentration of samples was determined by HPLC.2.11.In vitro cytotoxicity of excipients and blank DMNPs were studied by MTT assay. Fibroblast synovial cells from human rheumatoid arthritis were incubated with DMEM medium containing drug-blank DMNPs, HA, and PVP at different concentrations for 48 h. MTT and DMSO were successively added into each well to form and dissolve bluish violet formazan crystals, respectively. The absorbance was determined via a microplate reader . The cell viability (%) was calculated according to 2.12.2.12.1.n = 6). The drug groups were administrated MTX (0.2 mg) at different formulations every there days for seven times. DMNPs were inserted into the prepared hairless skin of thigh by thumb and immobilized for 30 min by medical tape to ensure the complete dissolution of needles.AIA model was established to evaluate the therapeutic potency of MTX-DMNs in vivo. Bacillus Calmette-Guerin was thoroughly ground with the sterile liquid paraffin to produce complete Freund adjuvant . The rats were injected subcutaneously at the right hind paw with 80 μL of CFA to build AIA model on day 0. On day 21, the successful modeling of rats was randomly divided into four groups: model group, MTX cream group, MTX oral administration group, and MTX-loaded DMNPs group. Normal rats without induced by CFA were put in normal group  = 2.12.2.The severity of swelling of soft tissue and narrowing of joint space was measured using radiological examination. After the last dose, the rats were anesthetized and subsequently performed the examination of ankle and hind limb by X-ray imaging.2.12.3.After blood collection, the rats were sacrificed on day 45. The synovium of ankle join was immediately removed and fixed in 4% paraformaldehyde for further study. After one week, the fixed tissue was embedded and sliced to sections, which were stained with H&E and observed by optical microscopy for histopathological analysis.Meanwhile, the sliced samples were labeled with protein according to the immunohistochemical protocol. After DAB staining, the brown area was observed as positive expression under the microscope to analyze the immunohistochemical result.2.12.4.To determine the serum cytokine level, blood samples (3.5 mL) were collected from the oculi vein after last drug administration. After standing at 4 °C for 1 h, the samples were centrifuged at 5000 rpm for 15 min to obtain the supernatant serum and stored for biochemical assay. The serum level of interleukin-1beta (IL-1β) and TNF-α were measured using enzyme-linked immunosorbent assay  in accordance with the manufacturer’s protocol.2.13.p < .05 was considered statistically significant, and p < .01 represented extremely significant.The data were performed by GraphPad Prism software (Version8.0.2) with one-way ANOVA and expressed as the mean ± standard deviation. 33.1.Supplementary Figure S1(A–C)).MTX-loaded DMNPs were prepared using micromolding with a two-step process as shown in As shown in 3.2.The morphology of MTX-loaded and blank DMNPs before and after pressure was shown in In order to study drug and excipients compatibility, FTIR and XRD were used to analyze the structure and morphology of MTX and auxiliary materials, respectively. Considering that a two-step process was used to prepare MTX-loaded DMNPs, it was mainly studied the interaction between MTX and needle materials.Supplementary Figure S1(D)) showed that the characteristic band of MTX at 3300–3400 cm−1 is the stretching vibration of N-H. The characteristic band at 1641 cm−1 corresponded to -CONH. The characteristic band at 1601 cm−1 belonged to the C = C stretching vibration of the benzene ring, while the band at 1496 cm−1 was due to the symmetric stretching mode of COO-. The peaks centered at 1208 cm−1 represented C-N stretching. Remarkably, the characteristic spectrum of MTX was also existed in PM and MNP. Therefore, the chemical structure of MTX did not alter during the preparation of microneedles.The FTIR spectrum of MTX , the effect of excipients on the crystal state of MTX was further studied by XRD analysis. The characteristic peaks of MTX were still present in PM and MNP, indicating that the addition of HA and sodium hydroxide did not affect the crystal form of MTX. Meanwhile, the characteristic peak of sodium hydroxide in the mixture was not obvious, which may be due to the tiny proportion.As shown in 3.3.R2 = 0.9998). The retention time of MTX was 6.2 min without interference from excipients . The intra-day and inter-day average precisions were 0.7 and 0.97 respectively (Supplementary Table S1). The results demonstrated a good recovery in the range of 95.19%–−101.07% with RSD < 2% (Supplementary Table S2).It had been validated that HPLC-UV method was reliable for the determination of MTX content in DMNPs, with good linearity in the concentration range of 5–220 μg/mL ). Considering the needle shape and needle loading capacity of MTX-DMNPs, HA was selected as the needle material of DMNPs for subsequent study.The excipients of DEX, HA and CS were chosen to prepared MTX-loaded DMNPs and the drug loading was detected by HPLC. The results showed that the drug contents of the needle part in three prescriptions of MTX-loaded DMNPs were 82.33 ± 3.36, 205.08 ± 3.77, and 167.67 ± 2.07 μg, respectively . The results showed that the insertion ratios of 100 and 200 mg/mL HA were about 72% and 85% respectively, while the ratios of 300 mg/mL HA were more than 95%, which was therefore adopted to fabricate DMNPs for further studies.To examine whether the microneedles could successfully penetrate the skin, in vitro and in vivo insertion experiments were performed. In in vitro experiments, the isolated dorsal skin of SD rats was penetrated using TB-loaded DMNPs for 5 min and removed afterward. Trypan blue was adopted because it stains internal tissues of skin through micropores and could not penetrate the The puncture performance in vivo and skin recovery were also investigated in rats using MTX-loaded DMNPs. As shown in In the experiment of skin recovery , the adm3.5.To evaluate the bio-safety of excipients, fibroblast synovial cells were incubated with blank DMNPs or mixture of excipients and cell viability was detected via MTT assay. The results showed that more than 80% of cells were viable at experimental concentrations , which i3.6.To further determine the dissolution time of MTX-loaded DMNPs in skin, the remaining height of the microneedles were observed by a microscope after being pressed on the legs of rats for different time , and the3.7.To study and compare the transdermal delivery performance of MTX cream and MTX-loaded DMNPs, the isolated hairless dorsal skin of rats and Franz diffusion cells  were app3.8.3.8.1.AIA model is always applied in pharmacodynamic study as it has similar immunological and pathological features with human . Compared with model group, the paw thickness of rats treated with drug formulations was markedly decreased after the last administration , indicating that long-term MTX treatment reduced the paw swelling degree and relieved inflammation. Simultaneously, the paw swelling degree among different MTX preparations was also analyzed. The swelling volume of MTX DMNP group was significantly reduced compared to that of cream group (p < 0.01) or oral group (p < 0.05) at the same dosage of MTX. The paw images further confirmed that the paw swelling of RA rats treated with MTX formulations were significantly relived on day 42  and thus enhanced the inflammatory response, resulting in the damage of synovial tissue. Therefore, the serum level of pro-inflammatory cytokines determined by ELISA kit was often used to evaluate the efficacy of RA treatment. TNF-α and IL-1β are frequently used to evaluate RA treatment. IL-1β is a vital cytokine that promotes inflammation, regulates chondrocyte apoptosis and accelerates joint destruction via increasing osteoclast absorption , suggesting that cytokines content in the AIA model rats was closely related to the progression of RA. The serum levels of TNF-α and IL-1β in AIA rats treated with different MTX formulations were decreased, especially in oral and DMNP group. Compared with cream group, the concentrations of TNF-α and IL-1β in MTX DMNP group were significantly reduced (p < .01), which may be attributed to the penetration promotion of MTX by DMNP. Likewise, DMNP group showed significant decrease level of TNF-α (p < .05) compared to oral group. The level of IL-1β in DMNP group was lower than that of oral group, but there was no significance difference, which may be due to the low concentration and considerable individual differences. In conclusion, MTX-loaded DMNPs could alleviate inflammation of RA effectively by decreasing the serum concentrations of pro-inflammatory cytokines.As shown in 4.As far as we know, there was few reports about the effect of MTX-loaded dissolving microneedles on RA. The delivery of MTX via microneedles was mainly studied for psoriasis treatment (Tekko et al., Click here for additional data file.


Endometrial cancer (EC) is the most common malignancy of the female reproductive system. Cancer antigen 125 (CA125) is a serum tumor marker widely reported in EC patients, particularly those with poor prognostic factors such as grade 3 tumors, deep myometrial invasion, lymph node metastasis (LNM), and extra-uterine disease. This retrospective study stratifies pre-operative CA125 levels to evaluate odds ratios (OR) and relative risk (RR) between CA125 levels and the likelihood of +LNM, lymphovascular space invasion (LVSI), grade, and stage. Patient charts for women 18 years or older with a diagnosis of EC and pre-operative or within one week CA125 measurement from January 2000 to January 2015 at a regional hospital were reviewed. OR and RR were determined by unconditional maximum likelihood estimation for CA125 levels as the predictor with staging, grade, +LVSI and +LNM as outcomes. The largest increase in risk for patients having stage I/II/III disease was 52% greater (1.52-fold risk) while largest increase in risk for patients having stage III/IV disease was 67% greater (1.67-fold risk), both at CA125 ≥ 222U/ml. Patients with CA125 ≥ 122U/ml had significantly increased risk of +LNM, with maximum increase in risk of 98% (1.98-fold risk) at 222U/ml. Patients with CA125 ≥ 175U/ml had significantly increased risk of +LVSI, with maximum increase in risk of 39% (1.39-fold risk) at 222U/ml. This study shows that elevated CA125 levels correspond to increased stage, +LVSI, and +LNM in patients with EC. A rising incidence of EC has been reported partly due to the obesity epidemic in the United States2. Consequently, the number of women diagnosed with EC is expected to significantly increase in coming years.Endometrial cancer (EC) is the most common malignancy of the female reproductive system in developed countries. According to the American Cancer Society, approximately 66,570 new cases of cancer of the body of the uterus were diagnosed in 2021, and almost 12,940 women will die from the disease3 has undergone several iterations of the surgical staging of EC. In an effort to achieve postoperative pathological staging, the primary surgical procedure for patients with all stages is a total hysterectomy with bilateral salpingo-oophorectomy and some form of lymph node sampling, whether sentinel lymph nodes or systematic lymphadenectomy. The addition of pelvic and or para-aortic lymph node dissection depending on various factors, such as tumor size, grade, subtype, myometrial invasion (MI), lymphovascular space invasion (LVSI), cervical involvement, and extrauterine metastasis is also performed5. Despite this, extensive lymphadenectomy has been linked to an increased incidence of surgical related complication such as lymphedema, deep vein thrombosis (DVT), intraoperative hemorrhage, and lymphocyst formation5. Further, lymph node sampling does not necessarily improve survival6. Several studies have argued whether a lymphadenectomy should be performed in patients with tumor size < 2 cm, tumor grade 1 or 2, endometrioid subtype, and < 50% myometrial invasion7, since the chance of lymph node metastasis (LNM) is < 5%8. While routine lymphadenectomy does not improve disease-free or overall survival, positive lymph nodes do aid in the prognosis and management of the disease. Knowledge of patients with a higher risk of lymph node involvement preoperatively would be beneficial when planning for surgery and discussing prognosis and surgical procedure with patients.The identification of those at highest risk of recurrence is vital in treatment planning. Since 1998 the International Federation of Gynecology and Obstetrics (FIGO)9. Ever since it was found that serum CA125 levels increased with the clinical course of some malignancies10, its use as a tumor marker has been reported in EC patients, particularly those with poor prognostic factors such recurrences, grade 3 tumors, deep myometrial invasion, LNM, and extrauterine disease. Previous work has evaluated preoperative serum CA125 levels as a predictor of metastatic disease and survival12. While studies have attempted to stratify CA125 utility, its clinical value in the evaluation of EC remains ambiguous since it can be mildly elevated in other non-malignant conditions14. Its use either in combination with imaging or alone may be able to help predict or stratify patients in a preoperative setting. Further, the marker is considered partially helpful in evaluating patients with significant comorbidities and perioperative risk who may not be candidates for comprehensive staging15. Additionally, CA125 has been incorporated into several preoperative predictive models with varying degrees of success. Clinically, CA125 can be a sensitive marker when using a cutoff of 35U/ml since only around 1% of healthy women have greater levels16. Furthermore, 6% of women with benign disease and 23% of women with no gynecological cancers have levels ≥ 35U/ml16, with elevated levels in over 80% of women with non-mucinous ovarian cancer.The use of cancer antigen 125 (CA125) as a predictor of endometrial cancer lymphovascular space invasion and lymph node metastasis remains understudied. CA125 is a serum tumor marker widely used in epithelial ovarian cancersIdeally, to minimize overtreatment and complications following a lymphadenectomy in EC patients, risk factors for + LNM could be used to distinguish patients more likely to benefit from lymphadenectomy versus those who would not. Therefore, the purpose of this study was to identify odds ratios (OR) and relative risks (RR) between pre-operative CA125 levels and the likelihood of + LNM, + LVSI, high grade, and advanced stage.A total of 890 patients with a diagnosis of endometrial cancer were included in the primary analysis, out of which 413 had pre-operative staging information, 414 had pre-operative grade information, 449 had LVSI information, and 411 had LNM information  fold risk of having stage III/IV disease. Since a proportion of EC patients are first identified and some incidentally treated by general gynecologists, being able to risk-stratify patients preoperatively into low-risk versus high-risk is of utmost importance to ensure not only accurate patient referral to gynecologic oncologists but also to select appropriate surgical procedures.In contrast to previous work, this study stratifies specific CA125 ranges at which there is an increase in relative risk of having a more advanced stage, higher grade, + LNM, and + LVSI. In this way, patients can be better risk-stratified in the preoperative period, allowing for more informed discussions regarding treatment options and having the improved knowledge of these risks during surgery. Previous studies have found that patients with deep myometrial invasion, + LVSI, or lymph node metastasis can have significantly elevated CA125. As such, CA125 levels are a marker in both preoperative and recurrent settings. If CA125 is elevated in the preoperative workup, it may be able to predict the need for further imaging, tests or biopsies prior to surgery, or better prepare patients for potential procedures to be incurred intraoperatively in the event of advanced malignancies.Limitations of this study include a single-center retrospective study and lack of evaluation of a specific age associated with CA125. In addition, since elevated CA125 levels can occur in various benign gynecologic conditions and other disease states, such as leiomyoma and endometriosis, elevated CA125 may not be an ideal serum biomarker in premenopausal patients.17. Logistic regression models were used in Yang et al.18 and Anton et al.19 to predict + LNM in EC patients. CA125 was found to be useful for predicting high-risk patients, including + LNM, by Zamani et al.20. Preoperative CA125 levels were significantly associated with deep myometrial invasion, advanced stage, and extrauterine and node metastasis in a study by Chen et al.21. Our study confirmed similar findings with a CA125 level of ≥ 222U/ml having up to a 38%  increase in the risk of LVSI. Also, using the same level gives a significantly increased risk of + LNM with the maximum increase in the risk of 98%  for a CA125 of ≥ 222U/ml. These data are further supported by the study by Antonsen et al.22, showing CA125 as a strong predictor for the presence of + LNM.Endometrial cancer is staged according to well defined and evidence-based surgical staging protocols, as clinical staging is less accurate and cannot assess LVSI, grade, or LNM. Proper surgical staging including lymph node assessment is crucial, especially in those with high-risk preoperative features to provide the best prognostic information. Previous studies have examined the predictive accuracy of high-risk features in endometrial cancer and serum markers such as CA125. Levels of CA125 ≥ 21.2 U/mL predicted + LVSI in women with EC in a study by Zhou et al.Our study was able to stratify CA125 levels with respect to increases in stage, + LVSI, + LNM, and grade in patients with EC. Further, we were able to quantify specific values at which the risk for the presence of high-risk features in the preoperative patient with EC could be utilized to better risk-stratify patients before surgery. These data could also be used to minimize potential postoperative risks in patients who are poor surgical candidates. Evaluation of CA125 in relation to other potential clinical variables to enable a multivariable analysis will be considered in follow-on work.This is a large single-center retrospective chart review of 890 patients diagnosed and surgically staged for EC between January 2000 to January 2015 at WellSpan York Hospital. Female patients over the age of 18 years old with a diagnosis of endometrial cancer and a pre-operative or within one week CA125 level were eligible for inclusion. All research was performed in accordance with the relevant institutional guidelines and regulations.23. CA 125 measurements were obtained with electrochemiluminescence Elecsys CA 125 II assay  and quantified by Labcorp . Currently, the standard method for assessing LVSI is light microscopic examination of haematoxylin and eosin (H&E) stained sections as the presence of tumor emboli within vascular channels lined by endothelial cells. The majority of patients had either endometroid adenocarcinoma (75.2%), serous (5.5%), mucinous (1.3%), clear cell (1.3%), mixed cell (4.2%) or carcinosarcoma (3.0%) histology, while the remainder had other histology subtypes (9.5%). Patients were subjected to either systematic lymphadenectomy or sentinel node sampling as determined by the operating physician in accordance with standard clinical protocol. Both pre-operative and post-operative pathology were analyzed by the same clinical oncology/pathology team.Data were abstracted using the PowerChart and E-care electronic medical records systems. Collected data included patient age at diagnosis, preoperative CA125 value, the procedure performed , both preoperative as well as postoperative histology, cancer grade (1/2/3), positive/negative lymph nodes, positive/negative lymphovascular invasion, pathologic stage (I/II/III/IV), pelvic/abdominal metastasis, race/ethnicity, physical exam findings, radiological imaging, and laboratory findings. The reference range of CA 125 is 0–35 units/mL (0–35 kU/L). The cutoff of 35 kU/L for CA 125 was determined from the distribution of values in healthy individuals to include 99% of the normal populationPatients with a historical diagnosis of endometrial cancer, those who did not have a documented CA1-25 level, pregnant women, or those who had been previously staged surgically were excluded from the study.The primary outcome was to determine OR and RR between a preoperative CA125 level and the likelihood of lymph node metastases, lymphovascular invasion, advanced stage, and high grade. Based on CA125 levels, a secondary outcome was to identify CA125 ranges that correspond to these variables.24, with scripts that utilized functions from the following R packages: Odds ratios (OR) and relative risk (RR) were calculated by unconditional maximum likelihood estimation  using oddsratio and riskratio functions from epitools package25. In this retrospective study, both RR and OR were calculated to improve data interpretation and avoid exaggerated estimates, as OR has the possibility to overestimate event odds when there is a relationship between patient groups and outcomes26. For CA125 averages and medians, 95% confidence intervals were produced with a non-parametric bootstrap method using boot function from the boot package where the number of resampling iterations was 1000 and the statistic was the average or median, respectively. P values for comparing medians and means were calculated using Wilcoxon rank-sum test and unpaired student’s T-test, respectively. P values for OR and RR were calculated with Fisher's exact test using a two-sided 95% CI, where the CI was calculated using a normal approximation.Data analyses were conducted in the R programming language (version 4.0.2)OR and RR were determined for CA125 levels as the predictor with staging, grade, + LVSI, and + LNM as outcomes. All RR and OR values were calculated relative to a CA125 level of 35U/ml or below being considered normal. Staging was subclassified as follows: stage I versus stage II/III/IV; stage I/II versus stage III/IV; stage I/II/III versus stage IV. Grade was subclassified as follows: grade 1 versus grade 2/3; grade 1/2 versus grade 3. CA125 thresholds were incremented by 1U/ml from 1 to 222 with OR and RR re-calculated at every threshold. A CA125 level of 35 U/ml was used as a baseline, as values at or below this level are considered normal.The study was approved by Institutional Review Board (IRB) at York Hospital Human Subjects Research Protection Program Office (IRB number 731831-1). The study was exempt from informed consent.


Our pathogenicity investigation showed that GS01 could cause an inflammatory reaction and induce serious diarrhea in neonatal piglets. To our knowledge, this is the first isolation and characterization of PSV in western China. Our results demonstrate that the PSV GS01 strain is destructive to neonatal piglets and might show an expanded role for sapeloviruses.Diarrhea is one of the most important problems associated with the production of piglets, which have a wide range of possible pathogens. This study identified a strain of porcine sapelovirus (PSV) by using next-generation sequencing (NGS) technologies as the pathogen among fecal samples in a pig herd. Phylogenetic analysis showed that the PSV isolates shared a unique polyprotein and clustered with Chinese isolates identified before 2013. The PSV strain was then isolated and named GS01. The IMPORTANCE Porcine sapelovirus (PSV) infection leads to severe polioencephalomyelitis with high morbidity and mortality, resulting in significant economic losses. In previous studies, PSV infections were always subclinical or only involved a series of mild symptoms, including spinal cord damage, inappetence, diarrhea, and breathless. However, in our study, we isolated a novel PSV by virome analysis. We also determined the biological characteristics of this virus in vitro and in vivo. Our study showed that this novel PSV could cause an inflammatory response and induce serious diarrhea in neonatal piglets. To our knowledge, this is the first isolation and characterization of PSV in western China. These findings highlight the importance of prevention for the potential threats of PSV. Diarrhea is a common but serious gastrointestinal disease in pigs, especially for suckling piglets. The main clinical symptoms include diarrhea, dehydration, and poor growth, even upon recovery. The morbidity and mortality vary among different causative agents. To date, with the development of drugs and facilities, bacteria and parasites are hardly the main factors contributing to diarrhea. On the contrary, more and more viruses are identified to be associated with enteric diseases. The most destructive enteric viruses, including porcine epidemic diarrhea virus (PEDV), swine acute diarrhea syndrome coronavirus (SADS-CoV), transmissible gastroenteritisvirus (TGEV), porcine rotavirus (PRoV), and porcine deltacoronavirus (PDCoV), can result in a death rate from 40% to 100% 14. Moreov5–The metagenomics approach, based on next-generation sequencing (NGS) technology, is an effective strategy for the detection of all potential pathogens in a sample. It was used in various independent studies for the detection of viruses in pig samples and many others –16. The –In this study, diarrhea samples containing unknown pathogens were collected from a pig farm in Gansu Province, China. After the failure of target detection of PEDV, TGEV, PDCoV, SADS-CoV, and PRoV, which was performed by reverse transcription-PCR (RT-PCR), the samples were then subjected to NGS. PSV was identified as the causative agent and successfully isolated. The molecular and biological characterization of this PSV isolate are systematically described here.In March 2021, diarrhea of unknown pathogens broke out in a pig farm in Gansu Province, China. Although no obvious clinical symptoms were observed in sows, suckling piglets ≤21 days old displayed serious watery diarrhea and dehydration. The death rate of suckling piglets was about 10%. The recovered pigs presented with growth retardation. None of the viruses PEDV, TGEV, PDCoV, SADS-CoV, PCV2, PCV3, PCV4, or rotavirus were detected in samples via PCR analysis. Therefore, collected samples were prepared for NGS, analyzed, and searched against the NCBI NT database to figure out the potentially associated pathogens . The res7.5 50% tissue culture infective doses (TCID50), suggesting the PSV strain could efficiently replicate in PK1 cells (PK1 cells grown to 85% confluence were inoculated with filtered samples and replaced with fresh medium at 1 h postinfection (hpi). The cells displayed visible cytopathic effect (CPE) from 36 hpi. CPE included shrinking, rounding, and detachment of all cells from 48 hpi. An immunofluorescence assay (IFA) confirmed the existence of PSV-VP1 in inoculated cells . The supK1 cells . To deteK1 cells . All theOM328111). The full genome of PSV GS01 was 7,530 bp in length, contained a 5′ untranslated region (UTR) of 474 bp, a polyprotein gene of 6,996 bp, and a 3′-UTR of 60 bp. Compared with other PSV strains, GS01 exhibited shorter 3′-UTR sequences. The length of the 5′-UTR was 489 to 491 bp, which was 80 to 120 bp shorter than the usual 3′-UTR for other strains. The phylogenetic tree based on the sequence of the polyprotein gene was then constructed with reference strains from China and other countries.For further analysis of the genomic characteristics of PSV GS01, its complete genome sequence was assembled by NGS and later submitted to GenBank  as the control. All piglets were observed daily for clinical scores and body temperature. The clinical symptoms were evaluated using 5 grades . The fecal swabs were also collected daily to determine virus shedding. The results showed that no significant clinical symptoms were observed among the challenged piglets compared to control piglets during the first 3 days. The clinical scores demonstrated that the physical situations of piglets became polarized after 3 days postinfection (dpi)  . At 4 dpon (dpi) . At 6 dpon (dpi) . Therefoon (dpi) . In conton (dpi) . Moreoveon (dpi) . In addion (dpi) . All theIn this study, NGS methods were used to detect unknown pathogens of porcine diarrhea samples. PSV was the only identified virus among the samples. The PSV GS01 strain was then successfully isolated. Phylogenetic analysis demonstrated that the isolate could be classified into a Chinese subcluster that contained PSV strains rarely identified since 2013. A new mutant cluster was identified within the VP1 of GS01 strain. The pathogenic examination further showed this virus could induce serious diarrhea threatening to suckling piglets.Sapelovirus in the family Picornaviridae, according to the taxonomic criteria system of the International Committee on Taxonomy of Viruses  and reverse transcribed used random hexamers, following DNase treatment and second-strand synthesis. Nextera XT reagents (Illumina) were used for library preparation. Briefly, DNA was first tagged and fragmented by using the Nextera XT transposon, followed by the addition of adapter sequences to the ends. The tagmented DNA was then amplified via a limited cycle PCR program. AMPure XP beads were used to remove the very short library fragments to purify the library DNA. The library was finally normalized and loaded for sequencing. Sequencing was performed with the NovaSeq 6000 system (Illumina) by Shanghai Tanpu Biotechnology Co., Ltd. . Bioinformatics analysis of the data was completed using the pipeline described in LLC-PK1 cells were purchased from the China Center for Type Culture Collection and maintained in our laboratory. The cells were cultured in MEM 199  supplemented with 0.1 U/mL bovine insulin and 10% fetal bovine serum . For virus isolation, LLC-PK1 cells were inoculated with the filtered samples and observed daily for 3 days to monitor the development of CPE. At 3 dpi, cells were subject to three freeze-thawing cycles. The supertanant was then collected. After five passages in LLC-PK1 cells with CPE appearance, the viral stock was used for purification. Briefly, confluent monolayers of PK1 cells were incubated with viral stock for 1 h at 37°C on a rocker. After washing with phosphate-buffered saline (PBS), the cells were overlaid with 0.75% low-melting-point agarose  in MEM 199 containing 5% FBS and incubated at 37°C for 72 h. To obtain purified virus, the agarose within a single plaque was collected and used for viral amplification. The primers used for PSV detection are listed in The culture medium of PSV-infected cells was collected and centrifuged at 12,000 rpm for 5 min to remove cell debris. The supertanant was then concentrated at 10,0000 x g for 2 h, at 4°C. The pellet was resuspend in PBS and purified by sucrose density gradient centrifugation. The sucrose cushion was 20% and 40%. PSV particles were below the 40% sucrose cushions. The pellet was resuspended in PBS and spotted onto Formvar-coated grids for 10 min followed by negative staining with phosphotungstic acid for another 10 min at room temperature. The grids were finally subjected to observation with a Hitachi electron microscope (HT7700) at 80 kV.LLC-PK1 cells were seeded in 12-well plates and infected with filtered samples at 37°C. At different time points, cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 at room temperature. The cells were then blocked with 5% skim milk and incubated with rabbit anti-PSV-VP1 polyclonal antibody , followed by Alexa Fluor 488 goat anti-rabbit IgG antibody  for 1 h. The cell nuclei were stained with 4′,6-diamidino-2-phenylindole  and examined with a fluorescence microscope  with a video documentation system.−1 and 10−10 and inoculated in LLC-PK1 cells. After incubation at 37°C for 1 h, the cells were washed with PBS three times and incubated for another 48 h. Then, the cells were fixed and subjected to an immunofluorescence assay. Finally, the results were calculated based on the Reed-Muench method.PSV was made into serial 10-fold dilutions between 10OM328111) and reference strains were aligned with the Molecular Evolutionary Genetics Analysis (MEGA) 7.0 tool  (The sequences of PSV GS01 (GenBank accession 6 TCID50/pig), while five pigs belonging to the control group were orally inoculated with an equal volume of MEM 199. Clinical symptoms were observed daily. At 7 dpi, all pigs were euthanized and different organs were collected for viral load determinations. Fecal swabs and tissues were also collected for viral load determinations. The small intestine was analyzed for microscopic lesions. All animal experiments were performed in accordance with the Experimental Animal Ethics Committee of Lanzhou Veterinary Research Institute.For pathogenic examination, 10 pigs of 12 days old were purchased from a farm negative for PSV, PDCoV, PEDV, TGEV, and SADS-CoV. Five pigs were orally inoculated with PSV GS01 . Briefly, the reaction mixtures were incubated at 95°C for 30 s, followed by 40 cycles at 95°C for 5 s and 60°C for 30 s. The sequences of primers and probe were as follows: PSV-F (5′-GGATTACACCAATGCACAG-3′), PSV-R (5′-CCAGTATGTAAAGGCTCT-3′), probe (5′–6-carboxyfluorescein–TCCTTGCTGTGGTCCTCTGC–6-carboxytetramethylrhodamine–3′).For the quantitation of viral shedding and viral loading among different tissues, the real-time PCR analysis was performed in an Applied Biosystems QuantStudio 5 system with Premix PRJNA813904.The complete genome data used in this study are available in the NCBI Sequence Read Archive (SRA) database under BioProject number