Twelve pre-processing protocols for formalin-fixed paraffin-embedded(FFPE)tissue samples were developed by orthogonal experimental design,incorporating different dewaxing buffers(Triton X-100 and xylene),lysis buffers(TFE and RapiGest),and enzyme digestion methods(iST,SP3,and FASP)to explore the optimal experimental conditions.These protocols were assessed based on protein and peptide identification depth,identification stability,and quantitative levels of protein abundance.The results indicated that Triton X-100 and xylene minimally impacted proteomics identification,whereas the TFE lysis buffer and iST digestion method significantly enhanced the proteomics analysis of FFPE samples.Considering the potential toxicity of xylene,the TTI protocol based on Triton X-100,TFE,and iST was determined to be the optimal choice.This protocol exhibited the best repeatability and stability,and a higher number of proteins associated with significant biological functions were identified.In conclusion,the established TTI protocol offered an efficient and comprehensive approach for proteomic analysis of FFPE samples,significantly enhancing the repeatability and stability of protein identification.

With the continuous rise in the incidence of colorectal cancer and the trend towards younger patient population,the existing treatment options,while able to prolong survival,are difficult to avoid significant side effects.It is imperative to develop new treatment strategies.Luteolin(LUT),as a natural herbal active ingredient,has been proved to have broad-spectrum anti-tumor effects in studies of multiple cancer types.However,the mechanism of LUT action in colorectal cancer has not been systematically elucidated.In this study,for the first time,the molecular mechanism of LUT on colorectal cancer SW620 cells from the perspective of proteomics-glycoproteomics co-regulation was revealed.Proteomic analysis identified 472 differentially expressed proteins.Functional enrichment analysis showed that down-regulated proteins were mainly involved in oxidative stress response,mRNA processing,RNA splicing,and actin filament organization among key biological processes,involving oxidative phosphorylation and peroxisome pathways.Up-regulated proteins were mainly involved in DNA replication,protein folding,and rRNA metabolism,closely related to DNA replication and protein processing pathways in the endoplasmic reticulum.At the level of glycoproteomics,231 differentially expressed intact N-glycopeptides were identified.Functional enrichment analysis of corresponding glycoproteins indicateed that LUT might exert biological effects by regulating biological processes such as nuclear organization,nuclear membrane organization,and Fc receptor-mediated signaling pathways,as well as endoplasmic reticulum protein processing and N-glycan biosynthesis pathways.Analysis of key interaction networks revealed 5 core target proteins namely RPS15A,WDR43,FBL,UTP18,and UTP11.The loss of these proteins had been confirmed to inhibit the proliferation and migration of various tumor cells.Notably,altered glycosylation modifications of the lysosome-associated membrane proteins LAMP1 and LAMP2 suggested that LUT might affect tumor metastatic potential by regulating organelle dynamics.It was found that LUT could inhibit the malignant phenotype of colon cancer cells through a dual mechanism of specifically regulating protein expression networks and glycosylation modification patterns,providing new molecular targets and theoretical basis for precise treatment of colorectal cancer based on natural products.

Programmed cell death 1 ligand 1 (PD-L1) is an important immunosuppressive molecule, which inhibits the function of T cells and other immune cells by binding to the receptor programmed cell death-1. The PD-L1 expression disorder plays an important role in the occurrence, development, and treatment of sepsis or other inflammatory diseases, and has become an important target for the treatment of these diseases. Mesenchymal stem cells (MSCs) are a kind of pluripotent stem cells with multiple differentiation potential. In recent years, MSCs have been found to have a strong immunosuppressive ability and are used to treat various inflammatory insults caused by hyperimmune diseases. Moreover, PD-L1 is deeply involved in the immunosuppressive events of MSCs and plays an important role in the treatment of various diseases. In this review, we will summarize the main regulatory mechanism of PD-L1 expression, and discuss various biological functions of PD-L1 in the immune regulation of MSCs.

Since Curcumae Radix decoction pieces have multiple sources, it is difficult to distinguish depending on traditional cha-racters, and the mixed use of multi-source Curcumae Radix will affect its clinical efficacy. Heracles Neo ultra-fast gas phase electronic nose was used in this study to quickly identify and analyze the odor components of 40 batches of Curcumae Radix samples from Sichuan, Zhejiang, and Guangxi. Based on the odor fingerprints established for Curcumae Radix decoction pieces of multiple sources, the odor components was identified and analyzed, and the chromatographic peaks were processed and analyzed to establish a rapid identification method. Principal component analysis(PCA), discriminant factor analysis(DFA), and soft independent modeling cluster analysis(SIMCA) were constructed for verification. At the same time, one-way analysis of variance(ANOVA) combined with variable importance in projection(VIP) was employed to screen out the odor components with P<0.05 and VIP>1, and 13 odor components such as β-caryophyllene and limonene were hypothesized as the odor differential markers of Curcumae Radix decoction pieces of diffe-rent sources. The results showed that Heracles Neo ultra-fast gas phase electronic nose can well analyze the odor characteristics and rapidly and accurately discriminate Curcumae Radix decoction pieces of different sources. It can be applied to the quality control(e.g., online detection) in the production of Curcumae Radix decoction pieces. This study provides a new method and idea for the rapid identification and quality control of Curcumae Radix decoction pieces.
Drugs, Chinese Herbal/analysis* ; Electronic Nose ; China ; Plant Roots/chemistry* ; Limonene/analysis* ; Chromatography, High Pressure Liquid

Aging can cause degenerative changes in the function of multiple tissues and organs in the body. Gastrointestinal diseases and intestinal dysfunction are very common in the elderly people. The purpose of this study is to explore the effect of the total extract of Astragalus membranaceus (Fisch.) Bge. on intestinal function and gut microbiota homeostasis in natural aging mice, which will provide clues for further mechanism study. The natural aging mice model is established and animal experiments follow the regulations of the Animal Ethics Committee of Nanjing University of Traditional Chinese Medicine. The overall health of the mice was evaluated by the "frailty index" scoring method. The intestinal absorption and transport function were measured by detecting intestinal glucose absorption capacity, transport time, lipase and amylase activities of aging mice. Intestinal inflammation was assessed by detecting inflammatory cytokines by enzyme-linked immunosorbent assay (ELISA). The pathological changes in the intestines of aging mice were tested by hematoxylin-eosin (H&E) staining and alizarin blue (AB) staining. The qRT-PCR method was used to explore the gene transcription level related with the proliferation and differentiation of intestinal stem cells. Microbiota analysis based on 16S rDNA were used to evaluate the composition of gut microbiota. The results showed that Astragalus had a tendency to reduce the "frailty index" of aging mice, but did not show a significant difference. In some indicators of aging phenotype, Astragalus has the most significant effect on hair loss and physical fitness. In terms of intestinal function, Astragalus could increase intestinal glucose absorption capacity, shorten intestinal transportation time and promote lipase secretion in aging mice. The levels of inflammatory cytokines such as tumor necrosis factor-α (TNF-‍α) in the aging intestinal tissue were reduced after Astragalus administration. Astragalus also ameliorated the pathological degeneration of the intestinal tissue of aging mice by increasing the length of small intestinal villi, the thickness of colonic mucosa and goblet cell number. In addition, Astragalus elevated the expression of genes associated with the proliferation and differentiation in jejunum and modulated gut microbiota, especially restoring the abundance of Lachnospiraceae. Taken together, the above research results demonstrate the total extract of Astragalus as a key factor improving the intestinal function and gut microbiota homeostasis of aging mice.

The purpose of this research is to study the effect of small molecule compound piceatannol (PIC) on host inflammation in adenine induced chronic kidney disease (CKD) mice, and then to explore its mechanism based on the regulation of gut microbiota. All procedures were approved by the Institutional Animal Care and Use Committee of the Nanjing University of Chinese Medicine. The level of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) was detected by enzyme linked immunosorbent assay (ELISA); UPLC-TQ/MS technology was used to monitor the level of proinflammatory uremic toxin indoxyl sulfate (IS) and p-cresol sulfate (PCS); the expression of occludin was tested by Western blot; in vitro anaerobic culture of gut bacteria was used to produce indole; the abundance of gut microbiota was evaluated by 16S rDNA sequencing. The results showed that PIC had no effect on inflammatory infiltration in kidney tissue of CKD mice, but could decrease IL-6 level in blood and IL-6/TNF-α level in colon tissue. PIC did not improve intestinal occludin protein expression in CKD mice; while it could significantly reduce the levels of IS and PCS in blood and liver of CKD mice. Further mechanism studies showed that PIC could inhibit the synthesis of IS precursor indole in gut bacteria. Moreover, PIC could decrease the abundance of gut bacteria which producing uremic toxin, such as reducing the abundance of indole and p-cresol producing gut bacteria. In conclusion, PIC could regulate gut microbiota and inhibit the synthesis of uremic toxin precursor, thereafter reducing the accumulation of IS and PCS in vivo, ultimately relieving the inflammation of CKD mice.

Hepatic encephalopathy is a common metabolic neuropsychiatric syndrome in the development of end-stage liver disease. Since the concept of intestinal-liver-brain axis was proposed, the relationship between the pathogenesis of hepatic encephalopathy and the gut microbiota has been a hot research topic. In recent years, studies have confirmed that gut microbiota is involved in and affects various pathological processes of hepatic encephalopathy. This article combines the latest research progress at home and abroad to elaborate on the research status of regulating gut microbiota and thus interfering with the pathological process of hepatic encephalopathy, hoping to provide new ideas and methods for the intervention of hepatic encephalopathy based on the regulation of gut microbiota.

Elaphurus davidianus( Milu),a rare animal unique to China,has been used as medicine for more than a thousand years,but the extinction of Milu in modern times resulted in the unavailability of related medical products. Today,the reintroduction of Milu population makes it possible to restore its medicinal usage. The resource reserves of Cervi Cornu,the natural shedding product from Milu,are increasing with the expansion of the population,allowing it to be fully utilized in the medical field. Mijiao Pills,first recorded in Important Prescriptions Worth a Thousand Gold for Emergency( Bei Ji Qian Jin Yao Fang) by Sun Simiao in the Tang Dynasty,is the first Chinese medicinal prescription with Cervi Cornu as the sovereign medicinal,which is effective in tonifying. Its composition,preparation,efficacy and indications,and administration are described in detail in the Important Prescriptions Worth a Thousand Gold for Emergency,which however,have changed significantly over the thousands of years,seriously affecting the clinical application of this classical prescription and related product development. Therefore,the key information of this prescription should be systematically collated and summarized. According to the principles of textual research on key information in ancient classical prescriptions promulgated by relevant authorities,this paper reviewed ancient Chinese medical books of the past dynasties,modern literature,as well as the Pharmacopoeia of the People's Republic of China( 2020 Version) to figure out such key information as the source,historical evolution,original plants and animals and their processing,dosage,preparation,and usage of Mijiao Pills. This paper aimed to provide a basis for the clinical application of Mijiao Pills and subsequent product development,thus facilitating the development and utilization of this precious medicinal animal resource.
Books ; China ; Cornus ; Humans ; Medicine, Chinese Traditional ; Prescriptions

Objective:To explore the effect and mechanism of Tuina on denervated skeletal muscle atrophy. Methods:A total of 77 male Sprague-Dawley rats were randomly divided into sham group (n = 7), model group (n = 35) and Tuina group (n = 35). The latter two groups were established skeletal muscle atrophy model by exposing and cutting off the common tibial nerve of rats. One day after modeling, the lower limbs of the surgical side received Tuina in Tuina group. Separately, the surgical side of gastrocnemius muscle were sampled on the 0th, 7th, 14th, 21st and 28th^{day after modeling, and measured the wet mass ratio. The cross-sectional area and diameter of muscle fiber were measured after HE staining. The mRNA expression of autophagy-realated factor Beclin-1, vacuolar protein sorting (Vps34) and microtubule-associated protein light chain 3 (LC3) were tested with reverse transcription real-time quantitative polymerase chain reaction. Results:There was no statistical difference in the ratio of gastrocnemius wet weight, the cross-sectional area and diameter of muscle fiber, and the mRNA expression of Beclin-1, Vps34 and LC3 among three groups on the 0th day (F < 1.321, P > 0.05). Compared with the sham group, the ratio of gastrocnemius wet weight, the cross-sectional area and diameter of muscle fiber decreased at different time points in the model group and Tuina group (P < 0.05), the ratio of gastrocnemius wet weight was higher, and the cross-sectional area and diameter of muscle fiber were bigger, both except on the 21st day, in Tuina group than in the model group (P < 0.05). Compared with the sham group, the mRNA expression of Beclin-1, Vps34 and LC3 increased at different points in the model group than in Tuina group (P < 0.05), and all the mRNA expression was higher, except on the 14th day, in Tuina group than in the model group (P < 0.05). The ratio of gastrocnemius wet weight, the cross-sectional area and diameter of muscle fiber showed a trend of progressive decrease with time in the model group and Tuina group (P < 0.05). The mRNA expression of Beclin-1 and Vps34 increased (P < 0.05), and the mRNA expression of LC3 increased in the model group 21 days after intervention (P < 0.05). The mRNA expression of Beclin-1, Vps34 and LC3 increased first and then decreased, except the mRNA expression on the 14th day in Tuina group (P < 0.05). Conclusion:Tuina may promote the activation of autophagy by up-regulating the expression of autophagy-realated factor Beclin-1, Vps34 and LC3, remove the damaged organelles and proteins, provide certain synthetic substrate and energy for muscle fiber regeneration, thereby reduce the loss of degree of denervated skeletal muscle atrophy.}

The skeletal muscle atrophy could be induced by the injury of nerve. According to the source of denervated skeletal mus-cle atrophy, it could be divided into exogenous muscle atrophy and endogenous muscle atrophy. In recent years, the ex-ogenous muscle atrophy models are mainly established by operating, physically injuring or chemically injuring, while the endogenous muscle atrophy models are mainly established by the transgenic animals of amyotrophic lateral sclero-sis. The selection and optimazation of animal models are crucial for the basic studies of denervated skeletal muscle atro-phy.