AIM: To identify the primary active components and underlying mechanisms of Yijing Decoction(YJD)in treating early diabetic retinopathy(DR)based on liquid chromatography-mass spectrometry and network pharmacology.METHODS: Active components of YJD were characterized through LC-MS. Components with optimal ADME(absorption, distribution, metabolism, excretion)properties were selected as key bioactive candidates. Network pharmacology approaches were employed to predict YJD-DR therapeutic targets. Protein-protein interaction(PPI)networks, gene ontology(GO)enrichment analysis, and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis were subsequently conducted to predict core targets and networks. Critical targets and pathways were experimentally validated through Western blot.RESULTS: Ten core therapeutic targets were identified, including TNF, Alb, EGFR, STAT3, PTGS2, ESR1, PPAR, MMP9, TLR4, and MAPK. YJD was related to cancer-related signaling, fluid shear stress and atherosclerosis, and neurodegenerative diseases, encompassing key biological processes such as inflammatory response regulation, programmed cell death activation, and enhanced cell migration. Furthermore, Western blot analysis confirmed that YJD significantly inhibited high glucose-induced phosphorylation of STAT3(P-STAT3/STAT3)and ERK(P-ERK/ERK)in rat retinal microvascular endothelial cells.CONCLUSION: This study revealed YJD's pharmacodynamical basis and its multi-component, multi-target, and multi-paths pharmacology. YJD exerts therapeutic effects on DR by coordinately regulating critical signaling pathways and alleviating intraocular inflammation, thus preserving retinal vascular endothelial cells, maintaining blood-retinal barrier integrity, and facilitating retinal neurovascular repair.

ObjectiveTaking the oligosaccharides in Rehmanniae Radix（RR） as the research object， the content determination method based on high performance liquid chromatography-evaporative light scattering detection（HPLC-ELSD） and thin layer chromatography（TLC） identification method were established to explore the content and distribution of oligosaccharides in different RR herbs and decoction pieces. MethodA total of 10 batches of fresh and raw RR， 12 batches of RR decoction pieces and Rehmanniae Radix Praeparata（RRP） were collected. A TLC identification method for fructose， sucrose， manninotriose， raffinose and stachyose in RR was established by using silica gel G thin-layer plates with ethyl acetate-water-anhydrous formic acid-glacial acetic acid（12∶6∶5∶4） as the developing agent and 10% sulfuric acid-ethanol solution as chromogenic agent. A HPLC-ELSD was used to determine the contents of fructose， glucose， sucrose， melibiose， raffinose， manninotriose and stachyose in different RR herbs and decoction pieces. Then principal component analysis（PCA） and partial least squares-discriminant analysis（PLS-DA） were used to analyze the contents of 7 kinds of saccharides in RR herbs and decoction pieces， and the differential components were screened with the value of variable importance in the projection（VIP）>1. ResultThe results of TLC identification showed that fresh RR， raw RR and its decoction pieces showed spots of the same color on the corresponding positions with the control products of stachyose， raffinose and sucrose， while the TLC of RRP showed spots of the same color at corresponding positions to manninotriose and fructose controls. The results of methodological investigations of 7 analytes met the requirements of determination. Only glucose， sucrose， raffinose and stachyose were detected in 10 batches of fresh RR and 10 batches of raw RR herbs， the average contents of which were 0.84%， 4.62%， 2.42% and 57.90% in fresh samples， while those were 3.16%， 9.36%， 7.05% and 38.10% in raw samples， respectively. In 12 batches of RR decoction pieces， the contents of the above seven sugars（fructose， glucose， sucrose， melibiose， raffinose， manninotriose and stachyose） were 1.68%， 4.27%， 9.96%， 0.53%， 6.85%， 3.05% and 37.52%， respectively. In 12 batches of RRP， the contents of the above seven sugars were 10.62%， 11.01%， 1.25%， 3.35%， 1.12%， 28.16% and 6.39%， respectively. The results of multivariate statistical analysis showed that fresh RR， raw RR and RRP could be distinguished from each other by the contents of the 7 sugars， and the main differential components were stachyose， sucrose， raffinose and manninotriose. ConclusionIn terms of oligosaccharides， the contents and types of saccharides in different herbs and decoction pieces of RR are quite different， and the TLC identification method based on this can be used to distinguish raw RR from RRP， which can lay a foundation for improving the quality standard of RR and developing and applying oligosaccharides in different processed products of RR.

Objective To observe the effect of tuina on glutamate uptake and synaptic cleft in the spinal dorsal horn of rats with neuropathic pain through astrocyte NDRG2/GLT-1 pathway,and to explore the potential analgesic mechanism of tuina on neuropathic pain.Methods A total of 54 SD rats were randomly divided into naive group,model group and tuina group(n=18).The CCI model was established in the model group,and the tuina group was treated with acupressure at"Weizhong"(BL 40)from the 4th day after CCI model was successfully established for 14 days.The changes of paw withdrawal threshold at different time points were observed to evaluate the analgesic effect of tuina.Immunofluorescence was used to observe the co-expression of NDRG2?GLT-1 and astrocytes in the spinal dorsal horn.The mRNA levels of NDRG2 and GLT-1 in astrocytes were detected by quantitative real time polymerase chain reaction.The concentrations of glutamate in synaptic cleft were measured by liquid chromatography coupled to tandem mass spectrometry.The width of the synaptic cleft was observed by transmission electron microscopy.Results Compared with the naive group,the paw withdrawal threshold in the CCI group decreased continuously(P<0.01).The number of NDRG2 and GFAP co-labeling positive cells in the spinal dorsal horn increased significantly(P<0.01),and the number of GLT-1 and GFAP co-labeled positive cells was significantly reduced(P<0.01).NDRG2 mRNA expression was up-regulated and GLT-1 mRNA expression decreased in astrocytes(P<0.01).The concentrations of glutamate increased significantly(P<0.01);The synaptic cleft was significantly narrowed(P<0.05).After tuina intervention,the above trend was significantly reversed.Compared with the model group,the paw withdrawal threshold of the tuina group increased from 11 days after CCI(P<0.01),the number of NDRG2 and GFAP co-labeling positive cells in the spinal dorsal horn was significantly reduced(P<0.01),and the number of GLT-1 and GFAP co-labeled positive cells increased significantly(P<0.01);down-regulated the expression of NDRG2 mRNA and restored GLT-1 mRNA expression in astrocytes(P<0.01);decreased glutamate concentration in synaptic cleft(P<0.05),the synaptic cleft was relatively widened(P<0.05).Conclusion Tuina alleviates pain in CCI rats at the spinal cord level possibly by promoting the uptake of glutamate by NDRG2/GLT-1 pathway in astrocytes,restoring the width of synaptic cleft and reversing synaptic plasticity.

The theory of "liver opens at the eyes" was first seen in Yellow Emperor's Internal Canon of Medicine， which is the ancient people's summary of the connection between the liver and the eyes. The theory of "liver opens at the eyes" suggests the characteristic of "co-damage and co-recover of liver and eyes". It has been found in clinical practice that liver diseases and eye diseases often occur together， and "liver and eyes co-recover" is an ideal choice. The key to achieving "liver and eyes co-recover" is to analyze its pharmacological material basis and mechanism. With the development of modern medicine， more and more evidence indicates that the liver and eyes have complex and close relationships in physiological and pathological aspects. In a pathological state， there is a phenomenon of "liver and eyes co-damage"， and after the intervention of traditional Chinese medicine， "liver and eyes co-recover" occurs. "Liver and eyes co-damage and co-recover" can be explained through the "co-material basis and co-action mechanism". On this basis， the research group tentatively proposed that the liver and eyes had "four common characteristics" （4CCs）， namely "co-damage， co-recover， co-material basis， and co-action mechanism" from the theoretical connotation of traditional Chinese medicine， clinical practice， and molecular biology. Additionally， the group also took the intervention of Prunella vulgaris， traditional Chinese medicine， for removing liver fire and improving eyesight on immune liver injury （ILI） and allergic conjunctivitis （AC） as examples to analyze 4CCs. This project aims to deeply analyze the scientific connotation of the theory of "liver opens at the eyes"， reveal the common characteristics and biological essence of liver and eyes， explore a new research paradigm of "liver and eyes co-recover"， and provide a reference for the study of common problems of multi-organ associated diseases.

Objective:To investigate the expressions of tissue inhibitor of matrix metalloproteinase-1 (TIMP1) and fibronectin 1 (FN1) in pregnancy associated breast cancer (PABC) and their correlations with expression of E-cadherin (E-cad).Methods:The clinicopathological data of 55 PABC patients in Binzhou People's Hospital Affiliated to Shandong First Medical University from January 2011 to December 2020 were retrospectively analyzed. Immunohistochemistry was used to detect expressions of TIMP1, FN1 and E-cad in cancer tissues and corresponding paracancerous tissues (>3 cm from the edge of the tumor foci). The expressions of TIMP1 and FN1 proteins in fresh intraoperative frozen cancer tissues and paracancerous tissues of 10 PABC patients were detected by Western blotting. The correlations of TIMP1 and FN1 expressions with clinicopathological characteristics of patients were analyzed by χ2 test, the correlation of TIMP1 and FN1 expressions with E-cad expression was analyzed by Spearman method, and the correlation of TIMP1 and FN1 expressions with survival was analyzed by Kaplan-Meier method. Results:The positive rates of TIMP1 and FN1 in PABC tissues were 72.7% (40/55) and 58.2% (32/55), and 25.5% (14/55) and 18.2% (10/55) in paracancerous tissues, and the differences were statistically significant ( χ2 values were 24.59 and 18.64, both P < 0.001). The results of Western blotting showed that the relative expressions of TIMP1 and FN1 proteins in the fresh cancer tissues of 10 PABC patients was higher than those in the corresponding paracancerous tissues (1.60±0.76 vs. 0.62±0.29, 1.31±0.62 vs. 0.44±0.15), and the differences were statistically significant ( t values were 5.92 and 4.86, both P < 0.001). The expressions of TIMP1 and FN1 in PABC tissues were correlated with estrogen receptor expression, Ki-67 positivity index, TNM stage and lymph node metastasis (all P < 0.05). The expressions of TIMP1 and FN1 were negatively correlated with expression of E-cad in PABC ( r values were -0.471 and -0.432, both P < 0.001). Five cases were lost to follow-up, and the remaining 50 cases had a median follow-up time of 43 months (12-90 months). Among the 50 cases, 36 cases were TMP1-positive and 29 cases were FN1-positive. The overall survival of TIMP1-negative group and FN1-negative group were better than those of the corresponding positive group ( χ2 values were 4.49 and 6.06, both P < 0.05); the median overall survival time of TIMP1-positive group and FN1-positive group were 51 months (95% CI 37-65 months) and 43 months (95% CI 32-53 months), while that of TIMP1-negative group and FN1-negative group were 89 months (95% CI 84-93 months) and 87 months (95% CI 85-92 months). Conclusions:TIMP1 and FN1 expressions are elevated in PABC tissues and negatively correlated with E-cad expression, TIMP1 and FN1 may be involved in PABC invasion through epithelial-mesenchymal transition and affect the prognosis of patients.

Objective:To analyze the urine of normal healthy left-behind children under 1 year old and left-behind children with zinc deficiency under 1 year old in Zunyi area using hydrogen nuclear magnetic resonance ( 1HNMR), thus providing a new biomarker for the early diagnosis of zinc deficiency. Methods:From January to August 2018, a total of 40 normal healthy left-behind children under 1 year old in Zunyi area(healthy control group)[22 males and 18 females, average age of (7.78±3.62) months, average height of (65.01±2.67) cm and average body mass of (7.15±1.59) kg] and 40 age-matched left-behind children with zinc deficiency in the same region(zinc deficiency group)[19 males and 21 females, average age of (7.89±3.57) months, average height of (64.25±2.95) cm and average body mass of (7.02±1.68) kg] were included for a cross-sectional study by stratified sampling.The urine 1HNMR spectra of children in the 2 groups were measured, and the age, height, body mass and serum zinc content of children in the 2 groups were compared.The metabolites of the 2 groups were compared by metabono-mics technology combined with multivariate statistical analysis, and the differential metabolites of children with zinc deficiency were screened out. Results:There were no significant differences in age, height and body mass between the 2 groups (all P>0.05). The serum zinc level of healthy control group was significantly higher than that of zinc deficiency group [(54.3±3.06) mmol/L vs.(39.2±3.77) mmol/L, t=22.65, P<0.05]. Urine 1HNMR spectrogram results showed that compared with healthy controls, 4-hydroxyphenylpyruvic acid, phenyl acetyl glycine, and hippuric acid salt water were significantly lower in zinc deficiency group ( r=-0.620, -0.689, and -0.721, respectively, all | r|>0.602, all P<0.05). Conclusions:Zinc deficiency in left-behind children under 1 year old in Zunyi area is mainly manifested by decreased metabolites of 4-hydroxyphenylpyruvic acid, phenylacetyl glycine and horse-urate, suggesting metabolic disorder of intestinal flora.Differentially expressed metabolites have a potential application value in the early diagnosis of zinc deficiency.

Objective:To study the types of pathogenic gene mutations and their main clinical characteristics in children with glucose-6-phosphate dehydrogenase (G6PD) deficiency in Zunyi area.Methods:Children with clinical manifestations of "yellow staining" or "suspected yellow staining" who were admitted to Guizhou Children's Hospital, Affiliated Hospital of Zunyi Medical University, from September 13, 2018 to September 13, 2020 were selected for G6PD gene mutation detection by multicolor probe melting curve analysis, and the pathogenic gene mutation types and clinical characteristics of children with G6PD deficiency were analyzed.Results:The results of G6PD gene mutation detection showed that among the 1 740 children tested, 119 were positive for gene mutation, and the positive detection rate was 6.84%. The proportion of male infants was higher than that of female infants, and the difference was statistically significant (91 males and 28 females, χ 2 = 15.10, P < 0.001); infancy accounted for 63.87% (76/119), and early childhood accounted for 18.49% (22/119). A total of 11 known pathogenic gene mutation types and 1 unknown mutation were detected. Among the top 4 pathogenic gene mutations, the overall was c.1024 C>T, c.1376 G>T, c.1388 G>A and c.95 A>G, male was c.1376 G>T, c.1388 G>A, c.1024 C>T and c.95 A>G; female was c.1024 C>T, c.95 A>G, c.1388 G>A and c.519 C>T. Among the 119 children with G6PD gene mutation, 90 cases had varying degrees of jaundice, including 36 cases of severe and more severe jaundice (including 2 cases of extremely severe neonatal bilirubin encephalopathy), and 54 cases of mild to moderate jaundice; 37 cases had anemia of different degrees, including 6 cases of mild anemia, 12 cases of moderate anemia, and 19 cases of severe or more severe anemia (including 1 case of extremely severe anemia). Conclusions:There are 12 types of gene mutations in children with G6PD deficiency in Zunyi area, and the most common mutation types are c.1024 C>T, c.1376 G>T, c.1388 G>A and c.95 A>G. Children with G6PD deficiency are often accompanied by varying degrees of jaundice and anemia.

OBJECTIVE: To investigate the antitumor activity of decoction and study its liver and kidney toxicity and its effect on the immune system in a tumor-bearing mouse model. METHODS: Hepatoma H22 tumor-bearing mouse models were randomized into model group, cyclophosphamide (CTX) group, and low-, moderate-, and high-dose decoction groups (JW-L, JW-M, and JW-H groups, respectively). The antitumor activity of decoction was assessed by calculating the tumor inhibition rate and pathological observation of the tumor tissues. Immunohistochemistry was used to detect the expressions of Bax, Bcl-2, Bax/Bcl-2 and caspase-3 in the tumors. The liver and kidney toxicity of decoction was analyzed by evaluating the biochemical indicators of liver and kidney functions. The immune function of the tumor-bearing mice were assessed by calculating the immune organ index, testing peripheral blood routines, and detection of serum IL-2 and TNF-α levels using enzyme-linked immunosorbent assay. RESULTS: Compared with that in the model group, the tumor mass in CTX, JW-M and JW-H groups were all significantly reduced ( < 0.05) with cell rupture and necrosis in the tumors. Immunohistochemistry revealed obviously up-regulated expressions of Bax and caspase-3 and down- regulated expression of Bcl-2 protein with an increased Bax/Bcl-2 ratio in CTX, JW-M and JW-H groups. Treatment with decoction significantly reduced Cr, BUN, AST and ALT levels, improved the immune organ index, increased peripheral blood leukocytes, erythrocytes and hemoglobin levels, and up-regulated the levels of TNF-α and IL-2 in the tumor-bearing mice. These changes were especially significant in JW-H group when compared with the parameters in the model group ( < 0.01). CONCLUSIONS decoction has a strong anti-tumor activity and can improve the liver and kidney functions of tumor-bearing mice. Its anti-tumor effect may be attributed to the up-regulation of Bax, caspase-3, TNF-α and IL-2 levels and the down-regulation of Bcl-2 expression as well as the enhancement of the non-specific immune function.
Animals ; Antineoplastic Agents, Phytogenic ; pharmacology ; Carcinoma, Hepatocellular ; drug therapy ; immunology ; metabolism ; pathology ; Drugs, Chinese Herbal ; pharmacology ; Kidney ; drug effects ; Liver ; drug effects ; pathology ; Liver Neoplasms ; drug therapy ; immunology ; metabolism ; pathology ; Mice ; Necrosis ; Neoplasm Proteins ; metabolism ; Random Allocation ; Up-Regulation

Objective: To explore the characteristic changes in urinary metabolites in left-behind children with vitamin D deficiency under 1 year old in Zunyi area by metabolomic nuclear magnetic resonance (NMR) in order to provide new biomarkers for early diagnosis of vitamin D deficiency. Methods: From January to August 2018, blood tests and urine collection were carried out on the left-behind children under 1 year old in Fenggang county, Bozhou district and Zheng′an county under Zunyi city by stratified sampling.Forty children diagnosed as a vitamin D deficiency were selected as a vitamin D deficiency group, and 40 children with normal urine test were selected as a healthy control group.For urine sampling, SIMCA-P+ software was applied to analyze the integral value of hydrogen spectrogram by principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) was used to distinguish the difference in urine metabolites between two groups of the left-behind children.Orthogonal partial least squares discriminant analysis (OPLS-DA) was used to screen different metabolites. Results: The serum level of 25-hydroxy vitamin D[25-(OH)D][(32.0±3.6) nmol/L ] in the healthy control group was higher than that in the vitamin D deficiency group[(15.8±2.3) nmol/L], and the difference was statistically significant (P<0.05). PCA and PLS-DA analysis showed significant differences in urine metabolites between the healthy control group and the vitamin D deficiency group (P<0.05). OPLS-DA indicated R²X=0.365, Q²=0.978, which further verified the difference of metabolites.Compared with the healthy control group, the urine of methyl malonic acid, 3-hydroxy butyrate, N-acetyl glycoprotein signal, glutamic acid, dimethyl glycine, 2-ketone glutaric acid, taurine, fumaric acid salt level increased significantly in the vitamin D deficiency group, and the differences were statistically significant (|r|>0.602, all P<0.05, df=39). However, the levels of ethyl malonic acid, creatine, choline, glycerophosphalocholine and equine were significantly decreased, and the differences were statistically significant (|r|>0.602, all P<0.05, df=39). Conclusions The left-behind children under 1 year old with vitamin D deficiency in Zunyi region are mainly characterized by disorder in energy metabolism, lipid metabolism, amino acid metabolism and intestinal microbial meta-bolism disorders, and their differential metabolites have potential application value in early diagnosis and pathogenesis of vitamin D deficiency.

OBJECTIVE： To investigate the effects of Lindera aggregate of stir-baking with vinegar-Aucklandia lappa on gastric emptying and gastrointestinal hormones in functional dyspepsia liver depression and qi stagnation （FDLDQS） model rats. METHODS： Totally 60 SD rats were randomly divided into blank group （n＝10） and modeling group （n＝50）； FDLDQS model was induced by chronic stress restraint or food deprivation or excessive fatigue in modeling group. After modeling， model rats were randomly divided into model group （normal saline）， L. aggregate of stir-baking with vinegar group （1.62 g/kg， calculated by crude drug）， A. lappa group （1.62 g/kg， calculated by crude drug）， L. aggregate of stir-baking with vinegar-A. lappa group （1 ∶ 1， m/m，1.62 g/kg， calculated by crude drug） and mosapride group （positive control， 1.35 mg/kg）， with 10 rats in each group. Administration groups were given relevant medicine intragastrically once a day； blank group and model group were given constant volume of normal saline intragastrically， for consecutive 14 d. 2 h after last medication， gastric emptying rate and small intestinal propulsion rate of rats in each group were measured by phenol red content method. After HE staining， the morphological changes of antrum tissue were observed under microscope. The contents of motilin （MTL）， gastrin （GAS） and cholecystokinin （CCK） in serum were determined by ELISA method. RESULTS： Compared with blank group， gastric emptying rate and small intestinal propulsion rate of rats were decreased significantly in model group （P＜0.01）； the serum contents of MTL and GAS were decreased significantly （P＜0.01）， while the content of CCK was increased significantly （P＜0.01）. No organic damage was found in all model groups. Compared with model group， L. aggregate stir-baking with vinegar group and A. lappa group， gastric emptying rate and small intestinal propulsion rate of rats were increased significantly in L. aggregate of stir-baking with vinegar-A. lappa group and mosapride group （P＜0.05 or P＜0.01）； serum contents of MTL and GAS were increased significantly （P＜0.01）， while the content of CCK was decreased significantly （P＜0.01）. CONCLUSIONS： L. aggregate of stir-baking with vinegar-A. lappa can accelerate gastric emptying and small intestinal propulsion rate of FDLDQS rats， increase serum contents of MTL and GAS but decrease the content of CCK； its effects are better than that of them alone.