Single-cell transcriptome analysis uncovers underlying mechanisms of acute liver injury induced by tripterygium glycosides tablet in mice / 药物分析学报

Tripterygium glycosides tablet(TGT),the classical commercial drug of Tripterygium wilfordii Hook.F.has been effectively used in the treatment of rheumatoid arthritis,nephrotic syndrome,leprosy,Behcet's syndrome,leprosy reaction and autoimmune hepatitis.However,due to its narrow and limited treatment window,TGT-induced organ toxicity(among which liver injury accounts for about 40％of clinical reports)has gained increasing attention.The present study aimed to clarify the cellular and molecular events underlying TGT-induced acute liver injury using single-cell RNA sequencing(scRNA-seq)technology.The TGT-induced acute liver injury mouse model was constructed through short-term TGT exposure and further verified by hematoxylin-eosin staining and liver function-related serum indicators,including alanine aminotransferase,aspartate aminotransferase,alkaline phosphatase and total bilirubin.Using the mouse model,we identified 15 specific subtypes of cells in the liver tissue,including endothelial cells,hepatocytes,cholangiocytes,and hepatic stellate cells.Further analysis indicated that TGT caused a significant inflammatory response in liver endothelial cells at different spatial locations;led to marked inflammatory response,apoptosis and fatty acid metabolism dysfunction in hepatocytes;activated he-patic stellate cells;brought about the activation,inflammation,and phagocytosis of liver capsular macrophages cells;resulted in immune dysfunction of liver lymphocytes;disturbed the intercellular crosstalk in liver microenvironment by regulating various signaling pathways.Thus,these findings elaborate the mechanism underlying TGT-induced acute liver injury,provide new insights into the safe and rational applications in the clinic,and complement the identification of new biomarkers and ther-apeutic targets for liver protection.

Mechanism of Glycyrrhizae Radix et Rhizoma Alleviating Tripterygium wilfordii Polyglycoside Tablets-induced Liver Injury / 中国实验方剂学杂志

ObjectiveTo investigate the protective effect of cytochrome P4502D6 （CYP2D6） and cytochrome P4503A4 （CYP3A4）， key enzymes of drug metabolism in liver， on acute liver injury in water extract of Glycyrrhizae Radix et Rhizoma （WEOGRR）. MethodHealthy male Kunming mice were divided into normal group， model group， WEOGRR low-， medium- and high-dose groups （5， 10， 15 g·kg-1·d-1） and positive drug group （diammonium glycyrrhizinate， 75 mg·kg-1·d-1）， with 10 in each group. One week after preventive administration， acute liver injury model was induced by single intragastric administration of 270 mg·kg-1 Tripterygium Glycosides tablets， and samples were collected after 18 h. The pathological changes of liver were observed by hematoxylin-eosin （HE） staining. Serum liver function indexes including alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， γ-glutamyl transpeptadase （γ-GT）， alkaline phosphatase （ALP）， and total bilirubin （TBIL） as well as the levels of oxidative stress indexes including malondialdehyde （MDA） and superoxide dismutase （SOD） in hepatocytes were determined by biochemical method. Real-time polymerase chain reaction （Real-time PCR） and Western blot were performed to detect the mRNA and protein expression levels of CYP2D6 and CYP3A4， respectively. ResultCompared with normal group， model group had significant hepatocyte swelling and inflammatory cell infiltration （P<0.01）， increased AST， ALT， γ-GT， ALP and TBIL （P<0.05）， elevated MDA and decreased SOD （P<0.01） as well as down-regulated mRNA and protein expression levels of CYP2D6 and CYP3A4 （P<0.05）. Compared with the model group， the normal group had intact liver structure without obvious abnormality， and the WEOGRR groups and positive drug group presented alleviated hepatocyte swelling and inflammatory cell infiltration （P<0.01）， reduced AST， ALT， γ-GT， ALP and TBIL （P<0.01）， lowered MDA and increased SOD （P<0.01） as well as up-regulated expression levels of CYP2D6 and CYP3A4 （P<0.01）. ConclusionThe protective effect of WEOGRR on acute liver injury induced by Tripterygium glycosides tablets may be related to reducing the contents of AST， ALT， γ-GT， ALP and TBIL in serum， inhibiting MDA and increasing the activity of SOD in liver cells， and enhancing the activities of CYP2D6 and CYP3A4， thus accelerating the metabolism of toxic substances.

A highly efficient protein corona-based proteomic analysis strategy for the discovery of pharmacodynamic biomarkers / 药物分析学报

The composition of serum is extremely complex,which complicates the discovery of new pharmaco-dynamic biomarkers via serum proteome for disease prediction and diagnosis.Recently,nanoparticles have been reported to efficiently reduce the proportion of high-abundance proteins and enrich low-abundance proteins in serum.Here,we synthesized a silica-coated iron oxide nanoparticle and devel-oped a highly efficient and reproducible protein corona(PC)-based proteomic analysis strategy to improve the range of serum proteomic analysis.We identified 1,070 proteins with a median coefficient of variation of 12.56％using PC-based proteomic analysis,which was twice the number of proteins iden-tified by direct digestion.There were also more biological processes enriched with these proteins.We applied this strategy to identify more pharmacodynamic biomarkers on collagen-induced arthritis(CIA)rat model treated with methotrexate(MTX).The bioinformatic results indicated that 485 differentially expressed proteins(DEPs)were found in CIA rats,of which 323 DEPs recovered to near normal levels after treatment with MTX.This strategy can not only help enhance our understanding of the mechanisms of disease and drug action through serum proteomics studies,but also provide more pharmacodynamic biomarkers for disease prediction,diagnosis,and treatment.

Construction and anti-tumor efficacy of a pentameric peptide vaccine that targets S100A8 / 药学学报

Myeloid-derived suppressor cells (MDSC) play critical roles in immune escape of tumor. We hypothesized that elimination of tumor-induced MDSCs might help to block tumor growth. Therefore, we constructed a cholera toxin B based peptide vaccine that targets a MDSC surface marker S100A8. Immunized BALB/c mice with CTB-S100A8 plus aluminum hydroxide induced high titers of anti-S100A8 antibodies and reduced tumor burden significantly in 4T1 mice model. We also found the vaccination led to significant reduction of tumor-induced monocytic MDSC (M-MDSC), with no effect on innate MDSCs, dendritic cell (DC) and macrophage (Mφ), demonstrating that targeting tumor-induced MDSC may be a promising approach in cancer immunotherapy.

[Clinical effect of percutaneous poking and tension band splint fixation for treatment of the calcaneal fracture].

OBJECTIVE: To observe the clinical efficacy of percutaneous poking tension band splint fixation for the treatment of calcaneal fractures. METHODS: From September 2007 to February 2009,40 patients with calcaneal fractures (42 feet) were treated by percutaneous poking reduction, including 28 feet of male and 14 feet of female, with an average age of 34.2 years ranging from 18 to 55 years. The course was from 2 hours to 7 days. All fractures were fresh and closed intra-articular calcaneal fractures. Patients were divided into two groups according to fixation methods, plaster fixation in 20 feet, tension band splint in 22 feet. Three aspects including the heel of the foot and ankle width of recovery, functional recovery, complications were compared according to the United States Orthopedic Foot and Ankle Society clinical score. RESULTS: These 40 patients were followed-up for 6 months to 2 years with an average of 9 months. The calcaneal width of the recovery, the functional recovery and force line of tension band splint group were all better than that of plaster fixation group (P < 0.05); There were no significant differences in pain between two groups (P > 0.05). Occurrence of complications in tension band splint group were lower than that of plaster fixation group (P < 0.05). CONCLUSION: Percutaneous poking tension band splint have advantage of stable calcaneal width and fewer complications on treatment of calcaneal fractures.

Clinical effect of percutaneous poking and tension band splint fixation for treatment of the calcaneal fracture / 中国骨伤

<p><b>OBJECTIVE</b>To observe the clinical efficacy of percutaneous poking tension band splint fixation for the treatment of calcaneal fractures.</p><p><b>METHODS</b>From September 2007 to February 2009,40 patients with calcaneal fractures (42 feet) were treated by percutaneous poking reduction, including 28 feet of male and 14 feet of female, with an average age of 34.2 years ranging from 18 to 55 years. The course was from 2 hours to 7 days. All fractures were fresh and closed intra-articular calcaneal fractures. Patients were divided into two groups according to fixation methods, plaster fixation in 20 feet, tension band splint in 22 feet. Three aspects including the heel of the foot and ankle width of recovery, functional recovery, complications were compared according to the United States Orthopedic Foot and Ankle Society clinical score.</p><p><b>RESULTS</b>These 40 patients were followed-up for 6 months to 2 years with an average of 9 months. The calcaneal width of the recovery, the functional recovery and force line of tension band splint group were all better than that of plaster fixation group (P < 0.05); There were no significant differences in pain between two groups (P > 0.05). Occurrence of complications in tension band splint group were lower than that of plaster fixation group (P < 0.05).</p><p><b>CONCLUSION</b>Percutaneous poking tension band splint have advantage of stable calcaneal width and fewer complications on treatment of calcaneal fractures.</p>

Synthesis of 1,5-bis(4,6-dichloro-1,3,5-triazinylamino)naphthalene and its application to spectrofluorimetric determination of aniline.

A new fluorescent reagent 1,5-bis(4,6-dichloro-1,3,5-triazinylamino)naphthalene (DTAN) was synthesized. The optimum conditions of fluorescent reaction of this reagent with aniline (PA) were also investigated. Based on this reaction, a new spectrofluorimetric method was developed for the determination of aniline. The fluorescent intensity was directly proportional to the concentration of aniline in the ranges 0.05-2.0 microg mL(-1) and 2.0-50 microg mL(-1) with the detection limits of 34 ng mL(-1) and 90 ng mL(-1). This method is simple, practical and can afford good precision and accuracy and can be successfully applied to assess aniline in water samples. A possible mechanism of the change of fluorescence intensity introduced by putting the aniline into the system is also discussed.

Spectrofluorimetric determination of dopamine using 1,5-bis(4,6-dicholro-1,3,5-triazinylamino) naphthalene.

A new fluorescent reagent, 1,5-bis(4,6-dichloro-1,3,5-triazinylamino)naphthalene, containing two active chlorines, was synthesized by a one-step reaction. Under the optimum conditions for the determination of dopamine, the enhanced fluorescence intensity is proportional to the dopamine concentration. The fluorescence intensity was measured at lambda(ex/em) = 400/460 nm, with and without dopamine. The linear range and detection limit for the determination of dopamine were 1.0 x 10(-7) mol/L-5.0 x 10(-5) mol/L and 4.0 x 10(-8) mol/L. This method is simple, practical, can afford good precision and accuracy and can be successfully applied to assess dopamine in injections and human serum samples.

Highly sensitive spectrofluorometric determination of trace amounts of mercury with a new fluorescent reagent, 2-hydroxy-1-naphthaldehydene-8-aminoquinoline.

A new fluorescent reagent, 2-hydroxy-1-naphthaldehydene-8-aminoquinoline (HNAAQ), was synthesized. The fluorescent reaction of this reagent with mercury was also studied. Based on this chelation, a highly sensitive spectrofluorometric method was developed for the determination of trace amounts of mercury in a water-ethanol (5 + 1, v/v) medium at pH 8.0. Under these conditions, the Hg-HNAAQ complex has excitation and emission maxima at 406 and 445 nm, respectively. The linear range of the method is from 0 to 16 microg L(-1) and the detection limit is 0.056 microg L(-1) of mercury. The interference of other ions was studied. In order to enhance the selectivity in the determination of mercury by the present method, we also applied the separation of mercury by distillation. Thus, the selectivity of the method could be increased remarkably. The procedure can be easily performed, and affords good precision and accuracy. This method has been successfully applied to the determination of mercury in waste water and prawns.

Lysozyme-enhanced europium(III)-metacycline luminescence and its application to the determination of metacycline.

A new spectrofluorometric method is described for the determination of metacycline (MC), based on modified enzyme-amplified lanthanide luminescence. Under the optimum conditions, Eu3+-MC forms a ternary complex with lysozyme in close proximity. Then lysozyme can remarkably enhance the characteristic fluorescence intensity of Eu3+ at 612 nm in metacycline-Eu3+ binary complex. The enhanced fluorescence intensity is in proportion to the concentration of MC. The limit of detection is 1.6 x 10(-8) mol L(-1), with a linear range from 6.2 x 10(-6) to 1.7 x 10(-5) mol L(-1). Interferences of other coexisting substances were studied. The developed method was successfully applied to the determination of MC in serum and urine samples. The mechanism of fluorescence enhancement was also studied.

Enzyme-amplified lanthanide luminescence based on complexation reaction--a new technique for the determination of doxycycline.

A new spectrofluorimetric method is described for the determination of doxycycline, based on modified enzyme-amplified lanthanide luminescence. Under the optimum conditions, Eu(3+)-doxycycline forms a ternary complex with lysozyme in close proximity and lysozyme can remarkably enhance the characteristic fluorescence intensity of Eu(3+) at 612nm in doxycycline-Eu(3+) binary complex. The enhanced fluorescence intensity is in proportion to the concentration of doxycycline. The limit of detection is 1.28 x 10(-8) moll(-1), with a linear range from 1.7 x 10(-7) to 1.7 x 10(-6) moll(-1). Interferences of other coexisting substances were studied. The developed method was successfully applied to the determination of doxycycline in serum, urine and real samples. The mechanism of fluorescence enhancement was also studied.

Study of the interactions between tetracycline analogues and lysozyme.

The interactions between tetracycline analogues (oxytetracycline, doxycycline, methacycline) and lysozyme (LYSO) were studied by using UV-visible absorption and spectrofluorimetric method. Tetracycline analogues can make LYSO's fluorescence quenching. The quenching mechanism is static quenching. Absorption spectrum and quenching constant all support this conclusion. The binding constants of tetracycline analogues with LYSO were obtained at various temperatures. According to the Förster nonradiative energy transfer theory, we can get the distances of donators and acceptors. We also determined the main acting force between them is electrostatic gravitation according to the thermodynamic parameter.

[Spectrophotomatric determination of antioxidative activity of extractives of Chinese traditional medicine by supercritical fluid CO2 extraction technology].

Applying supercritical CO2 fluid extraction technology, flavonoids were extracted from Chinese traditional medicine asarum heterotropoides, atractylodes macrocephala, and rheum palmatun. Using the method of autoxidation pyrogallol (known as 325 nm method), the superoxide radical scavenging effect of the extraction was carried out in the buffer solution of HCl-tris (pH 8.2). With spectrophotometry, hydroxyl radical created by the system Co2+ + H2O2 in the reaction like Fenton reaction was eliminated by alizarin violet as the color developing agent in the buffer solution HCl-tris (pH 9.0) and the reaction condition was investigated. Result showed that these extractions are elimination agent for these radicals. Asarum heterotropoides is the best of the three.

Study of the interaction between daunorubicin and human serum albumin, and the determination of daunorubicin in blood serum samples.

The interactions between daunorubicin and human serum albumin (HSA) were studied using the spectrofluorimetric method. The binding constants of daunorubicin with HSA were obtained at different temperatures. The effects of various metal ions on the binding constants of daunorubicin with HSA were also studied. The optimum conditions of fluorometric determination of daunorubicin were studied and the developed method was successfully applied to the determination of daunorubicin in serum samples.

[Studies on the interaction mode between polymyxin B sulfate and DNA with EB as a fluorescence probe].

The interaction between polymyxin B sulfate and ct-DNA was studied with ethidium bromide (EB) probe by UV spectroscopy, fluorescence spectroscopy, fluorescent polarization etc. The phenomena of hyperchromism effect and fluorescence quenching proved that polymyxin B sulfate in buffer solution at pH 7.4 can intercalate into the base pairs of ct-DNA. This interaction can cause conformational change of ct-DNA double helix. This interaction can be inhibited in the presence of high concentration of ions. At the same time, PB can combine with the phosphate group of ct-DNA by non-characteristic static force. In this paper, the interaction of PB and ct-DNA was made further discussion.