Objective This study conducted molecular biological identification of the viruses carried by Culex pipiens pallens specimens collected in Shunyi District, Beijing in 2023, and observed the changes in the infection rate of the viruses carried by Cx. pipiens pallens at different collection times. Methods Cx. pipiens pallens were collected using carbon dioxide mosquito traps. The mosquito samples were ground in batches and analyzed by molecular biology technologies. The virus infection rate at different collection times was analyzed statistically. Results 17 strains of Culex pipiens pallens densovirus(CppDNV)were identified from Cx. pipiens pallens samples collected in Shunyi District, Beijing, in 2023. The nucleotide sequence analysis of the virus genome coding region showed that CppDNV was a single-stranded DNA virus with a total length of 3 335 nt, encoding 2 non-structural proteins(NS1, NS2)and 1 capsid protein(VP). The nucleotide(amino acid)sequence lengths of the three proteins were 2 376 nt(791 aa),1 092 nt(363 aa)and 1 071 nt(356 aa), respectively. Phylogenetic analysis showed that CppDNV was located in genus Brevihamaparvovirus. Statistical analysis showed no significant difference in infection rates across collection times(χ2=4.429, P=0.194). Conclusions CppDNV was identified in Cx. pipiens pallens in Beijing, and it was stably maintained in this natural population.

Background/Aims: Metabolic dysfunction-associated steatohepatitis (MASH) is a significant risk factor for gallstone formation, but mechanisms underlying MASH-related gallstone formation remain unclear. Golgi membrane protein 1 (GOLM1) participates in hepatic cholesterol metabolism and is upregulated in MASH. Here, we aimed to explore the role of GOLM1 in MASH-related gallstone formation. Methods: The UK Biobank cohort was used for etiological analysis. GOLM1 knockout (GOLM1-/-) and wild-type (WT) mice were fed with a high-fat diet (HFD). Livers were excised for histology and immunohistochemistry analysis. Gallbladders were collected to calculate incidence of cholesterol gallstones (CGSs). Biles were collected for biliary lipid analysis. HepG2 cells were used to explore underlying mechanisms. Human liver samples were used for clinical validation. Results: MASH patients had a greater risk of cholelithiasis. All HFD-fed mice developed MASH, and the incidence of gallstones was 16.7% and 75.0% in GOLM1-/- and WT mice, respectively. GOLM1-/- decreased biliary cholesterol concentration and output. In vivo and in vitro assays confirmed that GOLM1 facilitated cholesterol efflux through upregulating ATP binding cassette transporter subfamily G member 5 (ABCG5). Mechanistically, GOLM1 translocated into nucleus to promote osteopontin (OPN) transcription, thus stimulating ABCG5-mediated cholesterol efflux. Moreover, GOLM1 was upregulated by interleukin-1β (IL-1β) in a dose-dependent manner. Finally, we confirmed that IL-1β, GOLM1, OPN, and ABCG5 were enhanced in livers of MASH patients with CGSs. Conclusions In MASH livers, upregulation of GOLM1 by IL-1β increases ABCG5-mediated cholesterol efflux in an OPN-dependent manner, promoting CGS formation. GOLM1 has the potential to be a molecular hub interconnecting MASH and CGSs.

Background/Aims: Metabolic dysfunction-associated steatohepatitis (MASH) is a significant risk factor for gallstone formation, but mechanisms underlying MASH-related gallstone formation remain unclear. Golgi membrane protein 1 (GOLM1) participates in hepatic cholesterol metabolism and is upregulated in MASH. Here, we aimed to explore the role of GOLM1 in MASH-related gallstone formation. Methods: The UK Biobank cohort was used for etiological analysis. GOLM1 knockout (GOLM1-/-) and wild-type (WT) mice were fed with a high-fat diet (HFD). Livers were excised for histology and immunohistochemistry analysis. Gallbladders were collected to calculate incidence of cholesterol gallstones (CGSs). Biles were collected for biliary lipid analysis. HepG2 cells were used to explore underlying mechanisms. Human liver samples were used for clinical validation. Results: MASH patients had a greater risk of cholelithiasis. All HFD-fed mice developed MASH, and the incidence of gallstones was 16.7% and 75.0% in GOLM1-/- and WT mice, respectively. GOLM1-/- decreased biliary cholesterol concentration and output. In vivo and in vitro assays confirmed that GOLM1 facilitated cholesterol efflux through upregulating ATP binding cassette transporter subfamily G member 5 (ABCG5). Mechanistically, GOLM1 translocated into nucleus to promote osteopontin (OPN) transcription, thus stimulating ABCG5-mediated cholesterol efflux. Moreover, GOLM1 was upregulated by interleukin-1β (IL-1β) in a dose-dependent manner. Finally, we confirmed that IL-1β, GOLM1, OPN, and ABCG5 were enhanced in livers of MASH patients with CGSs. Conclusions In MASH livers, upregulation of GOLM1 by IL-1β increases ABCG5-mediated cholesterol efflux in an OPN-dependent manner, promoting CGS formation. GOLM1 has the potential to be a molecular hub interconnecting MASH and CGSs.

Objective In this preliminary survey,we sought to determine the composition of mosquito species inhabiting the Emeifeng Nature Reserve,Fujian Province,China.Methods Mosquito larvae were collected by straw and spoon trapping,and adult mosquitoes were collected by lamp trapping at selected breeding sites in the reserve.The specimens were initially identified based on morphology,with subsequent verification using molecular biology methods.Results A total of 34 mosquito species in 13 genera were collected,among which,there were 4 species of Anopheles(Genus Anopheles Meigen,1818),2 species of Lutzia(Genus Lutzia Theobald,1903),15 species of Culex(Genus Culex Linnaeus,1758),4 species of Stegomyia(Genus Stegomyia Theobald,1901),and single species of Hulecoeteomyia(Genus Hulecoeteomyia Theobald,1904),Luius(Genus Luius Reinert,Harbach et Kitching,2008),Aedes(Genus Aedes Meigen,1818),Downsiomyia(Genus Downsiomyia Vargas,1950),Collessius(Genus Collessius Reinert,Harbach et kitching,2006),Uranotaenia(Genus Uranotaenia Lynch 1891),Armigeres(Genus Armigeres Theobald,1901),Toxorhynchites(Genus Toxorhynchites Theobald,1901),and pestle mosquito(Genus Tripteroides Giles,1904).Conclusions The species composition of mosquitoes sampled in the Emeifeng Nature Reserve will provide a basis for further research on mosquito vectors and contribute to measures for local mosquito control.

AIM To study the chemical constituents from the leaves of Drynaria fortunei(Kunze)J.Sm.and their antioxidant activity.METHODS ODS-AG-HG,Sephadex LH-20 and semi-preparative HPLC were used for separation and purification,then the structures of obtained compounds were identified by physicochemical properties and spectral data.The antioxidant activity was determined by DPPH mothod.RESULTS Fifteen compounds were isolated and identified as kaempferol-3-O-neohesperidoside(1),dihydrodehydrodiconiferyl alcohol(2),kaempferol-3,7-di-O-α-L-rahmnoside(3),astragalin(4),loliolid(5),trichothecene analogue(6),2,2-[bis-4-(2,3-dihydroxypropoxy)phenyl]propane(7),maculatin(8),trichothecin(9),4-[(Z)-but-2-enoyloxy]-8-chloro-12-hydroxy-7,13-epoxytrichothec-9-ene(10),8-deoxy-trichotecin(11),β-sitosterol(12),daucosterol(13),afzelin(14),samwinol(15).The IC50 values of the leaf and rhizome extracts against DPPH free radicals were(0.072±0.005),(0.287±0.012)mg/mL,respectively.CONCLUSION Compounds 1,2,5-11,15 are isolated from this plant for the first time.The leaves of D.fortunei exhibit strong antioxidant activity.

To explore the anti-depression effect of Suanzaoren Decoction(SZRD), the regulatory effects on endogenous metabolites in the liver of rats with depression induced by chronic unpredictable mild stress(CUMS) were analyzed by using LC-MS metabolomics. The rats were randomly divided into normal control group, model group, low-dose SZRD group, high-dose SZRD group, and positive drug group. The CUMS depression model was replicated by applying a variety of stimuli, such as fasting and water deprivation, ice water swimming, hot water swimming, day and night reversal, tail clamping, and restraint for rats. Modeling and treatment were conducted for 56 days. The behavioral indexes of rats in each group, including body weight, open field test, sucrose preference test, and tail suspension test, were observed. Plasma samples and liver tissue samples were collected, and the contents of 5-hydroxytryptamine(5-HT), dopamine(DA), and norepinephrine(NE) in plasma were measured using enzyme-linked immunosorbent assay(ELISA). Meanwhile, the regulatory effects of SZRD on the liver metabolic profile of CUMS model rats were analyzed by the LC-MS metabolomics method. The results show that SZRD can significantly improve the depression-like behavior of CUMS model rats and increase the neurotransmitter levels of 5-HT, DA, and NE in plasma. A total of 24 different metabolites in the rats' liver are identified using the LC-MS metabolomics method, and SZRD can reverse 13 of these metabolites. Metabolic pathway analysis indicates that nine metabolic pathways are found to be significantly associated with depression, and in the low-dose SZRD group, four pathways can be regulated, including pentose phosphate pathway, purine metabolism, inositol phosphate metabolism, and sphingolipid metabolism. In the high-dose SZRD group, two metabolic pathways can be regulated, including sphingolipid metabolism and glycerol glycerophospholipid metabolism. Sphingolipid metabolism is a metabolic pathway that can be regulated by SZRD at different doses, so it is speculated that it may be the primary pathway through which SZRD can alleviate metabolic disturbances in the liver of CUMS model rats.
Animals ; Rats ; Drugs, Chinese Herbal/administration & dosage* ; Metabolomics ; Depression/metabolism* ; Male ; Liver/drug effects* ; Rats, Sprague-Dawley ; Antidepressive Agents/administration & dosage* ; Serotonin/blood* ; Humans ; Disease Models, Animal ; Behavior, Animal/drug effects*

Based on copper death, this study investigates the effect and mechanism of Shenmai Injection on isoproterenol(ISO)-induced myocardial fibrosis(MF) in rats. SPF-grade male SD rats were randomly divided into a normal group, model group, captopril(5 mg·kg~(-1)) positive control group, and Shenmai Injection low(6 mL·kg~(-1)), medium(9 mL·kg~(-1)), and high(12 mL·kg~(-1)) dose groups. Except for the normal group, the rats in the other groups were subcutaneously injected with ISO(5 mg·kg~(-1)) once a day for 10 consecutive days to establish an MF model. Starting from the second day after successful modeling, intraperitoneal injections of the respective treatments were administered for 28 consecutive days. Hematoxylin-eosin(HE) and Masson staining were used to observe pathological changes and fibrosis levels in the myocardial tissue. Colorimetry was employed to detect serum Cu~(2+) concentration in rats. The levels of inflammatory cytokines interleukin-6(IL-6), interleukin-1β(IL-1β), interleukin-18(IL-18), tumor necrosis factor-α(TNF-α), as well as mitochondrial energy metabolites adenosine triphosphate(ATP), adenosine diphosphate(ADP), and adenosine monophosphate(AMP) in serum were measured using enzyme-linked immunosorbent assay(ELISA). Western blot was performed to detect the expression of collagen Ⅰ(Col-Ⅰ), collagen Ⅲ(Col-Ⅲ), and copper death-related proteins dihydrolipoamide acetyltransferase(DLAT), ferredoxin 1(FDX1), lipoic acid synthetase(LIAS), and heat shock protein 70(HSP70) in myocardial tissue. Immunofluorescence was used to detect the expression of DLAT, FDX1, and HSP70, while immunohistochemistry was conducted to examine the expressions of DLAT, FDX1, LIAS, and HSP70. The results showed that, compared to the model group, the myocardial structure disorder and collagen fiber deposition in the drug treatment groups were significantly improved, the cardiac index level was reduced, serum Cu~(2+), IL-6, IL-1β, IL-18, TNF-α, ADP, and AMP levels were significantly decreased, ATP levels were significantly increased, and the expressions of Col-Ⅰ, Col-Ⅲ, and HSP70 proteins in myocardial tissue were significantly reduced, while the expressions of DLAT, FDX1, and LIAS proteins were significantly elevated. In conclusion, Shenmai Injection effectively alleviates myocardial structure disorder and interstitial collagen fiber deposition in ISO-induced MF rats, promotes copper excretion, and reduces copper death in the ISO-induced rat MF model.
Animals ; Male ; Drugs, Chinese Herbal/administration & dosage* ; Rats, Sprague-Dawley ; Rats ; Myocardium/metabolism* ; Drug Combinations ; Fibrosis/metabolism* ; Copper/blood* ; Cardiomyopathies/genetics* ; Humans

This study aimed to investigate the correlation between changes in intestinal toxicity and compositional alterations of Euphorbiae Ebracteolatae Radix(commonly known as Langdu) before and after milk processing, and to explore the detoxification mechanism of milk processing. Mice were intragastrically administered the 95% ethanol extract of raw Euphorbiae Ebracteolatae Radix, milk-decocted(milk-processed), and water-decocted(water-processed) Euphorbiae Ebracteolatae Radix. Fecal morphology, fecal water content, and the release levels of inflammatory cytokines tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β) in different intestinal segments were used as indicators to evaluate the effects of different processing methods on the cathartic effect and intestinal inflammatory toxicity of Euphorbiae Ebracteolatae Radix. LC-MS/MS was employed to analyze the small-molecule components in the raw product, the 95% ethanol extract of the milk-processed product, and the milky waste(precipitate) formed during milk processing, to assess the impact of milk processing on the chemical composition of Euphorbiae Ebracteolatae Radix. The results showed that compared with the blank group, both the raw and water-processed Euphorbiae Ebracteolatae Radix significantly increased the fecal morphology score, fecal water content, and the release levels of TNF-α and IL-1β in various intestinal segments(P<0.05). Compared with the raw group, all indicators in the milk-processed group significantly decreased(P<0.05), while no significant differences were observed in the water-processed group, indicating that milk, as an adjuvant in processing, plays a key role in reducing the intestinal toxicity of Euphorbiae Ebracteolatae Radix. Mass spectrometry results revealed that 29 components were identified in the raw product, including 28 terpenoids and 1 acetophenone. The content of these components decreased to varying extents after milk processing. A total of 28 components derived from Euphorbiae Ebracteolatae Radix were identified in the milky precipitate, of which 27 were terpenoids, suggesting that milk processing promotes the transfer of toxic components from Euphorbiae Ebracteolatae Radix into milk. To further investigate the effect of milk adjuvant processing on the toxic terpenoid components of Euphorbiae Ebracteolatae Radix, transmission electron microscopy(TEM) was used to observe the morphology of self-assembled casein micelles(the main protein in milk) in the milky precipitate. The micelles formed in casein-terpenoid solutions were characterized using particle size analysis, fluorescence spectroscopy, ultraviolet spectroscopy, and Fourier-transform infrared(FTIR) spectroscopy. TEM observations confirmed the presence of casein micelles in the milky precipitate. Characterization results showed that with increasing concentrations of toxic terpenoids, the average particle size of casein micelles increased, fluorescence intensity of the solution decreased, the maximum absorption wavelength in the UV spectrum shifted, and significant changes occurred in the infrared spectrum, indicating that interactions occurred between casein micelles and toxic terpenoid components. These findings indicate that the cathartic effect of Euphorbiae Ebracteolatae Radix becomes milder and its intestinal inflammatory toxicity is reduced after milk processing. The detoxification mechanism is that terpenoid components in Euphorbiae Ebracteolatae Radix reassemble with casein in milk to form micelles, promoting the transfer of some terpenoids into the milky precipitate.
Animals ; Mice ; Milk/chemistry* ; Drugs, Chinese Herbal/chemistry* ; Male ; Tumor Necrosis Factor-alpha/immunology* ; Intestines/drug effects* ; Interleukin-1beta/immunology* ; Tandem Mass Spectrometry ; Female

This paper aimed to explore the intestinal toxicity of Euphoriae Ebracteolata Radix(EER) before and after being processed with Mongolian medicine Hezi Decoction(HZD) and the toxicity-reducing mechanism of this processing method. The intestinal toxicity in rats treated with unprocessed EER and HZD-processed EER extracts via 95% ethanol was compared. The comparison was based on several indicators, including fecal volume, serum diamine oxidase(DAO) and D-lactate(D-LA) levels, the water content of various intestinal segments and their contents, and inflammatory factor levels in intestinal segments. Tandem mass tag(TMT) quantitative proteomics technology was employed to analyze the key proteins associated with changes in intestinal toxicity between unprocessed EER and HZD-processed EER. The results indicated that compared with the blank group, unprocessed EER significantly increased the fecal volume, serum DAO and D-LA levels, water content of the ileal segment and its contents, as well as the release levels of inflammatory factors, including tumor necrosis factor(TNF-α) and interleukin-1 beta(IL-1β) in the ileal segment of rats(P<0.05), indicating that EER can cause diarrhea, increase intestinal permeability, and induce intestinal inflammation. Compared with those in the unprocessed EER group, all indicators in the HZD-processed EER group were significantly reduced(P<0.05). The TMT quantitative proteomics analysis revealed that a total of 6 487 proteins were identified in the rat ileum tissue. Compared to the blank group, 182 proteins exhibited significant changes in the unprocessed EER group, while 907 proteins in the HZD-processed EER group showed significant changes. The intersection of the differential proteins between the two groups identified 38 common proteins. Among them, the protein levels of intestinal barrier tight junction protein claudin3, squalene monooxidase(Sqle), clusterin, Na~+/H~+ exchange regulatory cofactor NHE-RF3(Pdzk1), and Y+L amino acid transporter 1(Slc7a7) exhibited significant changes before and after processing, and these changes were closely related to intestinal barrier function. Compared with the blank group, the expression of claudin3, Pdzk1, and Slc7a7 in the raw product group was significantly down-regulated(P<0.05),while the expression of Sqle and clusterin was significantly up-regulated(P<0.05).Compared with the raw product group, the expression of claudin3, Pdzk1, and Slc7a7 in the processed product group of HZD was significantly up-regulated(P<0.05), while the expression of Sqle and clusterin was significantly down-regulated(P<0.05). Western blot was used to detect the expression level of claudin 3 in the ileum of rats in each group. The results show that compared to that in the blank group, the expression level of claudin 3 in the unprocessed EER group was significantly reduced(P<0.01); compared to that in the unprocessed EER group, the expression level of claudin 3 in the HZD-processed EER group was significantly increased(P<0.01). This finding aligned with the proteomic outcomes, indicating that claudin 3 protein levels could serve as a crucial indicator for intestinal damage caused by EER. In summary, HZD-processed EER can reduce EER's intestinal toxicity, and the primary mechanism for its alleviation of intestinal barrier damage is the regulation of the intestinal barrier tight junction protein claudin 3 and other intestinal-related proteins.
Animals ; Drugs, Chinese Herbal/adverse effects* ; Proteomics ; Rats ; Male ; Rats, Sprague-Dawley ; Intestines/drug effects* ; Intestinal Mucosa/drug effects* ; Tumor Necrosis Factor-alpha/metabolism*