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.

The auxin/indole-3-acetic acid (Aux/IAA) gene family is an important regulator for plant growth hormone signaling, involved in plant growth, development, as well as response to environmental stresses. In the present study, we identified SmIAA7 which is potentially associated with Salvia miltiorrhiza leaf development through comparatively analyzed the transcriptome data from different pinnate leaves. SmIAA7 was successfully isolated from S. miltiorrhiza using the specific primers. Then subsequent bioinformatic analysis, prokaryotic expression and purification, subcellular localization, and induction expression analysis under auxin and abiotic stress were performed. The full-length of SmIAA7 contained an ORF of 684 bp encoding a protein of 227 amino acid with a molecular weight of 25.3 kD. Conserved domain analysis showed that SmIAA7 contains the conserved Aux_IAA domain (pfam02309). Sequence analysis and phylogenetic tree analysis results indicated SmIAA7 was phylogenetically close to IAA7 and IAA14 from other plants, suggesting SmIAA7 involved in plant growth and development as well as response to environmental stresses. The prokaryotic expression vector pET28a-SmIAA7 was constructed and SmIAA7 recombinant protein was successfully expressed in E. coli Rosetta (DE3) strain. Subcellular localization experiment demonstrated that SmIAA7 localized in the nucleus of plant cells. Real-time fluorescence quantitative PCR results showed that the expression level of SmIAA7 was upregulated in response to auxin. Drought, low temperature, and salt stress significantly increased the transcript level of SmIAA7 gene. This study lays a foundation for further elucidating the role of SmIAA7 in leaf development, signal transduction, and stress defense in S. miltiorrhiza.

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.

Aim To investigate the synergistic sensiti-zation effect of saikosaponin D(SSD)combined with temozolomide(TMZ)on glioblastoma cells(GBM)and its molecular mechanism.Methods The sensitiv-ity of RG-2,U251 and LN-428 GBM cell lines to SSD and TMZ was analyzed by CCK-8 method combined with HE staining,and the optimal compatible concen-tration was screened.The effect of HE staining com-bined with Hoechst fluorescence staining on the prolif-eration of GBM cell line was detected by clonal forma-tion experiment.The autophagosome formation of GBM cells was observed by monodansylcadaverine(MDC)staining.The expression and distribution of endoplas-mic reticulum stress-related factors and apoptosis and autophagy proteins were detected by Western blot and ICC.Results The sensitivity order of GBM cells to TMZ was RG-2＞U251＞LN-428.The results of com-bined administration showed the synergistic inhibitory effect of SSD combined with TMZ on proliferation of GBM cell lines,which was confirmed by cell cloning formation experiment.Compared with the TMZ group,Hoechst fluorescence staining showed a significant in-crease in the number of nuclear bright staining in the combined administration group.MDC fluorescence staining showed that there were more dense green parti-cles in the cytoplasm of SSD/TMZ plus group than that of TMZ group.Western blot results showed that com-pared with TMZ group,the expression of ER stress markers GRP78,CHOP,p-PERK and ATF6 signifi-cantly increased in SSD/TMZ group(P＜0.05).The expressions of apoptosis proteins caspase-12,caspase-9,caspase-3,cleaved caspase-3,Bax and autophagy proteins LC3 and Beclin-1 significantly increased(P＜0.05),which were verified by ICC test.Conclusions SSD can cooperate with TMZ to inhibit the prolifera-tion of GBM cells and induce apoptosis and autophagy,and enhance the sensitivity of GBM cells to TMZ by ac-tivating endoplasmic reticulum stress pathway.

Aim To investigate the effects of L-argi-nine on spontaneous contraction of rat colon and the relative mechanisms.Methods An organ bath system was use to measure the spontaneous contraction of both longitudinal smooth muscle strips(LMS)and circular muscle strips(CMS).Whole-cell voltage-clamp tech-niques were applied to observe the alterations in cur-rents of L-type voltage-dependent Ca2+channels(VD-CCs),and voltage-gated K+channel(Kv)in single smooth muscle cells(SMCs)from rat colon.Results L-Arginine(1 mmol·L-1)significantly enhanced the spontaneous contraction of both LMS and CMS.The excitatory response to L-arginine was remarkably attenuated by tetrodotoxin(1 mmol·L-1)and atro-pine(1 mmol·L-1),antagonist of muscarinic ACh receptor(mAChR).However,the inhibitor of nitric oxide(NO)synthesis L-NNA(1 mmol·L-1)failed to block the action of L-Arginine.Furthermore,whole-cell patch-clamp recordings showed that L-arginine ac-tivated VDCCs and inhibited Kv channels on SMCs.Conclusions L-Arginine exerts an excitatory effect on colonic motility in a nitric oxide(NO)-independent manner and the stimulatory action of L-arginine is part-ly mediated by mAChR.In addition,VDCCs and Kv channels are both involved in L-arginine-induced exci-tation.

Objective To investigate clinical effect of tendons pulling,poking and kneading for the treatment of external humeral epicondylitis.Metods From January 2018 to December 2021,a multicenter randomized controlled study was per-formed to collect 192 patients with external humeral epicondylitis in Wangjing Hospital,Beijing Dianli Hospital,and Beijing Fengsheng Osteotraumatology Hospital,respectively,and they were divided into treatment group and control group by random number table method.There were 96 patients in treatment group,including 36 males and 60 females,aged from 28 to 60 years old with an average of(41.20±5.50)years old;the course of disease ranged from 1 to 14 days with an average of(5.24±1.35)days;they were treated once every other day for 2 weeks.There were 96 patients in control group,including 33 males and 63 females,aged from 26 to 60 years old with an average of(43.35±7.75)years old;the course of disease ranged from 1 to 14 days with an average of(5.86±1.48)days;they were treated with topical voltaalin combined with elbow joint fixation for 2 weeks.Visual analogue scale(VAS)and Hospital for Surgery Scoring System(HSS)elbow pronation and supination angles,wrist metacarpal flexion and dorsal extension angles,elbow tenderness between two groups were compared before treatment and at 1,3,5,7,11 and 13 days after treatment;Hospital for Surgery Scoring System 2(HSS2)was compared before treatment and the final treatment.Results All patients were followed up for 10 to 14 days with an average of(12±1.6)days.VAS between treatment group and control group before treatment were 6.83±1.36 and 6.79±1.58,respectively,and decreased to 1.49±1.09 and 2.11±1.81 after the final treatment.VAS of treatment group were significantly lower than those of control group at 1,3,5,7,9,11 and 13 days after treatment(P＜0.05).HSS between two groups were 61.73±11.00 and 36.47±12.45 before treatment,respectively,and increased to 94.42±5.9 and 91.44±9.11 at the final treatment.HSS of treatment group were signifi-cantly higher than those of control group at 1,3,5,7,9,11 and 13 days after treatment(P＜0.05).On the 5th day after treat-ment,the external and internal rotation angles of elbow in treatment group were(66.41±12.69)° and(66.35±13.54)°,while those in control group were(62.08±16.03)° and(61.77±16.35)°.On the 7th day after treatment,the external and internal ro-tation angles of elbow were(69.79±12.64)° and(70.02±13.55)° in treatment group,and(65.28±15.86)° and(65.09± 16.67)° in control group.Elbow joint motion in treatment group was higher than that in control group(P＜0.05).On the 5th day after treatment,angles of wrist dorsiflexion and palm flexion were(39.43±15.94)°and(46.68±11.10)° in treatment group,and(38.51±18.49)° and(44.27±13.58)° in control group.On the 7th day after treatment,angles of wrist dorsiflexion and palm flexion were(42.52±16.50)° and(49.23±10.96)° in treatment group,and(41.18±20.09)° and(46.64±14.63)° in control group.The motion of wrist joint in treatment group was higher than that in control group(P＜0.05).On the 13th day after treatment,HSS2 in treatment group 93.61±6.32 were higher than those in control group 92.06±7.94(P＜0.05).There was no significant difference in elbow tenderness between two groups at each time point(P＞0.05).Conclusion Voltaren external treatment combined with elbow fixation and tendons pulling,poking and kneading could effectively improve symptoms of exter-nal humeral epicondylitis.Compared with voltaren external treatment,tendons pulling,poking and kneading has advantages of longer analgesic time and better elbow function recovery.

To clone and express the K26 gene of Leishmania isolated from three types of visceral leishmaniasis epidemic ar-eas in China and evaluate its effect on detecting specific antibodies against visceral leishmaniasis.The K26 fragments from Leishmania isolated KS-6,SC6 and JIASHI-1 was synthesized and cloned into pET32a vector.The recombinant plasmid pET32a-K26 was transformed into Escherichia coli BL21 strains and induced by isopropyl-β-D-thiogalactopyranoside(IPTG).The expressed recombinant protein was purified by the His-tagged affinity column(Ni-NTA).Serum samples of 110 visceral leishmaniasis patients were used for evaluating the sensitivity by ELISA.Serum samples from patients with malaria,schisto-somiasis japonica,cystechinococcosis,toxoplasmosis,paragon-imiasis,clonorchiosis and 40 healthy people were used for eval-uating the specificity.Detection results of ELISA were compared with that of rK39 strip of American InBios company.Comparation among three K26 antigens were given by x2 test.The sensitivity of the recombinant K26 protein of KS-6,SC6 and JIASHI-5 strains of Leishmania and rK39 strip test to detect the sera of patients with visceral leishmaniasis was 90.00％(99/110),92.73％(102/110),90.91％(100/110)and 93.64％(103/110),respectively.There was no cross reactivity with malaria(10),schistosomiasis japonica(10),cystechinococcosis(10),toxoplasmosis(5),paragonimiasis(5)and clonorchiosis(5),and 40 sera from healthy people were also negative.The specificity was 100.00％.There was no statistical difference in the sensitivity of the recombinant K26 protein of KS-6,SC6 and JIASHI-1 strains of Leishmania and rK39 strip test,x2 values are 0.97,0.07 and 0.57 respectively and the P values are 0.33,0.79 and 0.45,respectively.There was no statis-tical difference in the sensitivity of three K26 antigens(x2=0.53,P=0.97).Conclusion The recombinant K26 antigen has po-tential application value in the diagnosis of visceral leishmaniasis.

Objective:To investigate the relationship between mutated genes and clinical features in patients with essential thrombocythemia(ET).Methods:The clinical data of 69 patients with ET from October 2018 to March 2022 were retrospectively analyzed.According to driver mutation type,patients were divided into JAK2 group,CALR group and triple-negative group.The sex,age,cardiovascular risk factors,thrombosis,splenomegaly,routine blood test and coagulation status of patients in three groups were analyzed.Results:Among 69 ET patients,46 cases were associated with JAK2 mutation,14 cases with CALR mutation,8 cases with triple-negative mutation,and one with MPL gene mutation.There were no significant differences in age and sex among the three groups(P＞0.05).The highest thrombotic rate was 26.09％(12/46)in JAK2 group,then 12.5％(1/8)in triple-negative group,while no thrombotic events occurred in CALR group.The incidence of splenomegaly was the highest in JAK2 group(34.78％),while no splenomegaly occurred in triple-negative group.The white blood cell(WBC)count in JAK2 group was(9.00±4.86)× 109/L,which was significantly higher than(6.03±2.32)× 109/L in CALR group(P＜0.05).The hemoglobin(Hb)and hematocrit(HCT)in JAK2 group were(148.42±18.79)g/L and(0.44±0.06)％,respectively,which were both significantly higher than(131.00±15.17)g/L and(0.39±0.05)％in triple-negative group(P＜0.05).The platelet(PLT)in JAK2 group was(584.17±175.77)× 109/L,which was significantly lower than(703.07±225.60)× 109/L in CALR group(P＜0.05).The fibrinogen(Fg)in JAK2 and triple-negative group were(2.64±0.69)g/L and(3.05±0.77)g/L,respectively,which were both significantly higher than(2.24±0.47)g/L in CALR group(P＜0.05,P＜0.01).The activated partial thromboplastin time(APTT)in triple-negative group was(28.61±1.99)s,which was significantly decreased compared with(31.45±3.35)s in CALR group(P＜0.05).Conclusions:There are differences in blood cell count and coagulation status among ET patients with different driver gene mutations.Among ET patients,JAK2 mutation is most common.Compared with CALR group,the thrombotic rate,WBC and Fg significantly increase in JAK2 group,while PLT decrease.Compared with triple-negative group,the incidence of splenomegaly and HCT significantly increase.Compared with CALR group,Fg significantly increases but APTT decreases in triple-negative group.

Objective:To investigate the effect of selenium on cyclophosphamide(CTX)-induced spermatogenic impairment(SI)in mice and its underlying mechanism.Methods:We equally randomized 36 male KM mice into 3 SI model and 3 control groups,the first 3 treated by intraperitoneal injection of CTX at 100 mg/kg(the SI model control group),CTX plus SI model control group,selenium deficient model group(-Se SI),selenium supplemented model group(+Se SI),while latter 3 by intraperitoneal injection of normal saline(the normal control),selenium deficiency control group(-Se control),selenium addition control group(+Se control),respectively,all once a week for 6 successive weeks.Then we observed the histopathological changes in the testes of all the mice by HE staining,obtained the sperm count in the epididymides,determined the expressions of glutathione peroxidase 4(GPx4)and SLC7A11 proteins by Western blot and ferroptosis-related genes by RT-qPCR,and examined the changes in the expres-sions of ferroptosis-related proteins and genes in the GC2-spd cells treated with ferroptosis inhibitors and inducers in combination with different concentrations of inorganic sodium selenite(SeS)and organic selenomethionine(SeM).Results:Compared with the nor-mal controls,the SI model mice showed significantly decreased testicular and prostatic organ coefficients,reduced spermatogenic lay-ers,increased voids,decreased serum ferritin concentration(P<0.05),and elevated transferrin concentration(P<0.05).The or-gan coefficients were significantly higher in the+Se SI and+Se control than in the-Se SI and-Se control groups(P<0.05,P<0.01),with evident pathological improvement of the testis tissue in the+Se controls.The expressions of the GPx4 and solute carrier family 7 members 11(SLC7A11)genes in the testis were dramatically down-regulated in the SI model controls(P<0.01),but up-reg-ulated in the+Se SI and+Se control compared with those in the-Se SI and-Se control group(P<0.01 and P<0.05),but there were no statistically significant differences between their protein expressions.The results of in vitro GC2 spd cell experiments indicated that the GPx4 gene and GPx4 protein levels in the-Se group were significantly lower than those in the normal control group(P<0.05),while the SLC7A11 gene level decreased(P<0.01).Different doses of SeS and SeM significantly increased the GPx4 protein expression compared to the average Se group.Low doses of SeM promoted a significant increase in GPx4 gene levels,while high doses of SeS increased the expression levels of SLC7A11 gene and SLC7A11 protein(P<0.05,P<0.01).The Se group showed a signifi-cant decrease in the levels of acsl4 and ptgs2 genes compared to the normal control group.SeM promoted the expression of acsl4,while SeS promoted the expression of ptgs2 and fth1(P<0.01,P<0.05).The intervention results of GC2 spd showed that the Erastin group had a decrease in ptgs2 compared to the normal control group,while the SeS+Erastin and SeM+Erastin groups had an increase in ptgs2 gene expression compared to the Erastin group.However,the ptgs2 expression of Fer-1 was lower than that of the normal con-trol group,and the ptgs2 gene level of SeS+Fer-1 and SeM+Fer-1 groups was lower than that of Fer-1 group(P<0.05);The gene quantity of GPx4 in the SeM+Erastin and SeM+Fer-1 groups increased compared to the Erastin and Fer-1 groups(P<0.01,P<0.05);SeM+Erastin and SeS+Erastin showed a decrease in SLC7A11 compared to the Erastin group,as well as SeM+Fer-1 and SeS+Fer-1 groups compared to the Fer-1 group,accompanied by an increase in acsl4 and fth1(P<0.01).Conclusion:Selenium deficiency causes the reduction of the SLC7A11 and GPx4 gene levels,disorder of ferroptosis-related genes and down-regulation of the GPx4 protein expression in the mouse testis and spermatocytes.Selenium can promote the expression of GPx4,up-regulate the level of SLC7A11,and improve spermatogenesis in the testis of the mouse with SI.There are differences between organic SeM and inorganic SeS in regulating the ferroptosis pathway-related genes.