Inhibitory Effects of Bacillus subtilis on Staphylococcus aureus

[Objective] To produce drug resistance, seek non-toxic environmental so as to change the current biological drugs that did not excessive use of antibiotics. [Methods] A strain of Bacillus was purified and isolated from fresh and healthy in- testines of grass carps. Biochemical identification was carried out by conventional bacterial biochemical test method. Two pairs of primers were designed, 16S rRNA detection and sequencing analysis were carried out. Drug sensitive test was carried out by agar diffusion method. In vitro inhibition test on Staphylococcus aureus was carried out by Oxford cup method. [Results] The isolated bacterium had basically the same biochemical characters as Bacillus subtilis; and the homology reached 100%. Thus, the isolated bacterium was identified to be Bacillus subtilis. It was insensitive to amoxicillin, ampicillin, penicillin G and so on, but sensitive to amikacin, cefalexin, ciprofloxacin and cefradine. The inhibitory effects of Bacillus subtilis on Staphylococ- cus aureus were significant. The minimum inhibitory concentration （MIC） was 2.8×10^8×2^-5/ml and minimum bactericidal concentration （MBC） was 2.8×10^8×2^-2/ml. [Conclusions] The isolated Bacillus subtilis could be used to prevent and control diseases caused by Staphylococcus aureus, and reduce the abuse of antibiotics.

Inhibitory Effects of Original Nano-Cu_2O Drug and Nano-Cu_2O Suspension on Snake Melon Botrytis cinerea

The drug-containing culture medium method for the test of toxicity was adopted to compare inhibitive effects of original nano-Cu2O drug and nano-Cu2O suspension, and nano-Cu2O drug has better inhibitive effects on snake melon Botry- tis cinerea than original nano-Cu2O drug with the same mass concentration, and inhibitory effects are positively correlated with concentration. Correlation coefficients of the toxicity regression equation are 0.892 2 and 0.996 1, effective concentration EC50 of original nano-Cu2O drug and that of nano-Cu2O suspension are 3 948.9 and 167.9 mg/kg. Original nano-Cu2O drug has an inhibitive effect on snake melon Botrytis cinerea, but the inhibition of nano-Cu2O suspension is more obvious.

Inhibitory Effects of Extracts from Marigold(Tagetes patula) against Tomato Fusarium Wilt

[ Objective ] The study aimed to explore a new way for the control of Tomato Fusarium Wilt. [ Method ] Different solvents were used to prepare the ex-tracts of marigold, and the inhibitory effects of different extraction solvents and different extraction parts of marigold against Tomato Fusar/um Wilt were compared. [ Result ] Among different solvent extracts of marigold, chloroform extracts had the strongest inhibitory effects against the growth of the pathogen; among the chloro- form extracts from different parts of marigold, root extract had the most obvious inhibitory effect against the disease, followed by flower and leaf extracts, and the in- hibitory effect of stem extract was the weakest. [ Conclusion ] The active components of marigold have inhibitory effect against Tomato Fusarium Wilt, and the plant has good development prospects and application value.

Chemopreventive and Growth Inhibitory Effects of Selenium

There is very convincing evidence that a high dietary level of selenium substantially reduces the incidence of a wide variety of animal cancers. The human epidemiological evidence is less clear cut, but overall suggests that selenium may be protective: the evidence is strongest in men in relation to gastro-intestinal cancers. There is evidence that dietary selenium compounds reduce the formation of DNA adducts by carcinogens. Selenium compounds also inhibit growth in vitro and induce apoptosis. In general, there is a good correlation between the effectiveness of selenium compounds in chemoprevention and growth inhibition, implying that the mechanisms of growth inhibition and chemoprevention may be similar and that a major factor in the chemopreventive effects of selenium compounds in vivo is their ability to retard outgrowth of pre-malignant cells. Various hypotheses have been advanced as to how selenium compounds might prevent tumour cellgrowth. One is that they cause apoptosis by inducing oxidative stress. However, we have shown that the most potent selenium compound, selenodiglutathione (SDG), a natural metabolite of selenite, does not induce oxidative stress, at least not in the sarne way as other oxidants such as H2O2 and diamide. Firstly, a partially selenium-resistant variant cell line does not show increased resistance to H2O2. Moreover, SDG does not induce widespread tyrosine phosphorylation, including MAP and SAN kinases, like other oxidants such as H2O2 and diamide and its effects are not reversed by pretreatment with the tyrosine kinase inhibitor, herbimycin. Our experiments with the selenium-resistant variant suggest that a novel selenium-binding protein may be involved in growth inhibition by selenium

Growth Inhibitory Effects of Garlic Polysaccharide on Human HepG2 Cells

[Objective] The growth inhibitory effects of garlic polysaccharide(GPS) on human Hep G2 cells were evaluated in this paper. [Method] Hep G2 cells were treated with GPS for 48 h for morphology assay by transition electron microscope. Anti-proliferative effects with the same treatment for 24 hand 48 h were assayed by MTT method.Cell cycle distribution and apoptosis assay of treated cells were performed in flow cytometry. [Result] The results showed that GPS enhanced growth inhibitory effect on Hep G2 cells in a time-and dose-dependent manner. PI(Propidium iodide)/Annexin V staining analyzed by FCM(flow cytometry) demonstrated that GPS has a cytotoxic effect on tumor cells. Cell cycle arrest of Hep G2 treated with GPS occurred in G2 phase. [Conclusion] This study suggests that GPS could exert an antitumor effect and could be used as a therapeutic agent for live cancer.

Inhibitory effects of saikosaponin-d on CCl_4-induced hepatic fibrogenesis in rats

AIM： To investigate the suppressive effect of saikosaponin-d （SSd） on hepatic fibrosis in rats induced by CCh injections in combination with alcohol and high fat, low protein feeding and its relationship with the expression of nuclear factor-κB （NF-κB）, tumor necrosis factor-alpha （TNF-α） and interleukins-6 （IL-6）. METHODS： Hepatic fibrosis models were induced by subcutaneous injection of CCh at a dosage of 3 mL/kg in rats. At the same time, rats in treatment groups were injected intraperitoneally with SSd at different doses （1.0, 1.5 and 2.0 mg/kg） once daily for 6 wk in combination with CCh, while the control group received olive oil instead of CCh. At the end of the experiment, rats were anesthetized and killed （except for 8 rats which died during the experiment; 2 from the model group, 3 in high-dose group, 1 in medium-dose group and 2 in lowdose group）. Hernatoxylin and eosin （HE） staining and Van Gieson staining were used to examine the changes in liver pathology. The levels of alanine aminotransferase （ALT）, triglyeride （TG）, albumin （ALB）, globulin （GLB）, hyaluronic acid （HA） and larninin （LN） in serum and the content of hydroxyproline （HYP） in liver were measured by biochemical examinations and radioimmuneoassay, respectively. In addition, the expression of TNF-α and IL-6 in liver homogenate was evaluated by enzymelinked immunosorbent assay （ELISA） and the levels of NF-κBp65 and I-κBa in liver tissue were analyzed by Western blotting. RESULTS： Both histological examination and Van Gieson staining demonstrated that SSd could attenuate the area and extent of necrosis and reduce the scores of liver fibrosis. Similarly, the levels of ALT, TG, GLB, HA, and LN in serum, and the contents of HYP, TNF-α and IL-6 in liver were all significantly increased in model group in comparison with those in control group. Whereas, the treatment with SScl markedly reduced all the above parameters compared with the model group, especially in the medium group （ALT： 412 ± 94.5 IU/L vs 113.76 ± 14.91 IU/L, TG： 0.95 ± 0.16 mmol/L vs 0.51 ± 0.06 mmol/L, GLB： 35.62 ± 3.28 g/L vs 24.82 ± 2.73 g/L, HA： 42.15 ± 8.25 ng/mL vs 19.83 ± 3.12 ng/mL, LN： 27.56 ± 4.21 ng/mL vs 13.78 ± 2.57 ng/mL, HYP： 27.32 ± 4.32 ug/mg vs 16.20 ± 3.12 ug/mg, TNF-a： 4.38 ± 0.76 ng/L vs 1.94 ± 0.27 ng/L, IL-6：28.24 ± 6.37 pg/g vs 12.72 ± 5.26 pg/g, respectively, P 〈 0.01）. SSd also decreased ALB in serum （28.49 ± 4.93 g/L vs 37.51 ± 3.17 g/L, P 〈 0.05）. Moreover, the expression of NF-KB p65 in the liver of treated groups was lower than that in model groups while the expression of I-κBa was higher in treated group than in model group （P 〈 0.01）. The expression of NF-κBp65 and TNF-a had a positive correlation with the level of HA in serum of rats after treatment with CCh （r = 0.862, P 〈 0.01; r = 0.928, P 〈 0.01, respectively）. CONCLUSION： SSd attenuates CCh-induced hepatic fibrosis in rats, which may be related to its effects of hepato-protective and anti-inflammation properties, the down-regulation of liver TNF-a, IL-6 and NF-κBp65 expression and the increased I-κBa activity in liver.

Effects of leukemia inhibitory factor on endogenous neural stem cell proliferation and glycoprotein-130 expression in a mouse model of cerebral infarction

Leukemia inhibitory factor （LIF） has been shown to promote proliferation of endogenous neural stem cells. In this study, we treated mice with cerebral infarction using LIF to investigate whether the LIF receptor subunit glycoprotein （gp）130 is involved in neuroprotection. After LIF treatment, the motor function of model mice was significantly improved. Immunofluorescence histochemistry showed increased numbers of endogenous neural stem cells surrounding the infarct foci. Western blot analysis revealed that gp130 expression was significantly decreased surrounding the infarcted foci. Results demonstrated that LIF promoted the proliferation of endogenous neural stem cells by inhibiting gp130 protein expression.

Effects of leukemia inhibitory factor and basic fibroblast growth factor on free radicals and endogenous stem cell proliferation in a mouse model of cerebral infarction

The present study established a mouse model of cerebral infarction by middle cerebral artery occlusion, and monitored the effect of 25 tJg/kg leukemia inhibitory factor and （or） basic fibroblast growth factor administration 2 hours after model establishment. Results showed that following administration, the number of endogenous neural stem cells in the infarct area significantly increased, malondialdehyde content in brain tissue homogenates significantly decreased, nitric oxide content, glutathione peroxidase and superoxide dismutase activity significantly elevated, and mouse motor function significantly improved as confirmed by the rotarod and bar grab tests. In particular, the effect of leukemia inhibitory factor in combination with basic fibroblast growth factor was the most significant. Results indicate that leukemia inhibitory factor and basic fibroblast growth factor can improve the microenvironment after cerebral infarction by altering free radical levels, improving the quantity of endogenous neural stem cells, and promoting neurological function of mice with cerebral infarction.

Inhibitory effects of apigenin on the growth of gastric carcinoma SGC-7901 cells

AIM： To explore the growth inhibition and apoptosisinducing effect of apigenin on human gastric carcinoma SGC-7901 cells. METHODS： The effects of apigenin on the growth, clone formation and proliferation of human gastric carcinoma SGC-7901 cells were observed by N-rr, done-forming assay, and morphological observation. Fluorescent staining and flow cytometry analysis were used to detect apoptosis of cells. RESULTS： Apigenin obviously inhibited the growth, clone formation and proliferation of SGC-7901 cells in a dosedependent manner. Inhibition of growth was observed on d 1 at the concentration of 80 μmol/L, while after 4 d, the inhibition rate （IR） was 90%. The growth IRs at the concentration of 20, 40, and 80 μmol/L were 38%, 71%, and 99% respedJvely on the 7^th d. After the cells were treated with apigenin for 48 h, the number of clone-forming in control, 20, 40, and 80 μmol/L groups was 217±16.9, 170±11.1 （P〈0.05）, 98±11.1 （P〈0.05）, and 25±3.5 （P〈0.05） respectively. Typical morphological changes of apoptosis was found by fluorescent staining. The cell nuclei had lost its smooth boundaries, chromatin was condensed, and cell nuclei were broken. Flow cytometry detected typical apoptosis peak. After the cells were treated with apigenin for 48 h, the apoptosis rates were 5.76%, 19.17%, and 29.30% respectively in 20, 40, and 80 μmol/L groups. CONCLUSION： Apigenin shows obvious inhibition on the growth and clone formation of SGC-7901 cells by inducing apoptosis.

Synergistic growth inhibitory effects of Phyllanthus emblica and Terminalia bellerica extracts with conventional cytotoxic agents:Doxorubicin and cisplatin against human hepatocellular carcinoma and lung cancer cells

AIM: To examine the growth inhibitory effects of Phyllanthus emblica (P. emblica) and Terminalia bellerica (T. bellerica) extracts on human hepatocellular carcinoma (HepG2), and lung carcinoma (A549) cells and their synergistic effect with doxorubicin or cisplatin. METHODS: HepG2 and A549 cells were treated with P. emblica and T. bellerica extracts either alone or in combination with doxorubicin or cisplatin and effects on cell growth were determined using the sulforhodamine B (SRB) assay. The isobologram and combination index (CI) method of Chou-Talalay were used to evaluate interactions between plant extracts and drugs. RESULTS: P. emblica and T. bellerica extracts demonstrated growth inhibitory activity, with a certain degree of selectivity against the two cancer cell lines tested. Synergistic effects (CI < 1) for P. emblica /doxorubicin or cisplatin at different dose levels were demonstrated in A549 and HepG2 cells. The T. bellerica/ cisplatin or doxorubicin also showed synergistic effects in A549 and HepG2 cells. In some instances, the combinations resulted in antagonistic effects. The dose reduction level was different and specific to each combination and cell line. CONCLUSION: The growth inhibitory activity of doxorubicin or cisplatin, as a single agent, may be modified by combinations of P. emblica or T. bellerica extracts and be synergistically enhanced in some cases. Depending on the combination ratio, the doses for each drug for a given degree of effect in the combination may be reduced. The mechanisms involved in this interaction between chemotherapeutic drugs and plant extracts remain unclear and should be further evaluated.

Preparation and Inhibitory Effects of 20(S)-Ginsenoside Rh_2 on Hep-A-22 Cells

A modified method of preparing 20（S）-ginsenoside Rh2（G-Rh2） and the inhibitory effect of 20（S）-ginsenoside Rh2 on Hep-A-22 cells were investigated. The total saponins and strong alkali were dissolved in glycerol at the atmospheric pressure, and the degradation was performed at a high temperature. After G-Rh2 had been isolated and purified, MTT（methyl thiazolyl tetrazolium） assay was applied to evaluating the effect of 20（S）-ginsenoside Rh2 on the cells viability and morphological changes were observed. It was shown that 20（S）-ginsenoside Rh2 can reduce Hep-A-22 cells viability in dose-dependent manner and the cells took on cell shrinkage, membrane blebbing, chromosomal condensations, especially under the higher concentrations of it. In conclusion, 20（S）-ginsenoside Rh2 can be prepared effectively that not only decreases viability but also induces the apoptosis of Hep-A-22 cells.

Inhibitory Effects of Commonly Used Fungicides against Cercospora arachidicola Hori

Early leaf spot is an important disease of peanut, seriously affecting the yield of peanut. The inhibitory effects of different pesticides against early leaf spot have not been analyzed systematically. Based on screening of Cercospora arachidicola Hori in earlier period, the inhibition rates of different pesticides on C. arachidicola growth were studied, and pesticides with strong inhibitory effects against C. arachidicola were screened. The paper provided a reference for field imitation test.

Identifying Combinatorial Growth Inhibitory Effects of Various Plant Extracts on Leukemia Cells through Systematic Experimental Design

Plant extracts are widely studied for their anti-cancer and cancer preventive effects. In this study, we compared the leukemia growth inhibition effects of seven different plant extracts, theaflavin, epigallocatechin gallate (EGCG), epicathechin (EC), apigenin, quercetin, chrysin and tannic acid, in vitro using the K562 erythroleukemia cell line and application of the design of experiments (DoE) methodology. Our systematic approach enabled us to isolate the main factor contribution, two-factor interactions and produced interaction relationships and/or models to describe growth inhibitory effects of different plant extracts when they are used in combination. The results identified tannic acid as the most significant inhibitor in this group and had synergistic effects with EGCG at specific concentrations. The fitted model of their combined effects showed that the most potent combination is at low concentrations of tannic acid (10 - 20 μM) and high concentrations of EGCG (80 - 100 μM). We further showed that tannic acid induced both growth inhibition and apoptosis in K562 cells in ranges between 10 - 100 μM. The polyphenol caused cell cycle arrest at G2- phase under the higher concentrations. In summary, use of DoE techniques effectively identified the most prominent inducer in this group of plant bioactive compounds and produced combinatorial bioactivity of various polyphenols and flavonoids over the entire range of concentrations under study. This study exemplifies the usefulness of DoE and serves as a guide in its utility for in vitro assessment of bioactivity in plant constituents.

Angiotensin I converting enzyme(ACE)inhibitory activity and antihypertensive effects of rice peptides

In this paper,the antihypertension effect of rice peptide(RP)was studied.With spontaneously hypertensive rats(SHR)and Wistar Kyoto(WKY)as the research objects,RP disposable gastric and long-term gastric irrigation experiments were carried out and systolic blood pressure(SBP)was measured.At the end of the long-term gastric irrigation experiment,the content of nitric oxide(NO),angiotensin-converting enzyme(ACE),angiotensin II(Ang II)and renin in the plasma and the activity of ACE were determined.The results showed that RP could reduce systolic pressure of SHR and had time-dose dependence while high-dose RP signifi cantly reduced systolic pressure by 24.6 and 17.2 mm Hg,respectively after a single and long-term gastric irrigation test.RP also could inhibit the activity of ACE and increase the release of NO.These results suggested that the decompression mechanism of RP is likely to be related to the regulation of the renin-angiotensin system(RAS)and NO.

Inhibitory effects of Albizia lebbeck leaf extracts on germination and growth behavior of some popular agricultural crops

An experiment was conducted to observe the inhibitory effects of the leaf extracts derived from Albizia lebbeck （L.） Benth. On germination and growth behavior of some popular agricultural crops （receptor） of Bangladesh. Experiments were set on sterilized petridishes with a photoperiod of 24 h at room temperature of 27-30℃. The effects of the different concentrations of aqueous extracts were compared to distil water （control.）. The aqueous extracts of leaf caused significant inhibitory effect on germination, root and shoot elongation and development of lateral roots of receptor plants. Bioassays indicated that the inhibitory effect was proportional to the concentrations of the extracts and higher concentration （50%-100%） had the stronger inhibitory effect whereas the lower concentration （10%-25%） showed stimulatory effect in some cases. The study also revealed that, inhibitory effect was much pronounced in root and lateral root development rather than germination and shoot growth.

The roles of macrophage migration inhibitory factor in retinal diseases

Macrophage migration inhibitory factor(MIF),a multifunctional cytokine,is secreted by various cells and participates in inflammatory reactions,including innate and adaptive immunity.There are some evidences that MIF is involved in many vitreoretinal diseases.For example,MIF can exacerbate many types of uveitis;measurements of MIF levels can be used to monitor the effectiveness of uveitis treatment.MIF also alleviates trauma-induced and glaucoma-induced optic nerve damage.Furthermore,MIF is critical for retinal/choroidal neovascularization,especially complex neovascularization.MIF exacerbates retinal degeneration;thus,anti-MIF therapy may help to mitigate retinal degeneration.MIF protects uveal melanoma from attacks by natural killer cells.The mechanism underlying the effects of MIF in these diseases has been demonstrated:it binds to cluster of differentiation 74,inhibits the c-Jun N-terminal kinase pathway,and triggers mitogen-activated protein kinases,extracellular signal-regulated kinase-1/2,and the phosphoinositide-3-kinase/Akt pathway.MIF also upregulates Toll-like receptor 4 and activates the nuclear factor kappa-B signaling pathway.This review focuses on the structure and function of MIF and its receptors,including the effects of MIF on uveal inflammation,retinal degeneration,optic neuropathy,retinal/choroidal neovascularization,and uveal melanoma.

Combination of metformin and curcumin exhibits synergistic inhibitory effects on tumor growth and metastasis in HepG2 cells

OBJECTIVE Hepatocel ular carcinoma(HCC)is the most common cause of cancer-related mortality,with high incidence rates,robust metastatic propensity and acquired resistance to therapy.Metformin,an extensively prescribed and well-tolerated first-linetherapeutic drug for type 2 diabetes mellitus,has recently been identified as a potential and attractive anticancer adjuvant drug combined with chemotherapeutics to improve treatment efficacy and lower doses.Curcumin,a botanical extracts,has been shown antitumorigenic properties.This study aims to investigate the combinational effect of metformin and curcumin on inbibition of tumor growth and metastasis in Hep G2 cells and the possible underlying mechanisms.METHODS The cell proliferation was determined by MTT,CCK-8 and colony formation assay.The protein expression was detected by Western blotting.Activity of MMP-2 and MMP-9 was estimated by gelatin zymography.Flow cytometry analysis was used to evaluate the influence of metformin and curcumin on cell cycle arrest and apoptosis,and morphology observation of apoptosis was detected by Hoechst33342.Scratch and transwell assay was performed to detect the cell migration and invasion.The suppression of this combination therapy oncapillary tube formation was detected by tube formation assay.RESULTS Combination of metformin and curcumin induced stronger inhibition on Hep G2 cells proliferation than monotherapywhich related to induction of cell cycle arrest in G2/M phase and apoptosis through regulation of the protein expression of cyclin B and Bcl-2/Bax.Moreover,the co-treatment of metformin with curcumin exerted an enhanced inhibitory effect on Hep G2 cell metastasis and synergistically inhibited the tube formation of HUVEC cells.The suppression of PI3K/AKT/m TOR pathway and inhibition the protein expression of STAT3,MMP9,MMP2 and VEGF might involve in this synergistic effects of combination treatment.CONCLUSION Combination of metformin and curcumin inhibited Hep G2 cells proliferationmore effectively than monotherapy and synergistically induced a greater inhibition on migration and invasion of Hep G2 cells.

Inhibitory Effects of Six Fungicides against Neocordana musae

[Objective]The paper was to screen effective fungicides to control Cordana leaf spot caused by Neocordana musae.[Method]The toxicities of six fungicides against mycelial growth of N.musae were measured by the method of mycelium growth inhibition.[Result]The average EC50 values of six fungicides against mycelial growth ranged from 0.1029 to 504.065μg/mL.Among them,22.5%picoxystrobin SC and 12.5%epoxiconazole SC showed strong inhibitory activity on mycelial growth,with the average EC50 values of 0.1029 and 0.6851μg/mL,respectively;followed by 50%thiophanate-methyl SC,with an average EC50 value of 1.993.80%Azoxystrobin WG showed a relatively low sensitivity against N.musae,with an average EC50 value of 504.046.[Conclusion]The six fungicides tested have great value in preventing and controlling Cordana leaf spot.22.5%Picoxystrobin SC,12.5%epoxiconazole SC and 50%thiophanate-methyl SC have better inhibitory activities on mycelial growth of N.musae,and can be further used in field trials.

Inhibitory Effects of Sixteen Fungicides on Mycelial Growth and Spore Germination of Monilinia fructicola

[Objective]The paper was to compare the indoor toxicities of sixteen fungicides on mycelial growth and spore germination of Monilinia fructicola,to screen out effective fungicides and to discuss use characteristics of various types of fungicides.[Method]The inhibitory activities of 16 fungicides on mycelial growth and spore germination were determined by mycelial growth rate method and spore germination method.[Result]The EC50 values of 16 fungicides against mycelial growth ranged from 0.0184 to 61.5305 mg/L.Prochloraz,tetramycin,fenbuconazole and fludioxonil had strong inhibitory activities on mycelial growth,and their EC50 values were 0.0184,0.0456,0.0531 and 0.0814 mg/L,respectively,significantly lower than those of other 12 fungicides.The EC50 values of 16 fungicides against spore germination ranged from 0.0084 to 189.3938 mg/L.Tetramycin and chlorothalonil had strong inhibitory activities on mycelial growth,and their EC50 values were 0.0084 and 0.0378 mg/L,respectively,significantly lower than those of other 14 fungicides.[Conclusion]The 16 fungicides had great value in preventing and controlling peach brown rot.Benzimidazoles,diformimides and ergosterol inhibitors had good inhibitory activities on mycelial growth.Strobilurins,succinate dehydrogenase inhibitors and multiple-site protective fungicides had good inhibitory activities on spore germination.The agricultural antibiotics tetramycin,phenazine-1-carboxylic acid and pyrrole fungicide fludioxonil had good inhibitory activities on mycelial growth and spore germination.

Cumulative effects of excess high-normal alanine aminotransferase levels in relation to new-onset metabolic dysfunction-associated fatty liver disease in China

BACKGROUND Within the normal range,elevated alanine aminotransferase(ALT)levels are associated with an increased risk of metabolic dysfunction-associated fatty liver disease(MAFLD).AIM To investigate the associations between repeated high-normal ALT measurements and the risk of new-onset MAFLD prospectively.METHODS A cohort of 3553 participants followed for four consecutive health examinations over 4 years was selected.The incidence rate,cumulative times,and equally and unequally weighted cumulative effects of excess high-normal ALT levels(ehALT)were measured.Cox proportional hazards regression was used to analyse the association between the cumulative effects of ehALT and the risk of new-onset MAFLD.RESULTS A total of 83.13%of participants with MAFLD had normal ALT levels.The incidence rate of MAFLD showed a linear increasing trend in the cumulative ehALT group.Compared with those in the low-normal ALT group,the multivariate adjusted hazard ratios of the equally and unequally weighted cumulative effects of ehALT were 1.651[95%confidence interval(CI):1.199-2.273]and 1.535(95%CI:1.119-2.106)in the third quartile and 1.616(95%CI:1.162-2.246)and 1.580(95%CI:1.155-2.162)in the fourth quartile,respectively.CONCLUSION Most participants with MAFLD had normal ALT levels.Long-term high-normal ALT levels were associated with a cumulative increased risk of new-onset MAFLD.