Leishmanicidal activity in vitro of Musa paradisiaca L. and Spondias mombin L. fractions

Visceral leishmaniasis (VL) is a zoonotic disease characterized by infection of mononuclear phagocytes by Leishmania chagasi. The primary vector is Lutzomyia longipalpis and the dog is the main domestic reservoir. The control and current treatment of dogs using synthetic drugs have not shown effectiveness in reducing the incidence of disease in man. In attempt to find new compounds with leishmanicidal action, plant secondary metabolites have been studied in search of treatments of VL. This study aimed to evaluate the leishmanicidal activity of Musa paradisiaca (banana tree) and Spondias mombin (cajazeira) chemical constituents on promastigotes and amastigotes of L. chagasi. Phytochemical analysis by column chromatography was performed on ethanol extracts of two plants and fractions were isolated. Thin layer chromatography was used to compare the fractions and for isolation the substances to be used in vitro tests. The in vitro tests on promastigotes of L. chagasi used the MTT colorimetric method and the method of ELISA in situ was used against amastigotes besides the cytotoxicity in RAW 264.7 cells. Of the eight fractions tested, Sm1 and Sm2 from S. mombin had no action against promastigotes, but had good activity against amastigotes. The fractions Mp1 e Mp4 of M. paradisiaca were very cytotoxic to RAW 264.7 cells. The best result was obtained with the fraction Sm3 from S. mombin with IC(50) of 11.26 μg/ml against promastigotes and amastigotes of 0.27 μg/ml. The fraction Sm3 characterized as tannic acid showed the best results against both forms of Leishmania being a good candidate for evaluation in in vivo tests.     

Canine visceral leishmaniasis: comparison of in vitro leishmanicidal activity of marbofloxacin, meglumine antimoniate and sodium stibogluconate

The control of canine leishmaniasis largely depends on the success of treatment. Drugs currently available to treat this disease are toxic and partially effective. The curative effect of marbofloxacin, a third-generation fluoroquinolone developed for veterinarian individual treatment, was evaluated in vitro in the presence of Leishmania infantum promastigotes and dog-monocyte-derived macrophages; meglumine antimoniate and sodium stibogluconate were used as comparative treatments. We observed that the killing of Leishmania promastigotes and intracellular amastigotes by marbofloxacin was dose-dependent. We demonstrated that successful treatment of canine infected macrophages for 48 h was possible with 500 microg/ml of marbofloxacin. Leishmanicidal activity acted through a TNF-alpha and nitric oxide pathway and correlated with the generation of nitric oxide (NO(2)) production by monocytes derived macrophages from infected (23+/-5 microM) or healthy (21+/-6 microM) dogs, in comparison with NO(2) concentration in infected/non-treated macrophages (< 3 microM, P<0.01). This significant induced parasiticidal effect correlated with extensive elimination of amastigotes by macrophages derived from infected (11+/-5) and healthy dogs (6+/-2), when compared to infected/non-treated macrophages (530+/-105 and 472+/-86 amastigotes, respectively, P< 0.01). Marbofloxacin was shown to be non-toxic at 500 microg/ml in vitro and no cell apoptosis was observed. The molecule was able to induce a parasitic process after significant elimination of amastigotes in leishmania-infected dog macrophages. We propose that marbofloxacin, compared to standard chemotherapeutic agents (meglumine antimoniate and sodium stibogluconate), could be an effective and pragmatic oral route alternative to treat canine leishmaniasis.     

Reassortant H1N1 influenza virus vaccines protect pigs against pandemic H1N1 influenza virus and H1N2 swine influenza virus challenge

Influenza A (H1N1) virus has caused human influenza outbreaks in a worldwide pandemic since April 2009. Pigs have been found to be susceptible to this influenza virus under experimental and natural conditions, raising concern about their potential role in the pandemic spread of the virus. In this study, we generated a high-growth reassortant virus (SC/PR8) that contains the hemagglutinin (HA) and neuraminidase (NA) genes from a novel H1N1 isolate, A/Sichuan/1/2009 (SC/09), and six internal genes from A/Puerto Rico/8/34 (PR8) virus, by genetic reassortment. The immunogenicity and protective efficacy of this reassortant virus were evaluated at different doses in a challenge model using a homologous SC/09 or heterologous A/Swine/Guangdong/1/06(H1N2) virus (GD/06). Two doses of SC/PR8 virus vaccine elicited high-titer serum hemagglutination inhibiting (HI) antibodies specific for the 2009 H1N1 virus and conferred complete protection against challenge with either SC/09 or GD/06 virus, with reduced lung lesions and viral shedding in vaccine-inoculated animals compared with non-vaccinated control animals. These results indicated for the first time that a high-growth SC/PR8 reassortant H1N1 virus exhibits properties that are desirable to be a promising vaccine candidate for use in swine in the event of a pandemic H1N1 influenza.     

Sub-clinical infection as an effective protocol for obtaining anti-Leishmania chagasi amastigote antibodies of different animal species

This work aims at identifying an effective protocol to raise anti-Leishmania chagasi amastigote antibodies in different animal species. Protocols of immunization by subcutaneous injections of L. chagasi promastigote and amastigote lysates or by either intravenous or subcutaneous inoculation of live metacyclic promastigotes were assessed in mice, rabbits, and dogs. The immunization with live promastigotes produced a strong humoral immune response against L. chagasi amastigotes in all three animal species. The sera from animals immunized with the promastigote lysate did not react with amastigotes and, conversely, the sera from mice immunized with the amastigote lysate did not react with promastigotes. Taken all data together, the immunization through infection with metacyclic promastigotes was considered the most satisfactory way to immunize animals for obtaining anti-amastigote and anti-promastigote antibodies, since it did not only allowed the obtention of antibody against the two forms of the parasite, but it is also cheap, less laborious than carrying out the purification of amastigotes from infected tissues and avoid the use of a large number of hamsters for obtention the amastigotes, necessary to produce the immunogenic lysates. Furthermore, this immunization protocol was comparable to the amastigote lysate immunization protocol for the obtaining of mouse monoclonal antibodies (mAbs).     

Cell mediated immunity and specific IgG1 and IgG2 antibody response in natural and experimental canine leishmaniosis

In the present study, we have followed up Leishmania infantum infection in dogs: (1) naturally infected; (2) experimentally infected with amastigotes; and (3) experimentally infected with culture promastigotes. The main objective was to evaluate the differences of the humoral and cellular immune responses of each group. Sera from 12 beagle dogs were analysed for total anti-leishmanial antibodies and IgG1 and IgG2 subclasses by enzyme-linked immunosorbent assay (ELISA). Lymphoproliferation to L. infantum antigen was also performed. All naturally infected animals were symptomatic with a marked humoral response. Dogs inoculated with amastigotes were asymptomotic and presented lower antibody titres than naturally infected. Dogs inoculated with culture promastigotes were asymptomotic with no significant humoral response. Strong proliferative responses to Leishmania antigen was observed in dogs inoculated with promastigotes. In our experimental model, IgG1 antibody levels presented a similar pattern in all infected animals, and IgG2 reactivity was high in naturally infected dogs.     

In vitro effect of Aloe vera, Coriandrum sativum and Ricinus communis fractions on Leishmania infantum and on murine monocytic cells

In South America, visceral leishmaniasis is a zoonosis caused by the protozoan species Leishmania infantum (syn. L. chagasi) and is primarily transmitted through the bite of the female Lutzomyia longipalpis. Its main reservoir in urban areas is the dog. The application of control measures recommended by health agencies have not achieved significant results in reducing the incidence of human cases, and the lack of effective drugs to treat dogs resulted in the prohibition of this course of action in Brazil. Therefore, it is necessary to search new alternatives for the treatment of canine and human visceral leishmaniasis. The objectives of this study were to evaluate the in vitro effect of fractions from Aloe vera (aloe), Coriandrum sativum (coriander), and Ricinus communis (castor) on promastigotes and amastigotes of L. infantum and to analyze the toxicity against the murine monocytic cells RAW 264.7. To determine the viability of these substances on 50% parasites (IC50), we used a tetrazolium dye (MTT) colorimetric assay (bromide 3-4.5-dimethylthiazol-2-yl-2,5-dephenyltetrazolium), and on amastigotes we performed an in situ ELISA. All fractions were effective against L. infantum promastigotes and did not differ from the positive control pentamidine (p>0.05). However, the R. communis ethyl acetate and chloroform fractions, as well as the C. sativum methanol fraction, were the most effective against amastigotes and did not differ from the positive control amphotericin B (p>0.05). The R. communis ethyl acetate fraction was the least toxic, presenting 83.5% viability of RAW 264.7 cells, which was similar to the results obtained with amphotericin B (p>0.05). Based on these results, we intend to undertake in vivo studies with R. communis ethyl acetate fractions due the high effectiveness against amastigotes and promastigotes of L. infantum and the low cytotoxicity towards murine monocytic cells.     

Morphology and growth characteristics of cultured Leptomonas ctenocephali from Ctenocephalides felis felis (Siphonaptera: Pulicidae) of dogs in Brazil

To confirm the taxonomic identification of a trypanosomatid found in the hindgut, rectum and Malpighian tubules of dog fleas captured in Belo Horizonte, Minas Gerais, Brazil, between April and November of 2005, 910 specimens of Ctenocephalides felis felis were removed from street dogs and dissected, and isolates from their digestive tracts were cultivated in NNN-alpha-MEM medium. Four different morphological forms were observed in culture: long, slender, twisted promastigotes with a long flagellum; short, stubby, non-twisted promastigotes; rounded amastigotes; and cyst-like bodies. Twisted and non-twisted promastigotes were frequently seen forming rosettes, and these two forms presented significant differences (P<0.01) in terms of their morphological characteristics. Unlike the promastigote forms observed throughout the culture period, rounded amastigotes were seen only in the lag phase, and the cyst-like bodies were only seen in the decline phase. The trypanosomatid DNA obtained from the culture was analyzed by the polymerase chain reaction (PCR) method and found to be negative for Leishmania infantum chagasi. Based on the growth pattern, morphological parameters and molecular analysis, the flagellates were confirmed to be Leptomonas ctenocephali. The significance of this infection for animals is also commented.     

一株人源H1N1亚型猪流感病毒的进化分析与生物学特性研究

为了解猪流感病毒(SIV)的变异情况,我们2009年11月从河北某养殖场采集呈流感症状的猪鼻拭子40份,接种10日龄SPF鸡胚,分离到一株猪流感病毒,通过RT-PCR和血凝抑制试验鉴定为H1N1亚型,命名为A/swine/Hebei/15/2009(H1N1),其全基因序列测定及同源性分析发现,8个基因片段均与2000年左右H1N1人流感病毒有较高的同源性。系统遗传演化显示,该病毒分离株是由2000年人源H1N1流感病毒A/Dunedin/2/2000(H1N1)进化而来。抗原性分析显示该株与甲型H1N1流感病毒和经典H1N1病毒株抗原性差异较大。对小鼠致病性试验表明该病毒株可以直接感染小鼠并导致小鼠轻微临床症状和组织病理学变化,但不致死小鼠,表现为低致病性。     

Sequence and phylogenetic analysis of neuraminidase genes of H9N2 avian influenza viruses isolated from commercial broiler chicken in Iran (2008 and 2009)

A total of 512 tissue samples collected from 30 farms located in various states of Iran during 2008–2009 as part of a program to monitor avian influenza viruses (AIVs) infection in Iran’s poultry population. To determine the genetic relationship of Iranian viruses, neuraminidase (NA) genes from ten isolates of H9N2 viruses isolated from commercial chickens in Iran during 2008–2009 were amplified and sequenced. The viruses’ neuraminidase gene was >90% similar to those of A/Quail/Hong Kong/G1/97 (H9N2) sublineage. The neuraminidase stalk regions in these Viruses had no deletion as compared to that of chicken/Beijing/1/94 sublineage (Beijing-like viruses) and the two human isolates A/HK/1073/99, A/HK/1074/99. Phylogenetic analysis of neuraminidase (NA) gene showed that it shares a common ancestor A/Quail/Hong Kong/G1/97 isolate which had contributed the internal genes of the H5N1 virus. The results of this study indicated that No (Beijing-like) virus and (Korean-like) virus were found in chickens in Iran, and the NA genes of H9N2 influenza viruses circulating in Iran during the past years were well conserved and the earlier Iranian isolates may be considered to represent such a progenitor.     

Antibody responses of swine to type A influenza viruses in the most recent several years.

A serological survey was conducted on 4,080 swine sera collected for the years 1985-90. The swine sera positive to A/New Jersey/8/76 (swine type H1N1) strain were observed in annual (10-20%) and monthly (20-40%) incidences during the observation period except for occasional months. Antibodies to recent human H1N1 viruses in swine were recognized in relation to the human H1N1 influenza epidemics. Antibody responses of swine to human H3N2 strains appeared irrespective of human epidemics with the virus in the years 1985-87. However, in 1988 almost no antibodies to three human H3N2 isolates of 1983-88 were observed for this year except a few months though the human epidemic occurred in the area. Although in 1989-90 many swine had antibodies to the three strains in the percentage of 3 to 35, no antibody to the latest isolate, A/Hokkaido/20/89 (H3N2), was found for almost all the months of both years. These findings differed markedly from the possible relationship between the prevalence of H3N2 virus-antibodies in swine and the human influenza epidemics, which were described previously in many reports including our studies.     

Profile of Shiga toxin-producing Escherichia coli strains isolated from dogs and cats and genetic relationships with isolates from cattle, meat and humans

Pets can be reservoirs of Shiga toxin-producing Escherichia coli (STEC) strains. The aim of this study was to examine nine strains belonging to several serotypes (O91:H21, O91:H16, O178:H19, O8:H19, O22:H8, O22:HNT, ONT:H8), previously recovered from cats or dogs. To this end, we assessed a set of additional virulence genes (stx(2) subtype, subAB, ehxA, eae and saa), cytotoxic activity, and genetic relationships with strains isolated from cattle, meat and humans using pulsed-field gel electrophoresis (PFGE). Most of the isolates carried the stx(2) and/or stx(2vh-b) sequences, while only the O91:H21 isolate presented the mucus-activatable stx(2d) variant, as confirmed by sequencing the genes of subunits A and B. All the strains showed cytotoxic activity in cultured cells. One of the two O178:H19, selected for its high level of cytotoxicity in Vero cells, showed the ability to cause functional alterations in the human colon mucosa in vitro. None of the strains possessed the subAB, eae or saa genes and only the strains belonging to serotype O8:H19 carried the ehxA gene. The isolates shared 90-100% similarity by PFGE to epidemiologically unrelated strains of the corresponding serotypes recovered from cattle, meat or humans. Our results demonstrate that dogs and cats may have a role in the infection of humans by STEC, probably serving as a vehicle for bovine strains in the cycle of human infection, and thus emphasize the health risks for owners and their families.     

Pharmacokinetics of a single bolus intravenous, intramuscular and subcutaneous dose of disodium fosfomycin in horses

Pharmacokinetic parameters of fosfomycin were determined in horses after the administration of disodium fosfomycin at 10 mg/kg and 20 mg/kg intravenously (IV), intramuscularly (IM) and subcutaneously (SC) each. Serum concentration at time zero (C_S0) was 112.21 ± 1.27 μg/mL and 201.43 ± 1.56 μg/mL for each dose level. Bioavailability after the SC administration was 84 and 86% for the 10 mg/kg and the 20 mg/kg dose respectively. Considering the documented minimum inhibitory concentration (MIC₉₀) range of sensitive bacteria to fosfomycin, the maximum serum concentration (Cmax) obtained (56.14 ± 2.26 μg/mL with 10 mg/kg SC and 72.14 ± 3.04 μg/mL with 20 mg/kg SC) and that fosfomycin is considered a time-dependant antimicrobial, it can be concluded that clinically effective plasma concentrations might be obtained for up to 10 h administering 20 mg/kg SC. An additional predictor of efficacy for this latter dose and route, and considering a 12 h dosing interval, could be area under the curve AUC_0-12/MIC₉₀ ratio which in this case was calculated as 996 for the 10 mg/kg dose and 1260 for the 20 mg/kg dose if dealing with sensitive bacteria. If a more resistant strain is considered, the AUC_0-12/MIC₉₀ ratio was calculated as 15 for the 10 mg/kg dose and 19 for the 20 mg/kg dose.     

Isolation and Identification of one Strain of H9 Subtype Avian Influenza Virus and Study on its Biological Characteristics

In 2013,one case of suspected H9 subtype avian influenza occurred in a chicken farm of Jilin povince.Clinical samples were collected from the diseased farm,inoculated into the allantoic cavity of 9-day-old SPF chicken embryo,and then one strain of virus was isolated.The results of HA test,HI test and molecular biology test all showed that the isolate belonged to H9 subtype avian influenza virus (AIV).The HA cleavage site of the isolate was RSSR↓GLF,which was consisted with the molecular characteristic of low pathogenic AIV.The HA peptide chain had 9 potential glycosylation sites which were same as other isolates of recent years.The isolate had 8 receptor binding sites,including 234 receptor binding site by glutamine (Q) mutating into threonine (T).The phylogenetic tree revealed the isolate belonged to Eurasian lineages and it had far genetic relationship with the earliest domestic isolate (A/Chicken/Beijing/1/94(H9N2)),but had close genetic relationship with the representative strain (A/Chicken/Guangxi/55/2005(H9N2)) of major epidemic branch since 2007.We prepared an inactivated oil-emulsion vaccine of the isolate,and then vaccinated SPF chicken.21 days after vaccination,the HI titer of chicken serum antibody reached up to 10log2.The result suggested the isolate had good immunogenicity.     

Detection of Mycoplasma mycoides sub-species mycoides small colony by a specific capture/enrichment monoclonal antibody-based sandwich ELISA

The present study describes the development of a specific Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC) monoclonal antibody (MAb), 6E3, and its application in a sandwich ELISA (sELISA) format. Mab 6E3 reacted only to the 12 MmmSC within the 32 M. mycoides cluster strains and 12 representative strains of other bovine, ovine and caprine associated mycoplasmas examined. A capture/enrichment format of the sELISA that combined MAb 6E3 with a previously developed MAb 3H12 that cross reacted with Mmm Large Colony [Rodriguez, F., Ball, H.J., Finlay, D., Campbell, D., Mackie, D.P., 1996. Detection of Mycoplasma mycoides sub-species mycoides by monoclonal antibody-based sandwich ELISA. Veterinary Microbiology 51, 69–76], retained MmmSC specificity and improved the sensitivity from the 1.2 × 10⁷ cfu/ml for a standard 2 h capture stage sELISA down to as low as 2 cfu/ml for a 72 h capture. A low level of false positives (1%) was observed when this assay was applied to 200 bovine respiratory and milk samples submitted for diagnostic investigation. This simple and specific sELISA provides a suitable assay for screening large numbers of samples for CBPP.     

一株H9亚型禽流感病毒的分离鉴定及生物特性研究

2013年中国吉林某养鸡场发生疑似H9亚型禽流感疫情,采集该发病鸡场病料接种9日龄SPF鸡胚,分离得到一株病毒。经血凝(HA)试验、血凝抑制(HI)试验、测序分析,鉴定该毒株为H9亚型禽流感病毒(AIV)。对本试验分离株HA基因进行测序及序列分析,结果显示分离株HA基因的裂解位点为RSSR↓GLF,符合低致病性AIV的基因特征;HA肽链具有9个潜在糖基化位点,与近些年H9亚型AIV分离株的潜在糖基化位点相同;具有8个受体结合位点,其中234位受体结合位点由谷氨酰胺(Q)变异成苏氨酸(T);HA基因系统进化树结果显示本试验分离株属于欧亚进化分支,与中国最早分离株A/Chicken/Beijing/1/94(H9N2)亲缘关系较远,与2007年后中国H9亚型AIV主要流行分支的代表株A/Chicken/Guangxi/55/2005(H9N2)亲缘关系较近。将该毒株制成油乳剂灭活疫苗免疫SPF鸡,免疫后第21天免疫鸡血清抗体高达10log2,表明本试验分离株具有很好的免疫原性。     

Antigenic characterization of an H3N2 swine influenza virus isolated from pigs with proliferative and necrotizing pneumonia in Quebec.

A new strain of swine influenza A virus, designated A/Swine/Saint-Hyacinthe/150/90 has been isolated from pigs with severe proliferative and necrotizing pneumonia in Quebec. The antigenic characterization of the hemagglutinin was performed by hemagglutination inhibition test, immunoblot and indirect immunoprecipitation using polyclonal antisera. Only the last test was able to detect an antigenic relationship between the hemagglutinin of this isolate and an H3 subtype influenza virus. The immunoprecipitation test was a useful alternative for determining the hemagglutinin of influenza A virus subtypes. The neuraminidase inhibition test demonstrated a reactivity between the A/Swine/Saint-Hyacinthe/150/90 and antiserum against a N2 subtype influenza virus. Our results indicate that this new strain isolated for the first time in the porcine population of Canada is related to A/Sw/Hong Kong/76 H3N2 swine influenza virus.     

Identification of Leishmania donovani amastigotes in canine tissues by immunoperoxidase staining

This paper describes the demonstration of Leishmania donovani amastigotes in canine tissues by immunoperoxidase staining. An indirect immunoperoxidase method was applied to the organs of 20 dogs in which leishmaniasis was previously diagnosed. Haemosiderin pigment was eliminated with 5 per cent oxalic acid. Amastigotes of L donovani appeared as dark brown stained bodies which contrasted with haematoxylin stained host cells. No positively stained amastigotes could be seen in any of the sections incubated with control serum. The organs which more frequently showed leishmanids were: skin (macrophages and fibroblasts), liver, spleen, lymph nodes and bone marrow. In a few cases amastigotes were seen in kidneys, gut, adrenal glands, eyes and testicles. This technique is simple to perform, gives consistent results and allows unequivocal histopathological diagnosis of canine leishmaniasis.     

Characterization of an avian infectious bronchitis virus isolated in China from chickens with nephritis

One IBV isolate, SC021202, was isolated from the kidneys of the infected young chickens by inoculating embryonated eggs, and its morphology, physiochemical and haemagglutonating properties were detected. Virulence of the isolate SC021202 was determined with specific pathogen-free (SPF) chicken inoculation. Nucleotide acid sequence of S1 gene of the isolate SC021202 was further sequenced and analysed. The physiochemical and morphological properties of the isolate SC021202 were in accordance to that of typical infectious bronchitis virus (IBV). In a pathogenicity experiment, the clinical signs and related gross lesions resembling those of field outbreak were reproduced and the virus isolate SC021202 was re-isolated from the kidneys of the infected chicken. Sequence data demonstrated that the full length of the amplified S1 gene of the isolate SC021202 was composed of 1931 nucleotides, coding a polypeptide of 543 amino acid residues. Compared with IBV strains from GenBank, the nucleotide and deduced amino acid sequence of S1 gene of the isolate SC021202 shared 60.0-91.4% and 49.1-88.9% identities, respectively. A nucleotide fragment of 'CTTTTTAATTATACTAACGGA' was inserted at nucleotide site 208 in the S1 gene of the isolate. These results indicated that IBV isolate SC021202 was a new variant IBV isolate and responsible for field outbreak of nephritis.     

牛羊水间充质干细胞对小鼠酒精性肝病的影响

试验旨在分离培养牛羊水间充质干细胞（AF-MSCs），并研究其对酒精性肝病（ALD）小鼠治疗效果以及对肝组织中缺氧诱导因子-1α（HIF-1α）、血管内皮生长因子（VEGF）及Toll样受体4（TLR4）mRNA表达的影响。从4~5月龄雌性胎牛羊水中获取AF-MSCs，进行RT-PCR、免疫荧光及诱导成脂鉴定。将48只ICR小鼠（雄性）随机分为4组：空白对照组（BC组）、模型对照组（MC组）、AF-MSCs组（MC+AF-MSCs组）和水飞蓟宾胶囊（SC）组（MC+SC组），每组12只。除BC组灌胃0.2 mL生理盐水，其余各组均灌胃56度红星二锅头（5 g· kg ^-1，2次· d ^-1），连续4周；第29天MC+AF-MSCs组进行肝外注射Dil标记的AF-MSCs（5×10⁶个·mL ^-1），MC+SC组分2次灌胃SC 2.4 mg（溶于56度红星二锅头）；试验结束后，测定小鼠血清谷丙转氨酶（ALT）、谷草转氨酶（AST）和甘油三酯（TG）活性。HE染色、免疫组化法观察造模情况，Dil示踪AF-MSCs在肝内定植分布；qRT-PCR法检测HIF-1α、VEGF及TLR4基因表达情况。RT-PCR、免疫荧光结果表明，分离得到牛AF-MSCs，且有诱导成脂分化能力。血清学结果显示，MC组ALT、AST和TG活性极显著高于BC组（P<0.01）。组织病理切片显示，MC组肝组织中出现脂肪性变、炎性细胞浸润及肝血窦充血等病理性变化，说明56度红星二锅头成功复制ALD小鼠模型；与MC+SC组相比，MC+AF-MSCs组ALT、AST活性显著降低（P<0.01），肝组织中脂肪性变减轻，肝血窦未见充血，说明AF-MSCs对肝功能的改善比SC效果显著；qRT-PCR结果显示，MC+AF-MSCs组下调HIF-1α、VEGF、TLR4基因的表达量较MC+SC组显著（P<0.01），说明AF-MSCs参与HIF-1α/VEGF信号通路的调控，抑制TLR4表达的效果优于SC；细胞示踪术证明，AF-MSCs定植在病变部位并趋化更多AF-MSCs向炎症部位迁移参与肝组织的修复。综上，本研究成功分离得到牛AF-MSCs，其能够参与调节氧化应激、血管生成及抑制炎性因子的释放，且改善ALD的效果优于SC。