K+ channel[1]

TGF-β1-induced α-SMA expression in HSC-T6 cells was considerably suppressed by apimin (0.5-2 μg/mL) treatment for 24 hours. The activation of p-Smad2/3 and Smad4 produced by TGF-β1 was eliminated by apimin administration [1].

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Apamin inhibits activation of HSCs through the Smad signaling pathway [1]
TGF-β1 is a Smad family member, and it is known to stimulate the activation of HSCs. We next investigated whether the inhibitory effect of apamin on the activation of HSCs by TGF-β1 is through the Smad signaling pathway. α-SMA and collagen I expression was increased by TGF-β1 and was decreased by apamin treatment in the HSC-T6 cells (Fig. 7A). Also, immunofluorescence showed that TGF-β1 induced activation of HSC-T6 cells through increased α-SMA expression (Fig. 7C). However, apamin treatment markedly reduced the expression of α-SMA in the TGF-β1-induced HSC-T6 cells. Western blot analysis showed that phosphorylation of Smad2/3 and Smad4 were stimulated by 2 ng/ml TGF-β1 (Fig. 7B). Apamin treatment abrogated the activation of p-Smad2/3 and Smad4 induced by TGF-β1. These results indicated that apamin may attenuate TGF-β1-activated HSCs by inhibiting the Smad signaling pathway.

Treatment with apamin (0.1 mg/kg; twice weekly; intraperitoneal injection; 4 weeks; C57BL/6 male mice) caused liver damage and decreased proinflammatory cytokine levels. Apamin suppresses the expression of fibrotic genes, BEC proliferation, and collagen deposition in mice given 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) [1].

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Apaminameliorates liver damage and inflammatory hepatic injury [1]
To investigate the effects of apamin treatment on liver fibrosis, a mouse model induced by DDC diet feeding was used. When challenged with the DDC diet for 4 weeks, the structure of the hepatic lobule was clear in the NC group (Fig. 1A) and there was a large amount of bile duct proliferation, accompanied by inflammatory cell infiltration in the DDC-fed group as shown by H&E staining (Fig. 1C). In addition, the above pathological changes were reduced in the apamin-treated group (Fig. 1D) compared with these changes noted in the DDC-fed group. Masson's trichrome staining indicated collagen deposition surrounding the proliferated bile duct in the DDC-fed group (Fig. 2C). In contrast, apamin treatment resulted in diminished fibrosis and collagen deposition (Fig. 2D). In cholangiopathies, inflammation and reactive proliferation of bile ducts are closely related with the development of biliary fibrosis. ELISA and western blot analyses indicated that expression levels of IL-6, IFN-γ, TNF-α and IL-1β were significantly higher in the DDC-fed group compared with these levels in the NC group (Fig. 3). However, apamin treatment attenuated inflammatory cytokine expression, including IL-6, IFN-γ, TNF-α and IL-1β compared with expression levels in the DDC-fed group. Taken together, these data confirm the anti-inflammatory and moderate anti-fibrotic effects of apamin on the DDC-fed mice.

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Effect of Apamin on BEC proliferation in DDC-fed mice [1]
We next determined the effect of apamin on ductular reaction in the DDC-fed mice by immunofluorescence of CK19 expression. Chronic DDC feeding in mice was previously demonstrated to result in cholangitis and immune responses against BECs with the destruction of bile ducts and ductules (18). CK19 is regarded as a hallmark of bile epithelial cells (19). Immunofluorescence staining showed that CK19 was highly expressed in the BECs in bile ductules in enlarged portal tracts (Fig. 4). The DDC-fed group had significantly increased expression of CK19 compared with the NC group. In contrast, apamin treatment significantly reduced biliary activation and proliferation as evidenced by CK19 staining, indicating a defect in the ductular reaction. In addition, immunofluorescence staining of PCNA showed that apamin treatment suppressed the proliferation of BECs compared with the DDC-fed group. These results indicate that apamin may inhibit cholestatic liver fibrosis by suppressing BEC proliferation and ductular reaction induced by the DDC diet.

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Apamin inhibits ECM deposition in the livers of DDC-fed mice [1]
To investigate the anti-fibrotic effect of apamin on ECM deposition in the DDC-fed mice, we used western blot analysis, immunohistochemistry and immunofluorescence assay to determine the effects of this compound on ECM molecules. Liver fibrosis induced by DDC was confirmed by induction of fibrogenic genes, FSP-1, α-smooth muscle actin (α-SMA) and collagen I expression. Expression of α-SMA was strongly expressed in the myofibroblasts and HSCs around the proliferated bile duct in the DDC-fed group and clearly with the apamin treatment (Fig. 5). Moreover, expression of collagen I in the DDC-fed group was significantly increased, especially in the portal tracts. Compared to the DDC group, apamin treatment inhibited collagen I expression. During tissue remodeling in liver fibrosis, FSP-1 is considered as a marker of fibroblasts in the fibrotic liver. DDC feeding increased the number of cells positive for FSP-1 expression (Fig. 6C). In contrast, apamin treatment resulted in a reduction in FSP-1-positive cells (Fig. 6D). Furthermore, western blot results showed that the expression levels of TGF-β1, collagen I, and fibronectin were significantly higher in the DDC-fed group, whereas apamin treatment markedly decreased the protein level of TGF-β1, collagen I, and fibronectin compared with the DDC-fed group (Fig. 6F). Taken together, the data suggest that apamin may protect liver fibrosis during DDC feeding by suppressing fibrotic gene expression.

Western Blot Analysis[1]
Cell Types: HSC-T6 cells
Tested Concentrations: 0.5 µg/mL, 1 µg/mL and 2 µg/mL
Incubation Duration: 24 hrs (hours)
Experimental Results: Markedly decreased the expression of α-SMA in the TGF-β1- induced HSC-T6 cells. Abrogated the activation of p-Smad2/3 and Smad4 induced by TGF-β1.

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HSC-T6 cells, an immortalized rat hepatic stellate cell line, which has a stable phenotype and biochemical characteristics, was kindly provided by Dr S.L. Friedman. Cells were cultured at 37°C in a humidified incubator under a 5% CO2 atmosphere. HSC-T6 cells were seeded in complete medium for 24 h. The cells were changed to fresh serum-free media containing the indicated concentrations of Apamin (0.5, 1 and 2 µg/ml). After 24 h, the cells were replaced with fresh serum-free media containing 2 ng/ml of TGF-β1 for 24 h[1].

Animal/Disease Models: 8weeks old C57BL/6 male mice (20-25 g) with DDC feeding[1]
Doses: 0.1 mg/kg
Route of Administration: intraperitoneal (ip)injection; twice a week; for 4 weeks
Experimental Results: Resulted in diminished liver injury and proinflammatory cytokine levels. Suppressed the deposition of collagen, proliferation of BECs and expression of fibrogenic genes in the DDC-fed mice.

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DDC-induced mouse model of biliary fibrosis [1]
For induction of liver injury, 8-week-old C57BL/6 male mice (20–25 g) were selected. Male C57BL/6 mice were fed a control diet or a DDC supplemented diet (0.1%) for 4 weeks to induce advanced biliary fibrosis as previously described. The mice received an intraperitoneal injection of Apamin (0.1 mg/kg) dissolved in saline twice a week. Mice were sacrificed after 4 weeks from the first DDC diet administration.
Apamin was dissolved in 4 mM HCl containing 0.1% bovine serum albumin (BSA).

16133797 mouse LD50 intraperitoneal 3800 ug/kg Toxicon., 22(308), 1984 [PMID:6145236]
16133797 mouse LD50 subcutaneous 2900 ug/kg BEHAVIORAL: ATAXIA European Journal of Biochemistry., 56(35), 1975 [PMID:1175625]
16133797 mouse LD50 intravenous 4 mg/kg BEHAVIORAL: EXCITEMENT; BEHAVIORAL: CHANGES IN MOTOR ACTIVITY (SPECIFIC ASSAY) Naunyn-Schmiedeberg's Archives of Pharmacology., 300(189), 1977 [PMID:593441]
16133797 mouse LD50 intracrebral 1800 ng/kg Toxicon., 22(308), 1984 [PMID:6145236]
16133797 mouse LD50 parenteral 600 mg/kg Toxicon., 20(157), 1982

[1]. Apamin suppresses biliary fibrosis and activation of hepatic stellate cells. Int J Mol Med. 2017 May;39(5):1188-1194.

A highly neurotoxic polypeptide from the venom of the honey bee (Apis mellifera). It consists of 18 amino acids with two disulfide bridges and causes hyperexcitability resulting in convulsions and respiratory paralysis.

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Cholestatic liver disease is characterized by the progressive destruction of biliary epithelial cells (BECs) followed by fibrosis, cirrhosis and liver failure. Activated hepatic stellate cells (HSCs) and portal fibroblasts are the major cellular effectors of enhanced collagen deposition in biliary fibrosis. Apamin, an 18 amino acid peptide neurotoxin found in apitoxin (bee venom), is known to block Ca2+-activated K+ channels and prevent carbon tetrachloride-induced liver fibrosis. In the present study, we aimed to ascertain whether apamin inhibits biliary fibrosis and the proliferation of HSCs. Cholestatic liver fibrosis was established in mouse models with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) feeding. Cellular assays were performed on HSC-T6 cells (rat immortalized HSCs). DDC feeding led to increased hepatic damage and proinflammtory cytokine levels. Notably, apamin treatment resulted in decreased liver injury and proinflammatory cytokine levels. Moreover, apamin suppressed the deposition of collagen, proliferation of BECs and expression of fibrogenic genes in the DDC-fed mice. In HSCs, apamin suppressed activation of HSCs by inhibiting the Smad signaling pathway. These data suggest that apamin may be a potential therapeutic target in cholestatic liver disease. [1]
The principal finding of this study is the anti-fibrotic effects of apamin. Apamin suppressed the proliferation of BECs and activation of HSCs. In the present study, apamin significantly inhibited bile duct proliferation and reduced ECM deposition in the DDC-fed mice. Furthermore, apamin suppressed the protein expression of p-Smad2/3 and Smad4 induced by TGF-β1 in the HSCs. These results suggest that apamin inhibits the proliferation of BECs and activation of HSCs by suppressing the TGF-β1 signaling pathway in hepatic fibrosis. [1]

C81H132F3N31O26S4

2141.36

2139.87947

Apamin;24345-16-2

CNCKAPETALCARRCQQH-NH2 (Disulfide bridge: Cys1-Cys11;Cys3-Cys15); H-Cys(1)-Asn-Cys(2)-Lys-Ala-Pro-DL-Glu-Thr-Ala-Leu-Cys(1)-Ala-Arg-Arg-Cys(2)-Gln-Gln-His-NH2.TFA; L-cysteinyl-L-asparagyl-L-cysteinyl-L-lysyl-L-alanyl-L-prolyl-DL-alpha-glutamyl-L-threonyl-L-alanyl-L-leucyl-L-cysteinyl-L-alanyl-L-arginyl-L-arginyl-L-cysteinyl-L-glutaminyl-L-glutaminyl-L-histidinamide (1->11),(3->15)-bis(disulfide) trifluoroacetic acid

CNCKAPETALCARRCQQH

White to off-white solid powder

C[C@H]1C(=O)N[C@H](C(=O)N[C@H](C(=O)N[C@@H](CSSC[C@H]2C(=O)N[C@H](C(=O)N[C@H](C(=O)N3CCC[C@H]3C(=O)NC(C(=O)N[C@H](C(=O)N[C@H](C(=O)N[C@H](C(=O)N[C@@H](CSSC[C@@H](C(=O)N[C@H](C(=O)N2)CC(=O)N)N)C(=O)N1)CC(C)C)C)[C@@H](C)O)CCC(=O)O)C)CCCCN)C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC4=CN=CN4)C(=O)N)CCCNC(=N)N)CCCNC(=N)N.C(=O)(C(F)(F)F)O

GUBJIBRYPCCYMV-AHHFRWINSA-N

InChI=1S/C79H131N31O24S4.C2HF3O2/c1-35(2)26-49-70(127)107-51-31-136-135-30-41(81)63(120)105-50(28-57(84)114)71(128)108-53(73(130)99-42(12-7-8-22-80)64(121)96-38(5)77(134)110-25-11-15-54(110)75(132)102-47(18-21-58(115)116)69(126)109-59(39(6)111)76(133)95-37(4)62(119)104-49)33-138-137-32-52(106-66(123)44(14-10-24-92-79(88)89)98-65(122)43(13-9-23-91-78(86)87)97-61(118)36(3)94-72(51)129)74(131)101-45(16-19-55(82)112)67(124)100-46(17-20-56(83)113)68(125)103-48(60(85)117)27-40-29-90-34-93-40;3-2(4,5)1(6)7/h29,34-39,41-54,59,111H,7-28,30-33,80-81H2,1-6H3,(H2,82,112)(H2,83,113)(H2,84,114)(H2,85,117)(H,90,93)(H,94,129)(H,95,133)(H,96,121)(H,97,118)(H,98,122)(H,99,130)(H,100,124)(H,101,131)(H,102,132)(H,103,125)(H,104,119)(H,105,120)(H,106,123)(H,107,127)(H,108,128)(H,109,126)(H,115,116)(H4,86,87,91)(H4,88,89,92);(H,6,7)/t36-,37-,38-,39+,41-,42-,43-,44-,45-,46-,47?,48-,49-,50-,51-,52-,53-,54-,59-;/m0./s1

3-[(1R,4S,7S,13S,19S,22S,25S,28R,31S,34S,37S,40R,47S,50R)-50-amino-40-[[(2S)-5-amino-1-[[(2S)-5-amino-1-[[(2S)-1-amino-3-(1H-imidazol-5-yl)-1-oxopropan-2-yl]amino]-1,5-dioxopentan-2-yl]amino]-1,5-dioxopentan-2-yl]carbamoyl]-4-(4-aminobutyl)-47-(2-amino-2-oxoethyl)-34,37-bis(3-carbamimidamidopropyl)-19-[(1R)-1-hydroxyethyl]-7,22,31-trimethyl-25-(2-methylpropyl)-2,5,8,14,17,20,23,26,29,32,35,38,46,49-tetradecaoxo-42,43,52,53-tetrathia-3,6,9,15,18,21,24,27,30,33,36,39,45,48-tetradecazatricyclo[26.16.10.09,13]tetrapentacontan-16-yl]propanoic acid;2,2,2-trifluoroacetic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O :~50 mg/mL (~23.35 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)