Toll-like receptor 4 (TLR4) – Ulinastatin attenuates TLR4 expression and downstream NF-κB pathway activation [2].
Myeloid differentiation primary response gene 8 (MyD88) – involved in the TLR4/NF-κB pathway [1].
Nuclear factor kappa-B (NF-κB) – Ulinastatin reduces NF-κB activation [1][2].
Tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), interleukin-2 (IL-2) – Ulinastatin reduces their production/release [1][2].
Bax, Bcl-2, caspase-3 – Ulinastatin up-regulates Bcl-2, down-regulates Bax and caspase-3, reducing the Bax/Bcl-2 ratio [1].
Endogenous proteases (trypsin, chymotrypsin, elastase) – inhibited by Ulinastatin [1].
Heparanase – activity reduced by Ulinastatin [1].
Coagulation factors (thrombin, thrombin-antithrombin complex, P-selectin) – Ulinastatin inhibits excessive thrombin conversion and reduces TAT complex and P-selectin [1].
T regulatory cells (Tregs, CD4+CD25+), CD3+, CD4+, CD8+ lymphocytes – Ulinastatin increases CD3+/CD4+ ratio, decreases CD8+ percentage, and reduces Treg percentage [1].

In LPS-stimulated BEAS-2B cells, urinastatin (500–5000 U; 24 hours) can drastically reduce TLR4 expression and NF-κB activation [1]. Linstatin

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In human lung epithelial BEAS-2B cells stimulated with LPS (10 μL of 10 μg LPS in 100 μL PBS), treatment with Ulinastatin (10 μL, 50 μL, or 100 μL of 100,000 U in 2 mL) 1 h before and 6 h after LPS stimulation significantly decreased the protein levels of TLR4, p-65, and NF-κB in a dose-dependent manner as measured by western blot. The medium dose (50 μL) showed optimal effect. Ulinastatin also significantly reduced the secretion of pro-inflammatory cytokines IL-2, TNF-α, IL-1β, and IL-6 in the culture media measured by ELISA [2].
In BEAS-2B cells transfected with pPICZA-TLR4 plasmid to overexpress TLR4, the protective effect of Ulinastatin (50 μL) on LPS-induced inflammatory response was abrogated: TLR4 mRNA and protein levels, p-65 and NF-κB protein levels, and cytokine levels (IL-2, TNF-α, IL-1β, IL-6) were no longer suppressed by Ulinastatin [2].
Ulinastatin up-regulates Bcl-2 and down-regulates Bax, leading to decreased Bax/Bcl-2 ratio, reduced cytochrome c release, decreased caspase-3 activation, and decreased apoptosis response in various models [1].
Ulinastatin inhibits endogenous proteases (trypsin, chymotrypsin, elastase) and increases neutral protease activity, which enhances collagenase activity [1].
Ulinastatin reduces endothelial glycocalyx destruction, heparan sulfate production, and heparanase activity in ARDS models [1].
Ulinastatin reduces the thrombin-antithrombin (TAT) complex and P-selectin, thereby inhibiting blood coagulation [1].
Ulinastatin increases the ratio of CD3+ and CD4+ lymphocytes and reduces CD8+ lymphocytes, increasing the CD4+/CD8+ ratio, and also reduces the percentage of CD4+CD25+ regulatory T lymphocytes (Tregs) [1].

Animals exposed to LPS-induced acute lung injury (ALI) were significantly protected by ulinstatin (10000 U/kg; intravenous injection; twice); this was achieved by lowering lung wet/dry weight ratio, ALI score, total cells, neutrophils, macrophages, myeloperoxidase activity, and malondialdehyde content—factors linked to lung histological damage [1].

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In a mouse model of LPS-induced acute lung injury (ALI) (male C57BL/6 mice, 8-10 weeks old, 18-22 g), Ulinastatin (10,000 U/kg) administered intravenously 1 h before and 6 h after intratracheal LPS instillation (5 mg/kg) significantly improved 72-hour survival rate (approximately 50% survival in LPS+UTI group vs. 20% in LPS group) [2].
Ulinastatin treatment significantly decreased the lung wet/dry weight ratio and lung injury score (histological evaluation: alveolar congestion, hemorrhage, neutrophil infiltration, alveolar wall thickness) compared to LPS alone [2].
Ulinastatin significantly reduced the numbers of total cells, neutrophils, and macrophages in bronchoalveolar lavage fluid (BALF) of LPS-induced ALI mice [2].
Ulinastatin significantly decreased myeloperoxidase (MPO) activity and malondialdehyde (MDA) content in lung tissues of LPS-induced ALI mice [2].
Ulinastatin reduced mRNA expression of TNF-α, IL-1β, IL-6, IL-2, VCAM-1, ICAM-1, MCP-1, and COX-2 in lung tissues measured by RT-qPCR [2].
Ulinastatin attenuated LPS-induced TLR4 mRNA and protein expression, as well as p-65 and NF-κB protein levels in mouse lung tissues [2].
In septic liver injury models, Ulinastatin decreased TNF-α content and increased IL-10 content, alleviating septic injury [1].
In hepatic ischemia-reperfusion injury models, Ulinastatin increased SOD activity, decreased MDA and TNF-α concentrations, inhibited pro-apoptotic genes BAX and caspase-3, and promoted anti-apoptotic gene Bcl-2 [1].
In liver fibrosis studies, homozygous Ulinastatin-knockout mice showed significantly higher levels of hyaluronic acid (HA) and TGF-β and more severe liver fibrosis compared to heterozygous knockout mice, indicating that Ulinastatin reduces HA and TGF-β production and slows liver fibrosis [1].
In autoimmune liver disease with liver failure, Ulinastatin decreased IL-6 and IL-8 levels [1].
In acute liver failure models, Ulinastatin reduced AST, ALT, MDA, iNOS, TNF-α, and caspase-3, while increasing SOD and GSH-Px levels [1].

Myeloperoxidase (MPO) activity in lung homogenates was measured using a commercial kit. Lung tissues were collected, homogenized in potassium phosphate buffer (pH 6.0), and centrifuged at 500×g for 15 min at 4°C. Then 10 μL of the supernatant was transferred into a buffer containing 0.17 mg/mL 3,3'-dimethoxybenzidine and 0.0005% H2O2. The total protein content in each sample was analyzed. Ulinastatin treatment significantly reduced LPS-induced MPO activity [2].
Malondialdehyde (MDA) content in lung homogenates was measured using a commercial determination kit according to the manufacturer’s instructions. Ulinastatin significantly reduced LPS-induced MDA content [2].

Western Blot Analysis[1]
Cell Types: Human lung epithelial BEAS-2B cells
Tested Concentrations: 500 U, 2500 U, 5000 U
Incubation Duration: 24 hrs (hours)
Experimental Results: Markedly attenuated TLR4 expression and NF-κB activation in LPS-stimulated BEAS-2B cells.

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Human lung epithelial BEAS-2B cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin in a 5% CO2 atmosphere. For Ulinastatin treatment, cells were treated with Ulinastatin (10 μL, 50 μL, or 100 μL of 100,000 U in 2 mL) 1 h before and 6 h after LPS stimulation (10 μL of 10 μg LPS in 100 μL PBS). After 24 h, cells were harvested for analysis. Cell viability was determined by MTT assay. Ulinastatin did not show cytotoxicity at the tested doses [2].
Western blot analysis: Collected cells were lysed, and proteins were extracted using a protein extraction kit. After blocking with 5% milk in TBST for 2 h, membranes were probed with primary antibodies against TLR4, p-65, and NF-κB overnight at 4°C, then incubated with HRP-conjugated secondary antibody. Bands were visualized by enhanced chemiluminescence. Ulinastatin reduced TLR4, p-65, and NF-κB protein levels in a dose-dependent manner [2].
RT-qPCR: Total RNA was isolated from cells using TRIzol, reverse transcribed, and real-time quantitative PCR was performed using M-MLV Platinum RT-qPCR kit. Primers for TNF-α, IL-6, IL-1β, IL-2, VCAM-1, ICAM-1, MCP-1, COX-2, TLR4, and β-actin were used. Ulinastatin reduced mRNA expression of these inflammatory mediators [2].
ELISA: Levels of IL-2, TNF-α, IL-1β, and IL-6 in culture media were measured using cytokine-specific ELISA kits. Total amounts were normalized to total protein from viable cell pellets. Ulinastatin significantly decreased these cytokine levels [2].
TLR4 gene transfection: BEAS-2B cells were transfected with pPICZA-TLR4 plasmid DNA using Lipofectamine Plus Reagent to upregulate TLR4 expression. After 24 h transfection, cells were treated with LPS with or without Ulinastatin (50 μL). TLR4 overexpression abrogated the protective effects of Ulinastatin on TLR4/NF-κB signaling and cytokine production [2].

Animal/Disease Models: Male C57BL/6 mice (8-10 weeks old, 18-22 g)[1]
Doses: 10000 U/kg
Route of Administration: iv; twice (1 h before and 6 h after LPS treatment)
Experimental Results: Dramatically protected animals from LPS-induced ALI.

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Male C57BL/6 mice (8-10 weeks old, 18-22 g) were housed under controlled conditions (24±1°C, 40-80% relative humidity, 12-h light/dark cycle) with free access to food and water. The ALI model was induced by intratracheal instillation of LPS (5 mg/kg in 50 μL phosphate-buffered saline). Ulinastatin (10,000 U/kg) was administered intravenously via the caudal vein 1 h before and 6 h after LPS treatment. The sham group received saline only. Survival was monitored for 72 hours. For histological and biochemical analyses, mice were sacrificed under ether anesthesia 24 h after LPS treatment [2].
Lung wet/dry weight ratio: Lungs were obtained immediately after dissection, weighed (wet weight), dried in a constant temperature oven at 60°C, and weighed again (dry weight). The ratio was calculated [2].
Histological evaluation: Lungs were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). A pathologist blindly scored lung injury based on alveolar congestion, hemorrhage, neutrophil infiltration, and alveolar wall thickness/hyaline membrane formation using a 4-point scale (0=no injury to 4=diffuse injury). Lung injury score was the mean of four sections per animal [2].
Bronchoalveolar lavage fluid (BALF) collection: Ice-cold PBS was infused into the lungs twice and withdrawn via a tracheal cannula. Total leukocyte count was determined using a hemocytometer [2].
In septic liver injury mouse models, Ulinastatin was administered and liver tissues were analyzed for TNF-α and IL-10 levels [1].
In hepatic ischemia-reperfusion injury models, Ulinastatin was combined with dexmedetomidine or added to lactated Ringer's solution for liver soaking [1].

In rare cases, intravenous injection of high-dose Ulinastatin may cause adverse reactions such as dizziness, pain at the injection site, decreased white blood cell count, nausea, vomiting, and even allergic reactions. These adverse reactions are usually short-lived, and severe reactions occur in few instances. Ulinastatin is generally tolerable and safe for most patients at reasonable doses [1].
In a randomized, double-blind, placebo-controlled, ascending-dose study in healthy Chinese volunteers, high-dose Ulinastatin after 2-hour intravenous infusion was generally safe and tolerable [1].

[1]. Research progress of ulinastatin in the treatment of liver diseases. Int J Clin Exp Pathol. 2020 Nov 1;13(11):2720-2726.

[2]. Ulinastatin Protects Against LPS-Induced Acute Lung Injury By Attenuating TLR4/NF-κB Pathway Activation and Reducing Inflammatory Mediators. Shock. 2018 Nov;50(5):595-605.

Ulinastatin can treat septic liver injury, hepatic ischemia-reperfusion injury, hepatitis, hepatic fibrosis, autoimmune liver disease with liver failure, acute lung injury, acute respiratory distress syndrome, pancreatitis, sepsis, septic shock, systemic inflammatory response syndrome, acute circulatory failure, and cerebral edema [1][2].
Ulinastatin exerts its effects through multiple mechanisms: reducing inflammatory factor production (TNF-α, IL-6, IL-1β, IL-2) via TLR4/NF-κB pathway inhibition; anti-apoptosis via Bcl-2 upregulation and Bax/caspase-3 downregulation; regulating multiple enzyme activities (inhibiting trypsin, chymotrypsin, elastase, heparanase; activating neutral protease); regulating blood coagulation (reducing thrombin-antithrombin complex and P-selectin); and immune regulation (increasing CD4+/CD8+ ratio, decreasing Tregs) [1][2].
Ulinastatin improves survival in LPS-induced ALI mice (from ~20% to ~50% at 72 h) [2].
Ulinastatin reduces mechanical ventilation time, ICU hospitalization time, and mortality rate in patients [1].
The protective effect of Ulinastatin against LPS-induced ALI is mediated via attenuation of TLR4/NF-κB pathway activation [2].

C13H16O3

220.264344215393

220.109

80449-31-6

White to off-white solid powder

1.2±0.1 g/cm3

331.5±42.0 °C at 760 mmHg

171.0±16.8 °C

0.0±0.7 mmHg at 25°C

1.549

2.87

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

H2O : ~10 mg/mL
DMSO : ~1 mg/mL

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.)