TREATING LIVER INJURY WITH BEE VENOM

What Is Liver Injury?
Liver injury is also called liver laceration, which results due to any sort of trauma faced by the liver. It is also the most common type of abdominal injury. This can happen due to the penetration of any foreign object like a knife or a bullet or due to any massive impact like a car accident.

​The injury caused, can result in a liver hematoma or large tears – which go deep into the liver. A liver injury can become fatal if, severe bleeding occurs due to the damage done to the large blood vessels present in the liver. Any sort of bleeding that happens due to liver injury takes place inside the abdominal cavity. ​

​What are the symptoms of the liver injury?

1. Liver injury can be detected easily through the following symptoms:
2. The subject will demonstrate symptoms of shock
3. Skin color will become blue or pale
4. Heart rate will go up and will lead to rapid breathing
5. A Persistent abdominal pain
6. Swelling of the abdomen (due to accumulation of blood)
7. How are liver injuries characterized?
8. There are different ways of characterizing liver injury. They are stated below:
9. Medical professionals make use of ultrasonography or CT (computed tomography) scans to examine the liver injury.
10. Minor liver injuries take time, but they heal on their own – without any treatment. Only severe liver injuries require a surgical process.
    
    

Bee Venom Phospholipase A2 Protects against Acetaminophen-Induced Acute Liver Injury...

Introduction
Paracetamol is, also known as acetaminophen, is one of the most effective analgesics and antipyretic drugs that is used for treating different medical problems. Generally, it is a safe drug, when used therapeutically, but if taken in high doses – by humans or animals – it can lead to additional hepatic lesions, nephrotoxicity, severe hepatic necrosis, and even death. Several researchers have successfully managed to discover the mechanism responsible for acetaminophen encouraged acute injury, especially the signaling pathways that lead to toxicity and tissue damage in the liver.

It has been found that Tregs (Regulatory T Cells) play a vital role in maintaining overall tolerance of the immune system. Hence, any sort of Treg cell deficiency can lead to an autoimmune disease or further aggravate an existing infection, illness or disease. These cells play an important role in supporting the immune system of our body during any sort of infection, allergy, and tumor immunity and transplantation tolerance.
 
According to the different studies carried out earlier, it was established that the Tregs generate a therapeutic effect against an immune-mediated injury. The inflammatory factor having an expression of IL-10 was found to be elevated in response to the drug-induced liver injury. Increased susceptibility to the acetaminophen brought hepatic injury, demonstrated a correlation with an enhanced expression of IL-6 and YNF – the pro-inflammatory cytokines.

PLA2 obtained from honey bee venom is a prototypic group III enzyme, which is responsible for hydrolyzing fatty acids. Melittin is another essential component of honey bee venom that further boosts the effects of PLA2. Different clinical studies, conducted earlier, have proved that PLA2 helps in preventing spinal cord injury and neuronal cell death.
In this clinical examination, the focus has been placed on proving that PLA2 prevents hepatic dysfunction and produces anti-inflammatory cytokines in the acetaminophen injected mice. It can be supposed for now that PLA2 can produce therapeutic effects and offers protection against acetaminophen-induced hepatotoxicity.
 
Method
To conduct this experiment C57BL/6 male mice, nearly 6 to 8 weeks old, were obtained from the Charles River, Korea. Each mouse weighted 20 to 21gms. While the IL-10−/− mice (B10.129P2(B6)-Il10tm1Cgn/J, nearly 7 to 8 weeks old and Male Foxp3EGFPC57BL/6 mice (C. Cg-Foxp3tm2Tch/J, nearly 6 weeks old were acquired from the Jackson Laboratory, USA. All of the mice were kept in a proper air-conditioning system with pathogen-free conditions and a 12h light and 12h dark cycle. It was also made sure that all of the mice had ample food and water. The overall setup of this clinical experiment was formally approved by the Animal Care and Use Committee of the Kyung Hee University. Execution of the experimental mice, at the end of this clinical study, was done through CO2 asphyxiation.

Treatment
Acetaminophen and PLA2 were obtained from the Sigma Aldrich, USA. All of the mice were injected with 0.2mg/kg (according to the bodyweight of the mice) of PLA2, intraperitoneally, before giving them the acetaminophen injection. This treatment was given only once for 5 days. On the other hand, the control group also got an equal quantity of PBS. PBS and acetaminophen were mixed at a special concentration of 20mg/ml.

The last injection of acetaminophen, having a dose of 500mg/kg, was given to the mice intraperitoneally after two days had passed subsequently the final dose of PBS or PLA2. And right after 24 hours had passed after administering the acetaminophen injection to these mice, they were put down by CO2 asphyxiation. Liver, spleen and blood samples were acquired to conduct further analysis.

Analysis
To determine any sort of changes in the Tregs population, after PLA2 treatment, the splenocytes were removed from the Foxp3EGFP mice. Cells were treated with PLA2 (0.01µg/ml, 0.µg/ml 1, 1µg/ml and 10µg/ml) and then cultured in RPMI 1640 media consisting of 2µg/ml anti-mice CD28 antibody and 2.5µg/ml anti-mice CD3 antibody for a time period of 72h. Obtained cells were incubated with the fluorescently tagged Abs for CD25 and CD4 staining. Data were analyzed by the Cell Quest Pro and FACS data was acquired with the help of the FACS flow cytometer.

Assessment
After giving the acetaminophen injection to the mice, blood samples were obtained right after 0hrs, 6hrs and 24hr had passed. These samples would help in measuring hepatic dysfunction through the quantification of ALT and AST. All of the blood samples were stored at room temperature for an hour and later centrifuged for only 10 minutes at 1000g to ensure proper separation of the serum. ALT and AST levels were measured through the Fuji Dri Chem 3500i instrument obtained from Japan while, the IL-10 serum level was measured through ELISA, USA.
 
Staining
Separated livers were placed in 4% of PFA for 24hours and then later embedded in paraffin. Paraffin samples were cut into 5µm thick slices and subjected to deparaffinization. These samples were then stained in hematoxylin for the 90s and then dipped in eosin thrice. After this process, the liver samples were placed under running water for 10 minutes and finally covered with glass. Periportal and portal areas of the liver were assessed through microscopy.

Injecting
An Anti-mouse CD25 rat IgG1 antibody was created from the hybridomas obtained from ATCC, USA. For depleting Tregs, the anti-mouse CD25 was administered intraperitoneally, having a dose of 0.1mg per mouse. This was given before acetaminophen and PLA2 injections. According to Flow cytometry analysis, it was found that Tregs were being depleted after using FITC-anti-mouse CD4 antibodies PE-anti-mouse CD25.
 
Assessment
Separated livers were stored at -70 degrees centigrade to examine inflammation in the liver tissues after the acetaminophen injection. The frozen liver tissues went through homogenizes in a protein solution obtained from Intron Biotechnology, Korea. And after that, they were incubated for thirty minutes on ice and finally centrifuged at 13,000rpm at 4-degree centigrade for 10 minutes. IL-6 and TNF present in the liver were measured with an enzyme-linked assay obtained from BD Biosciences, USA. For measuring nitrite levels, liver samples were incubated with an equal quantity of Griess reagent (1% sulfanilamide/0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride/2.5% H3PO4) for 10 minutes at room temperature. Protein concentration was measured with the BCAth Protein Assay Kit obtained from Thermo Scientific, USA. Final concentrations were evaluated with the total quantity of protein and later expressed in the form of pmol/mg or pg/mg. 
   
Results
To examine the immune-modulating influence of PLA2 in the splenocytes, they were treated with PBS or PLA2 for 3 days. The Treg population was increased in a dose-dependent method in the PLA2 treated group linked with the PBS treated group.  
Protective effectAfter PLA2 pretreatment, the mice were administered a very high dose of acetaminophen at 200mg/kg. Samples of blood were obtained after 0hours, 6hours, and 24hours to measure the ALT and AST levels. For H and E staining, the liver tissues were dissected 24hours after giving the acetaminophen injection. Hepatic death of cells in the periportal area was managed with the help of PLA2 injection. ALT and AST were increased after administering acetaminophen, whereas the PLA2 injection suppressed the increase noticed in ALT and AST levels.

Hepatoprotective effect
PLA2 effects in the CD4+CD25+T cell diminution model were examined with an anti-CD25 antibody administered at 0.1mg/mouse through intraperitoneal injection. Levels of ALT and AST in the serum were also measured. It was found that PLA2 therapy did not affect liver histopathology, ALT and AST levels in the Treg depleted mice did not show any sort of change. The hepatoprotective effect of PLA2 was reduced in the Treg depleted mice. In light of these results, it can be said that due to Treg depletion hepatotoxic effects of PLA2 were eliminated and the hepatoprotective effect of PLA2 was found to be Treg dependent.
 
Cytokines in the liver
For examining the anti-inflammatory effect of PLA2 therapy in the acetaminophen-induced injury, levels of NO, IL-6, and TNF in the liver were calculated after 24hours of giving the acetaminophen injection. It was observed that the treated mice showed an increase in the levels of NO, IL-6, and TNF. On the other hand, PLA2 treated subjects showed an impressive decrease in the level of NO, IL-6 and TNF than the control group and depletion of Tregs brought down the ability of defensive behavior of PLA2.

IL-10 production delivered the effects of PLA2
It is a well-known phenomenon that IL-10 offers protection against acetaminophen-induced injury to the liver. But it was important to determine if the hepatoprotective effects of PLA2 are dependent on the IL-10 or not. For this purpose IL-10, deficient mice were chosen. Levels of ALT and AST were measured 24hours after administering the acetaminophen injection. Results obtained indicated that the PLA2 effect on the acetaminophen-induced hepatotoxicity was abolished in the IL-10 deficient mice. This suggests that IL-10 plays an important role in mediating the protective effects of PLA2 in the state of hepatotoxicity.
 
Besides, these clinical findings also suggest that Tregs are very important for the primary IL-10 augmentation and production in the mice. IL-10 also offers protection against acetaminophen-induced liver lethality and injury. Louis et al. showed that proper administration of IL-10 removes any chances of hepatotoxicity in a lipopolysaccharide and galactosamine mouse model.

Discussion
You must know that the liver is one of the vital organs that help with detoxification of various toxins and drugs from our system. Toxins and drugs can damage the liver to a great extent. It happens gradually, but the symptoms take a lot of time to surface. That is why, in this clinical experiment, it was recorded that a high dose of acetaminophen – an analgesic drug – leads to acute hepatotoxicity in the experimental mice.

According to a clinical study done earlier, it was concluded that Tregs helped in diminishing inflammation caused due to liver injury. Adoptive induction of Tregs in the mice successfully prevents any sort of liver injury. In this research, it was presented that PLA2 offsets inflammatory immune disease with the help of Treg regulation. PLA2 derived from the honey bee venom impressively enhanced the Treg population in the splenocytes. From these results, a hypothesis was established that PLA2 can deliver a protective effect on the acetaminophen-induced hepatotoxicity with a supplementary action of Tregs.
It can be proposed that PLA2 can be used as an alternative therapy for NAC or even used simultaneously with NAC to lessen inflammation caused due to liver injury through acetaminophen.

Conclusion
In the end, it can be declared that PLA2 encourages the secretion of IL-10 by Treg modulation and prevents acute liver injury. It is highly suggested that PLA2 retains a hepatoprotective effect in the acetaminophen-induced toxicity through modulation of IL-10 and Tregs in experimental mice. 
 
Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4257707/​