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Description

Liver function and protection

Good health is dependent upon proper liver function. The liver is a huge chemical plant of the human body. It has many major functions as shown in Fig.1. In the daily life, as shown in Fig.2, there are many reasons which overload liver to disease. Liver disease can be divided into viral liver and non viral liver diseases. Non viral liver diseases consist of alcoholic liver, drug or toxic liver disease, metabolic abnormal liver disease, fatty liver, etc.

Liver functions

Daily life overloads liver leading to liver diseases

https://www.clinicians.co.nz/

Protection agents

Liver disease is a common disease with great danger. And the incidence rate in East Asia is higher than the average in the world. Scientists have made great efforts to prevent or treat it by hepatitis virus vaccine inoculation, clinic operation, and medicine administration, etc. At present oral administration of two protective drugs (glutathione and S-adenosyl methionine) is very effective against a variety of liver diseases in hospital.

Glutathione

Glutathione (GSH) is biosynthesized from glutamic acid, cysteine and glycine, and distributes in many organs in human body, especially in the liver where the highest concentration of GSH exists. GSH plays an important role in liver biochemical metabolism. The liver therapeutical effects of glutathione are listed in the following:

Inhibit the formation of fatty liver.
Directly antagonize the toxicity of oxygen free radicals.
Preventing hepatocyte injury.
Treatment of fulminant hepatic failure caused by drug poisoning.

GlutathioneS-adenosyl methionine

Fig.3 Molecule structures of GSH and SAM

S-adenosyl methionine

S-adenosyl methionine (SAM) is an important intermediate product in liver metabolism. As a methyl donor and a precursor of physiological sulfhydryl compounds, SAM participates in important metabolic reactions in vivo. The liver therapeutical effects of SAM are listed in the following:

Delay the need for liver transplantation and reduce mortality in patients with alcoholic cirrhosis in a 24-month trial.
Significantly increase hepatic glutathione levels in nonalcoholic liver disease with oral administration of 1200mg SAM daily
(J Hepatol 1999, 30(6) 1081-1089).

Bacterial drug delivery system

Oral administration is the most attractive option for drug delivery due to convenience of administration, patient acceptance, and long-term compliance. However, this strategy is not feasible for many drugs, especially peptide or protein drugs, due to poor biochemical stability (degradation and denaturation) in the harsh gastrointestinal environment (Fig.3) and low epithelial permeability. Synthetic cell therapy is a ﬁeld that has broad potential for future applications in human disease treatment. In vivo synthesis and delivery via cell therapy has several important advantages over traditional oral systemic treatment.

The required dosage is reduced by several orders of magnitude.
The route of administration is less invasive than intravenous or subcutaneous injection.
Multiple therapeutic agents can be produced by the same cell simultaneously.
No need for puriﬁcation and formulation of the active compound.

Our objective

Lactococcus lactis is one of the most common organisms used in bacterial drug delivery system. Lactococcus lactis is a food-grade bacterium that is widely used in the dairy industry. It is a Gram-positive bacterium and therefore lacks endotoxic lipopolysaccharides (LPS) which are associated with commonly used Gram-negative bacteria. It is also non-invasive and non-commensal and thus has less potential to trigger immunotolerance or side effects upon prolonged use. The bacterium has an established safety profile through its long use in fermented dairy products and is considered as a GRAS (generally recognized as safe) microorganism. During the last two decades, significant advances have been made in the field of lactococcal genetics and protein expression systems. The most commonly used system is the nisin-controlled gene expression (NICE) system, containing the nisin promoter.

In the present project, since the oral table administration of GSH and SAM have some disadvantages, such as low stability and short life span, here we tried to develop a novel in-vivo strategy of produce and deliver them simultaneously by using NICE system. In the experiment, two-functional GSH synthetase gene (gshF) and SAM synthetase gene (metK) were in tandem inserted into the expression vector (pNZ8148), and the resulted plasmid (pNZ8148-SG) was employed to construct the target vector pNZ8148-SGC by introducing adhesion factor gene (cwaA). This target vector was transformed to get recombinant Lactococcus lacti, which was employed to produce our “smart yogurt”.