The Therapeutic effect of MKA on Bacterial Lipopolysaccharide (LPS) induced lipid peroxidation, cytosolic LDH leakage and mitochondrial membrane depolarization in RAW 264.7 Macrophages

The Polyherbal formulations are used as a potential target for treating various diseases due to its wide array of phytoconstituents with antioxidant potential. In the present study, the therapeutic effect of Polyherbal formulation (MKA) comprising of three plants Mimusops elengi L., Kedrostis foetidissima (Jacq.) Cogn. and Artemisia vulgaris L. were studied in LPS induced RAW 264.7macrophages. Four different concentrations (25, 50, 75 and 100μg/ml) of MKA were tested against control, LPS treatment and standard Quercetin in LPS induced RAW 264.7 macrophage cells. The rate of Lipid peroxidation was measured in terms of Malondialdehyde (MDA) levels. The cytosolic LDH leakage was determined by measuring NADH release at 340nm. The changes in mitochondrial membrane potential were studied by measuring red/green luorescent intensity of JC-1 stained cells in the low cytometer. It was found that MKA treatments signi icantly reduced the rate of Lipid peroxidation and LDH leakage compared to LPS treatment. The results of low cytometry revealed that the JC-1 green luorescent intensity decreased with increase in MKA concentration, in a dose-dependent manner. It is evident from the study results that, theMKAhas a therapeutic effect on LPS inducedRAW264.7 macrophages by protecting the cells from lipid peroxidation, restoring the cell membrane integrity and mitochondrial membrane potential.


Malondialdehyde,
In lammatory, Flow cytometry, depolarisation ABSTRACT The Polyherbal formulations are used as a potential target for treating various diseases due to its wide array of phytoconstituents with antioxidant potential. In the present study, the therapeutic effect of Polyherbal formulation (MKA) comprising of three plants Mimusops elengi L., Kedrostis foetidissima (Jacq.) Cogn. and Artemisia vulgaris L. were studied in LPS induced RAW 264.7 macrophages. Four different concentrations (25, 50, 75 and 100 µg/ml) of MKA were tested against control, LPS treatment and standard Quercetin in LPS induced RAW 264.7 macrophage cells. The rate of Lipid peroxidation was measured in terms of Malondialdehyde (MDA) levels. The cytosolic LDH leakage was determined by measuring NADH release at 340nm. The changes in mitochondrial membrane potential were studied by measuring red/green luorescent intensity of JC-1 stained cells in the low cytometer. It was found that MKA treatments signi icantly reduced the rate of Lipid peroxidation and LDH leakage compared to LPS treatment. The results of low cytometry revealed that the JC-1 green luorescent intensity decreased with increase in MKA concentration, in a dose-dependent manner. It is evident from the study results that, the MKA has a therapeutic effect on LPS induced RAW 264.7 macrophages by protecting the cells from lipid peroxidation, restoring the cell membrane integrity and mitochondrial membrane potential.

INTRODUCTION
Reactive oxygen species (ROS) is the main causative agent of diseases like cardiovascular diseases, cancer, pulmonary diseases, atherosclerosis and in lammatory diseases.
Oxidative stress occurs in a biological system as a result of an improper balance between formation and neutralization of ROS (Fridovich, 1999;Fang et al., 2002). Antioxidants can detoxify ROS by acting as radical scavengers, electron donors, singlet oxygen quenchers, synergists, hydrogen donors, metal-chelating agents, peroxide decomposers and enzyme inhibitors (Frei et al., 1988). Herbal medicines are a rich source of phytochemicals, which acts as potent antioxidants with free radical scavenging activity. The synergistic action of Polyherbal formulations renders a more therapeutic effect compared to single herbal preparation (Parasuraman et al., 2014).
The Lipopolysaccharide (LPS) is a bacterial endotoxin, which functions as a potent activator of RAW 264.7 macrophages and triggers the release of in lammatory mediators leading to acute and chronic in lammatory conditions (Veres et al., 2004;Troutman et al., 2012). There is an increase in the level of ROS and lipid peroxidation rate in LPS triggered macrophages (Ambrozova et al., 2011).
Excess of ROS production affects the function of Calcium (Ca 2+ ) regulating proteins and other electron-transport chain proteins in mitochondria, thereby altering the mitochondrial membrane potential (Guo et al., 2013).
The ROS acts on membrane lipids inducing lipid peroxidation which releases toxic lipid-derived aldehydes (LDAs) like malondialdehyde (MDA), acrolein and 4-hydroxy-trans-2-nonenal (HNE). The LDAs activate various kinases involved in redoxy signalling pathways leading to the cytotoxicity of the cell, ultimately causing cell death (Yadav, 2015). The excess of LDH leakage from the cytoplasm into the extracellular portion is indicative of the extent of cell membrane damage (Li et al., 2014).
In the present investigation, the protective action of MKA, the polyherbal formulation comprising of three selected plants Mimusops elengi (L.), Kedrostis foetidissima (Jacq.) Cogn. and Artemisia vulgaris (L.) was studied in LPS primed macrophage cells. The lipid peroxidation rate, cytosolic LDH leakage, changes in mitochondrial membrane potential were analyzed in LPS induced RAW 264.7 macrophages upon treatment with different concentrations of MKA. This study is the continuation of the previous research entitled "Evaluation of free radical scavenging capacity and reducing the power of polyherbal formulation comprising of three selected plants" (Poongodi and Nazeema, 2019).

Sampling and Preparation of Polyherbal formulation
The leaf sample of three plants Mimusops elengi (L.), Kedrostis foetidissima (Jacq.) Cogn and Artemisia vulgaris (L.) were collected from Sulur, Coimbatore. The Polyherbal formulation MKA was prepared using a leaf sample of three plants Mimusops elengi (L.), Kedrostis foetidissima (Jacq.) Cogn. and Artemisia vulgaris (L.) in ratio 1:1:1. The sample is extracted for phytoconstituents using 80% ethanol in Soxhlet apparatus. The obtained extract was evaporated in rotavaporator, and the resultant sample is dissolved in DMSO and stored for further studies (Poongodi and Nazeema, 2019).

RAW 264.7 Macrophage Cell Culture
RAW 264.7 macrophage cell line was purchased from NCCS, Pune. The macrophage cells were cultured in Dulbecco's modi ied Eagle medium (DMEM) containing 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin at 37 • C with 5% CO 2 . The cultured macrophage cells were washed in DMEM and detached the cells using 0.25% trypsin-EDTA solution (Lee and Park, 2011).

Cell treatment
The cell treatment was performed by the method of Khan et al. (1995) modi ied method. The macrophage cells were seeded in 6-well plate at a density of 5x10 3 cells per well and incubated for 24 hours at 37 • C in 5% CO 2 . The cells were washed with DMEM solution and added 1600µl of growth medium. Cells were treated with 200µl of MKA at four different concentrations 25, 50, 75 and 100 µg/ml and incubated for 1-2 hours before LPS treatment. Quercetin was used as standard at a concentration of 25µM. Then, added LPS (1µg/ml) and incubated for 24 hours at 37 • C in 5% CO 2 . The negative control (without LPS and MKA) and Positive LPS (without MKA) were also studied. All these mixtures were centrifuged at 2000xg for 10 minutes. The supernatant was used to study Lipid peroxidation and LDH leakage. The pelleted cells were used to study mitochondrial membrane potential.

Lipid Peroxidation
The lipid peroxidation was studied by the method of Draper and Hadley (1990). The culture supernatants were used to study the malondialdehyde (MDA) levels, which is an indicator of ROS mediated lipid peroxidation. To 0.5 ml of media supernatant, added 1ml of 30% trichloroacetic acid (TCA) and centrifuged at 3500xg for 10 minutes. Then, 1 ml of this supernatant was mixed with 1 ml of thiobarbituric acid (TBA) and heated this mixture at 90 • C for 10 minutes and cooled. The MDA levels were measured at 532 nm. The standard curve was plotted using 1,1,3,3 tetra ethoxy propane. The results are expressed as nanomoles of MDA equivalents.

Cell Membrane integrity assay (LDH leakage)
Membrane integrity was studied in terms of extra-cellular LDH leakage. The LDH in the cell supernatant was evaluated by monitoring the decrease in the level of NADH at 340nm in a microtitre plate reader (Anthos 2020, Austria). This decrease in NADH level is due to the conversion of pyruvate to lactate by LDH. The LDH leakage is directly proportional to the level of membrane damage (Pereira et al., 2015).

Flow cytometric analysis of mitochondrial membrane potential
The pelleted cells were incubated in media containing one µg/ml of JC-1 dye for 15 minutes at 37 • C in a CO 2 incubator. Then the cells were washed twice with phenol-red free media to remove the unbound dye. The mitochondrial membrane potential was immediately studied as the ratio of red and green luorescence in Flow cytometer (BD FACSverse) (Venkatesan et al., 2017).

Statistical analysis
All experiments were performed in triplicates, and the results were expressed as Mean±Standard Deviations (SD). Data were analyzed using one way ANOVA followed by post hoc Dunnett's multiple comparison test using SPSS software (Version 21). P<0.01 were considered statistically signi icant.

The effect of MKA on Lipid Peroxidation in RAW 264.7 macrophages
Lipid peroxidation was measured in terms of MDA levels, which is elevated in conditions of oxidative stress. In Figure 1, the effect of MKA on the rate of lipid peroxidation in LPS induced RAW 264.7 cells were illustrated. In LPS treated macrophages, MDA level was found to be 2.86±0.13 nM. In 25, 50, 75 and 100 µg/ml of MKA treatments, MDA levels reduced to 2.23±0.15, 1.45±0.24, 0.91±0.01, 0.73±0.05 nM respectively. The MDA level was 0.62±0.02 nM/mg in Quercetin treatment. Data are presented as Mean±SD of three replications; **represent signi icant difference (P<0.01) vs LPS; # represent signi icant difference (P<0.01) vs Control.
Reactive oxygen species (ROS) produced in the normal physiological process has an essential role in tissue homeostasis and cell signalling pathways. Excess ROS production damages the cell membrane by lipid peroxidation of membrane lipids (Ferreira et al., 2018;Lundgren et al., 2018;Que et al., 2018).
The polyherbal formulation MKA comprising of Mimusops elengi L., Kedrostis foetidissima (Jacq.) Cogn. and Artemisia vulgaris L. reduced the level of lipid peroxidation induced by bacterial endotoxin LPS in macrophages. In LPS treated macrophages, MDA level was found to be signi icantly higher compared to control, which is an indication of oxidative stress due to lipid peroxidation. In 25, 50, 75 and 100 µg/ml of MKA treatments, MDA level signi icantly reduced compared to LPS treatment. MKA has therapeutic potential which can reduce lipid peroxidation in a dose-dependent manner, thereby preventing further cell and organelle damage.

LPS Induced LDH Leakage in Cell Supernatant (Membrane integrity Assay)
LPS induced in lammation causes cell membrane damage, resulting in leakage of Lactate dehydrogenase enzyme. So, the measurement of LDH leakage is an indicator of the extent of in lammation in LPS induced RAW 264.7 cells. LDH leakage levels were measured in cell supernatant and illustrated in Table 1. Signi icant increase of 844.97±25.39 U/L of LDH was seen in LPS treated cells compared to control. Also, it was evident that MKA treatment signi icantly reduced LDH leakage. LDH release in 100 µg/ml of MKA treatment was 309.92±19.75 U/L. In Quercetin treatment, LDH release was found to be 298.86±11.99 U/L. This con irmed that MKA reduced the in lammatory damage, thereby reducing the LDH release from LPS induced RAW 264.7 cells.
The effect of MKA on cell membrane integrity was studied in terms of cytosolic LDH leakage. The study results revealed that MKA has a protective effect on the cell membrane, reducing LDH leakage signi icantly compared to LPS treatment.

The effect of MKA on LPS induced mitochondrial membrane depolarisation
LPS induction caused mitochondrial membrane damage in RAW 264.7 macrophages, which was studied in terms of mitochondrial membrane poten-  298.86 ± 11.99** tial using Flow cytometer. The mitochondrial membrane potential and energy production is affected due to excess ROS production by LPS, which cause systemic in lammation (Galley, 2011).
The effect of MKA on mitochondrial depolarization of LPS induced RAW 264.7 macrophages were illustrated in Figure 2, (1) (7) Quercetin standard (25µM) + LPS treatment. The JC-1 green luorescence intensity is enhanced in conditions of mitochondrial membrane damage due to membrane depolarization. In Figure 3, the mitochondrial membrane potential is represented in terms of green luorescent intensity. **represent signi icant difference (P<0.01) vs LPS; ## represent signi icantdifference (P<0.01) vs Control; # represent no signi icant difference(P<0.01) among the group.
It was found that there is a signi icant increase (p<0.01) in the mitochondrial membrane potential of LPS treated compared to control. Also, there is a signi icant decrease (p<0.01) in the mitochondrial membrane potential of MKA treatments 25, 50, 75 and 100 µg/ml compared to LPS treatment. It was also noted that there is no signi icant difference between 100 µg/ml MKA treatment and standard Quercetin. The MKA has therapeutic property in restoring the mitochondrial membrane damage caused by LPS in macrophages. The LPS induction shifts the luorescence signal to more green, indicating the high level of mitochondrial membrane depolarisation. The MKA treatment shifted the luorescent signal to normal, thereby reducing the membrane depolarisation and restoring the mitochondrial membrane damage in a dose-dependent manner.

CONCLUSION
The Polyherbal formulation MKA, reduced the rate of lipid peroxidation in LPS triggered cells, which proves its ef icacy in protecting the cells from oxidative damage. The LDH leakage was also reduced in MKA treatments, which validates its potential in protecting the cell membrane from oxidative damage. Also, the MKA restores the mitochondrial membrane potential signi icantly compared to LPS treated group. From these study results, it is evident that the MKA has a therapeutic role in protecting the macrophages from LPS induced oxidative stress.