The Effect of Miana (Coleus Scutellariodes [L]) on Vascular Endothelial Growth Factor Expression in Balb/C Mice Infected with Mycobacterium Tuberculosis

1Sulianti Saroso Infectious Diseases Hospital, Jakarta, Indonesia. 2Molecular Biology and Immunology Laboratory, Faculty of Medicine Hasanuddin University, Makassar, Indonesia. 3Pasar Rebo General Hospital, Jakarta, Indonesia. 4Department of Pulmonology and Respiratory Medicine, Faculty of Medicine Hasanuddin University, Makassar, Indonesia. 5Department of Physiology, Faculty of Medicine Hasanuddin University, Makassar, Indonesia. *Corresponding author E-mail: hattaram@yahoo.com

Tuberculosis (TB) is one of the major health problem in the world. It ranks tenth as the cause of death in the world. World Health Organization (WHO) reported 10 million cases of TB cases and 1.3 million deaths globally in 2018. It is estimated that two thirds of TB burden lies in eight countries which are India (27%), China (9%), Indonesia (8%), Philipphines (6%), Pakistan (5%), Nigeria (4%), Bangladesh (4%) dan South Africa (3%). WHO 2018 report estimated there were more than 1 million new TB cases in indonesia. 1,2,3 The problem in battling TB is the increasing antibiotic resistant TB cases in indonesia. Indonesia is ranked 8 th out of 27 countries with the highest cases of multi drug resistant (MDR) TB in the world. 4,5,6 This showed that there is a need for additional means to treat TB in addition to the administration of antibiotic. While antibiotic work to eradicate pathogen bacteria, it is important to increase host immune response to M. tuberculosis bacterial virulence to create a synergy between antibiotics and immunoregulators. 7,8 When M.tuberculosis invade the host cell, inflammation occurs as the host immune response. This inflammatory response creates local hypoxia due to increased metabolic activity and thus creating increased temperature in the inflamed tissue. The rise in local tissue temperature affect the amount and pressure of local oxygen and creates cellular hypoxia and inflammation. 9,10 Low oxygen level and inflammatory cytokines IL-1a and IL-6 induces the expression of VEGF mRNA. 11,12 Vascular endothelial growth factor (VEGF) plays a role in angiogenesis and angiogenesis increases blood flow to ischemic cells and tissues and corrects hypoxia. 13,14 The use of traditional herbal medicine widely in infectious diseases 15,16,17,18,19,20,21,22,23 and non infectious diseases such as has been a staple alternative to western medicine for indonesian people. 24,25,26,27,28 Recently, traditional herbal medicine may become an alternative or adjuvant to standard therapy. Miana (Coleus scutellariodes [L] Benth) is known as one of the traditional herbal medicine that is often used. It contains flavonoid, tanin, triterpenoid, steroid and atsiri oil that has antibacterial, antioxidant and antiinflammatory effects. 29,30,31,32 Miana leaves have endophyte bacteria that have the ability to synthesize antibacterial agent that contains phtalic acid derivate. In one in vitro study, Miana extract can suppress the growth of Candida albicans and Salmonella typhi. 33,34,35 A study found that Miana extract can improve host immunity by modifying the degree and quality of immune response from T cell, B cell, IL-10 and IL-37. Miana extract can affect proliferation of T cells in mice that was given 510mg/kgBW of Miana extract. Administration of Miana extract also increases the number of CD4 T cells, IFN-g levels, IL37 and TNF-a and also reduces the bacterial colonies in Wistar mice's lungs. 31,32,36,37 Other that immunostimulant and immunoregulatory effects, Miana also has antiinflammatory and antioxidant properties. In Salmonella typhi infected Balb/c mice model, Miana leaves extract can suppress the expression of toll like receptor-4 (TLR-4) m-RNA which is similar to the effect produced by antibiotics. 32 However, not much is known about the effect of Miana leaves extract on VEGF, one of the pivotal factor on combating cell hypoxia due to M. tuberculosis infection. This study aims to elucidate the effect of Miana on VEGF expression in Balb/c mice infected with M.tuberculosis.

experimental design
This study is an experimental study using 16 BALB/c mice conducted in July 2020 in Molecular Biology and Immunology Laboratory, Microbiology Division, Faculty of Medicine, Hasanuddin University (UNHAS), Makassar, Indonesia. The mice were grouped into four groups of four mice. Group 1 is a negative control group that was treated with placebo. Group 2 is a positive control group that was treated with antituberculosis drug. Group 3 is an intervention group that was treated with Miana extract. Group 4 is also an intervention group that was treated with Miana extract and antituberculosis drug.

Balb/c Mice
Sixteen 12 weeks old pathogen free BALB/c mice weighing 30-40 grams were uses in this experiment. The mice were granted by UNHAS Molecular Biology and Immunology Laboratory, Faculty of Medicine, Makassar, Indonesia.

Miana extract
Miana leaves were obtained from Toraja, South Sulawesi, Indonesia. The Miana extract was made using 10 grams of plucked Miana Leaves that were washed and dried in 50°C oven. The dried leaves were grinded using a grinder and sieved using size 100 mesh to achieve fine powder from. A total of 30 grams of Miana powder was diluted with ethanol with 1:10 ratio and mixed using a shaker for 24 hours in room temperature. The mixture was filtered using Whatman filter paper number 50. The filtrate was evaporated using a rotary evaporator in 50°C temperature until concentrated and then dried using a freeze dryer. The concentrated extract was made into a pellet.
The pellets were stored in a refrigerator until use. The dosage of Miana extract used in this experiment was 510 mg/kgBW. The pellets will be diluted with aquades and given to the mice using a nasogastric tube. 32,33 M. tuberculosis induction M. tuberculosis induction was done by injecting 10 3 CFU/ml of M. tuberculosis into the peritoneal cavity using a 0.6 ml syringe. 16,17,18

Miana treatment in mice
All 16 Balb/c mice were put into groups of 4 (group 1-4) randomly. On day 0, 0.2 ml of venous blood was drawn (before induction and before treatment blood sample) from all mice in all groups and M. tuberculosis induction was done on all mice. On day 1, the second blood draw (after induction before treatment blood sample) was done on all groups. Group 1 was given 10 mg/kgBW/ day of aquadest as placebo, group 2 was given 1.95 mg/day of rifampicin, group 3 was given 750 mg/ kgBW of Miana extract, group 4 was given 1.95 mg/day of rifampicin and 750 mg/kgBW of Miana extract. From day 1 to day 7, the treatment specific to each group was continued. On day 8, the third blood draw (after treatment blood sample) was done.
Venous blood sample was centrifugated to obtain the blood serum. The serum was stored in -20°C until analysis. VEGF protein level was measured using Mouse VEGF Elisa Kit Catalog No. LS-F978. Protein concentration was measured in pg/ml. VEGF protein level before induction, before treatment, and after treatment of all groups were done. All sample analysis was done in duplicate to ensure validity of ELISA analysis results.

statistical analysis
Normality test was done using Shapiro-Wilk test and Levene's test was done for homogeneity test. Protein levels of each groups were presented in means and standard deviation. One way ANOVA test was used to determine whether there are any statistically significant differences between the means of VEGF protein levels of the treatment groups as a whole. Post hoc test was done to determine which group is statistically significantly different from other groups. P value below 0.05 is determined as statistically significant. All statistical analyses were done using SPSS 20.0 software for Windows. From the description of the obtained data, it can be seen that there is a steep increase of VEGF protein levels after M. tuberculosis infection. The level of VEGF protein kept increasing after placebo treatment in group 1 while the VEGF levels in other groups decreased after treatment. One-Way ANOVA test was done to see whether there is a significant difference of means after treatment in general. The test result showed a significant difference of VEGF protein level means between groups after treatment in general ( Table 2; F = 105.710, p value = 0.000). To further analyse the difference of VEGF protein level means between groups, a post hoc analysis was done.

VeGF protein levels before induction, after induction, and after treatment
Post hoc analysis (Table 3) showed that VEGF protein level was significantly lower after administration of rifampicin (4,655.215 ± 1,731.676 pg/ml, p value = 0.000), Miana

discussion
Tuberculosis is infectious diseases which their pathomechanisms has been involve the several immune system both cellular and humoral system. 38,39 Serum VEGF levels is known to be elevated in patients with active pulmonary tuberculosis and lowered after successful treatment. 40 In this study, VEGF levels were also elevated after the mice was infected with M. tuberculosis and decreased after treatment whereas VEGF level in placebo treated mice kept increasing. This indicates the role of VEGF in M. tuberculosis infection.
This study found that there was a significant difference in VEGF level means between groups in general. Further investigation showed that VEGF levels were significantly lower in M. tuberculosis-infected mice treated with rifampicin, Miana, and rifampicin plus Miana compared to placebo. VEGF level was also lower in rifampicin group compared to Miana group. There was also a significantly lower VEGF level in rifampicin plus Miana group compared to Miana group. However, there was no significant difference of VEGF level between rifampicin group and rifampicin plus Miana group. This result indicate that Miana extract administration does have an effect on VEGF protein levels. Our result showed that rifampicin with Miana extract as adjuvant can be an acceptable Tuberculosis treatment.
However, the exact mechanism of how Miana extract affect VEGF in Tuberculosis infection remains unclear. To elucidate the possible mechanism of the effect of Miana extract on VEGF production, understanding of tuberculosis infection immune response pathway and the mechanism of Miana extract as antimicrobial, anti-inflammatory, and antioxidant are crucial.
During infection such as tuberculosis infection, inflamed site due to infection is relatively hypoxic. Hypoxic environment increases the cellular level of HIF-1a. The increase in HIF-1a will increase the level of VEGF. There are multiple other pathways of VEGF production induction in during infection. 40 Inflammation caused by infection stimulates the production of cyclooxygenase (COX) 1 and COX 2 which will induce the production of prostaglandin E2 (PGE2). PGE2 and HIF-1a upregulates the production of cellular VEGF. Upregulation of cellular VEGF enhances angiogenesis, vasodilatation, mediate extravasation of monocytes under hypoxic condition, and works together with histamine to increase vascular permeability which causes plasma exudation. 41 Miana is one of the traditional herbal medicine commonly used to treat infectious diseases and immune booster by the Toraja people in South Sulawesi, Indonesia. It is also commonly used as a complement to antituberculosis treatment. To be used, Miana leaves needs to be extracted. Miana leaves extract contains compounds such as alkaloids, tannins, flavonoids, saponins and terpenoids. Flavonoids have antimicrobial, antiinflammatory and antioxidant properties. One study stated that flavonoid content in miana extract is on average 8.59 mg/gram extract. Flavonoid antibacterial mechanism consists of inhibition of nucleic acid synthesis and causing damage to bacterial cell wall, microsomes, and lysosomes. Flavonoids also has very strong antioxidant activity. Flavonoid in Miana increases T lymphocytes, CD4 T cells, IFN-g, IL-37, TNF-a , TLR4 and NRAMP1. Tannin, saponin, and terpenoid also have antimicrobial activity. 31,32,33,36 From the explanation above, it is clear that M. tuberculosis infection causes inflammation and hypoxia and activates a cascade of immune response in which at one of the ends of the line increases VEGF protein levels to combat the bacteria as well as hypoxia. Miana extract, which contains flavonoids and others, directly acts on the bacteria as a type of antibacterial as well as immunoregulator to increase host immune response to combat the bacteria and anti-inflammatory action. The elimination or reduction of the cause of the inflammation diminishes the signal for the induction of upregulation of VEGF. This may explain the possible mechanism of the effect of Miana on VEGF levels. Further study is needed to elucidate the immunoregulatory pharmacodynamics of Miana leaves extract.

conclusion
There were significant differences in VEGF protein levels between treatment groups. VEGF protein levels in Balb/c mice treated with rifampicin, Miana, and rifampicin plus Miana were significantly lower than placebo. VEGF protein levels were significantly lower in rifampicin treatment group compared to Miana group; significant lower VEGF protein level in rifampicin plus Miana treatment group compared to Miana group; and no significant difference between rifampicin group and rifampicin plus Miana group.

acknowledGeMent
The author would like to thank the Molecular Biology and Immunology Laboratory staffs and consultants for their continuous help and support for the completion of this study.

Conflict of Interest
There are no potential conflict of interest to be disclosed.

Funding source
The study was funded privately by authors. There were no sponsors involvement in the study.