Schisandra chinensis polysaccharides attenuate the growth of tuberculosis bacilli in rats via immunity enhancement

Purpose: To evaluate the effect of Schisandrachinensis polysaccharides (SCPP11) on the growth of tuberculosis (TB) bacilli in a rat model. Methods: Tuberculosis (TB) was induced by administration of colonies of tubercle bacilli via the inhalation route. SCPP11 was administered at separate doses of 25, 50 and 100 mg/kg to different rat groups, p.o., for 4 weeks. Untreated rats served as TB control. At the end of treatment, assessments were made on the effect of SCPP11 on colony forming unit (CFU), cytokine levels, and population of immune cells present in lung tissue homogenates of the TB-infected rats. Results: The CFU of TB bacilli was significantly reduced in the lung tissues of SCPP11-treated group, when compared to untreated TB control group. Moreover, SCPP11 attenuated lung tissue levels of inflammatory cytokines, and significantly enhanced immunity, relative to the TB control group. Conclusion: These results indicate that SCPP11 ameliorates TB by suppressing the growth of tuberculosis bacilli.


INTRODUCTION
Tuberculosis (TB) is caused by tubercle bacilli infection, which causes the death of approximately 1.2 million people every year throughout the world [1].Available literature reveals that immune-compromised people are readily prone to TB reactivation [2].Microbicidal activity of alveolar macrophages are activated due to secretion of chemokines and TNF-α which recruit T-lymphocytes in the lung granulomas during respiratory infection of MT [3].Nonoxidative and oxidative mechanisms are used by alveolar macrophages to kill MT bacteria [4].Digestive enzymes and acidic pH within the lysosomes of macrophages also promote bacterial death [5].
In multiple drug-resistant TB (MDR-TB), the management of TB is made difficult due to development of resistance against anti-TB agents by tubercle bacteria [6].The MT bacteria resistance is developed by altering the acidification phagosome and fusion of phagolysosome [7].In view of the problems posed by bacterial resistance, there is need for development of new drugs for the management of MDR-TB.
In the last few decades, alternative medicines have shown great potential in the management of chronic disorders.Schisandra chinensis (Turcz.)Baillis traditionally used for medicinal purposes in China [8].It is used for enhancing the secretion of body fluids, and as a kidney tonic, and also for calming the mind [9].It is also used for the management of neurasthenia and hepatitis [10].Several pharmacologically active polysaccharides are present in Schisandra chinensis [11].These polysaccharides are known to possess broad spectrum therapeutic uses, which include immunomodulatory activities [11].Studies have revealed that Schisandra chinensis polysaccharides promote the secretion of inflammatory cytokines, leading to the activation of NK cells, macrophages and lymphocytes [12].Moreover, the polysaccharides enhance the activation of complement system and antibody production [13].The present study was carried out to evaluate the effect of Schisandra chinensis polysaccharides on Mycobacterium tuberculosis infection in a rat model of TB.

EXPERIMENTAL Animals
Female albino rats (6 -7 weeks old; weighing 200 -250 g) were procured from Shanghai Medical College, China.The animals were housed under standard conditions (temperature: 25 ± 2 °C, humidity: 60 ± 5 %, and 12 h light/dark cycle) as per standard guidelines.They were kept for the period of one week for the acclimatized to laboratory conditions.All the procedure involved in the study was approved by institutional ethical committee of Wuhan Red Cross Hospital, China (no.IAEC/WRCH/2016/19).The study followed the guidelines of Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC) for experimentation and animal use [14].

Induction of TB
M. tuberculosis H37Rv was isolated with nutrient broth for 14 days and Acrodisk filter no.4650 was used to filter the broth.The filtered bacillary solution was used for subsequent studies.A Glas-Colaerosol generator was used for preparing an aerosol form of the TB strain which was administered to the rats.Approximately, 500 bacilli were administered to each rat through the aerosol which contained 107 CFU of H37Rv bacilli/5 mL.Five groups of rats were used (10 rats/group): normal control, TB control, 25 mg/kg SCPP11, 50 mg/kg SCPPII, and 100 mg/kg SCPPII.The treatments were given for four weeks.

Colony-forming unit assay
The animals were sacrificed and lung tissue was excised from each rat and used to prepare a tissue homogenate in sterile saline solution.Ogawa slant medium (1%) was used to dilute 100 µL of the lung homogenate.Colonies of H37Rv bacilli were cultured in the same medium at 37 °C for 4 weeks, after which the colonies were counted.

ELISA assay
ELISA kits were used for the determination of concentration of inflammatory cytokines (IL-4, IL-12, TNF-α, iNOS and IFN-γ) in the lung tissue homogenate as per the manufacture of the kit.

Statistical analysis
All data are expressed as mean ± standard deviation (SD).Statistical analysis was performed using one way analysis of variance (ANOVA).Post-hoc comparison of means was carried out by Dunnett's post hoc test (Gradpad Prism 6.1, CA, USA).The level of statistical significance was set at p < 0.05.

Effect of Schisandra chinensis polysaccharides on M. tuberculosis colonies
The effect of Schisandra chinensis polysaccharides on the number of colonies of M. tuberculosis in the lung tissue homogenate of TB-infected rats is shown in Figure 1.The CFU of M. tuberculosis was significantly and dosedependently reduced by SCPP11 treatment, when compared to the untreated TB control (p < 0.01).

Effect of Schisandra chinensis polysaccharides on levels of inflammatory cytokines
The effect of Schisandra chinensis polysaccharides on levels of IL-4, IL-12, TNF-α, iNOS and IFN-γ in the lung tissue homogenate of TB-infected rats is shown in Figure 2.There was significant increase in the levels of IL-4, IL-12 and TNF-α, and significant decreases in IFN-γ level in TB control group, relative to the normal control group.However SCPPII treatment resulted in significant and dose-dependent reductions in IL-4, IL-12, TNF-α levels, while the level of IFN-γ was dose-dependently increased in the lung tissues, when compared to the TB control group.

Effect of Schisandra chinensis polysaccharides on immune cell counts
The effect of Schisandra chinensis polysaccharides on CD4, CD8, CD25, NK, α/β T cells and γ/δ T cellsin lung tissue homogenate of TB-infected rats is shown in Table 1.
It was observed that CD4, CD8, CD25, NK and α/β T cells were significantly enhanced in the lung tissue homogenate of the SCPP11-treated group, when compared to the TB control group of rats (p < 0.05; p < 0.01).However, SCPII had no noticeable effect on the number of γ/δ T cells in lung tissue homogenate of TB-infected rats.

DISCUSSION
The present study evaluated the effect of Schisandra chinensis polysaccharides on TBinfected rats.The TB was induced by administration of colonies of tubercle bacilli through inhalation, prior to a 4-week administration of SCPP11.The effects of  In the present study, it was observed that treatment with SCPP11 attenuated the altered levels of cytokines in the lung tissues of TBinfected rats.Moreover, immune cells were lower in the TB-infected rats, which is consistent with the immune cell profiles of TB patients [19].However, treatment with SCPP11 significantly boosted the populations of CD4, CD8, CD25, NK and α/β T-cells in the lung tissue, relative to the untreated TB control group.

CONCLUSION
The results of the present investigation provide evidence that Schisandra chinensis polysaccharides enhance cellular and humoral immunity, thereby reducing the growth of TB bacilli.Thus, these polysaccharides possess the potential for application in the clinical management of TB.

Figure 1 :
Figure 1: Effect of Schisandra chinensis polysaccharides on CFU of M. tuberculosis in the lung tissue homogenate of TB-infected rats.Values are expressed as mean ± SD (n = 10); **p < 0.01, compared to TB control group

Figure 2 :
Figure 2: Effect of Schisandra chinensis polysaccharides on inflammatory cytokines in the lung tissue homogenate of TB-infected rats.Values are expressed as mean ± SD (n = 10); ## p < 0.01, compared to normal control group; **p < 0.01, compared to TB control group

Table 1 :
Effect of Schisandra chinensis polysaccharides on the levels of immune cells in lung tissue homogenate of TB-infected rats Studies have shown that the expressions of the inflammatory cytokines IL-4, IL-12, TNF-α and IFN-γ are altered in TB [15].It is known that Th1cells mediate TB-associated immunological responses [16].They activate macrophages by secreting IFN-γ, thereby enabling them to destroy invading microorganisms [17].Moreover IL-10 and IL-4 which participate in humoral immunity are secreted by Th2 cells [18].