T-CELL ACTIVATION CONTROLLING EFFECTS OF ETHYL ACETATE FRACTION OF KALANCHOE PINNATA (LMK) PERS ON TMPD-TREATED LUPUS MICE

T-CELL ACTIVATION CONTROLLING EFFECTS OF ETHYL ACETATE FRACTION OF KALANCHOE PINNATA (LMK) PERS ON TMPD-TREATED LUPUS MICE

Niken Indriyanti ^{1, 2}, Joewono Soeroso ³ and Junaidi Khotib^*4

Department of Pharmacology ¹, Faculty of Pharmacy, Mulawarman University, Samarinda, East Kalimantan - 75119, Indonesia.

Faculty of Pharmacy ², Universitas Airlangga, Jl. Dharmawangsa Surabaya, East Java - 60115, Indonesia.

Department of Internal Medicine ³, Faculty of Medicine, Universitas Airlangga, Jl. Prof. Dr. Moestopo Surabaya, East Java - 60115, Indonesia.

Department of Clinical Pharmacy ⁴, Faculty of Pharmacy, Universitas Airlangga, Jl. Dharmawangsa Surabaya, East Java - 60115, Indonesia.

ABSTRACT: T-cell activation, with the low expression of CD62L (L-selectin) marker in the spleen, will increase B cell activity to produce lupus antibodies. Therefore, lupus patients need drugs which could increase the L- selectin expression in the spleen to reduce the auto-reactive T cell. The tested drug to increase the L-selectin in this research was the ethyl acetate fraction of Kalanchoe pinnata (Lmk) Pers leaves (EF-KP), based on its immunosuppressant and anti-inflammatory activities. The research was performed by means of 2, 6, 10, 14 tetramethylpentadecane (TMPD) induced lupus-like mice. The experimental groups consisted of negative control (NC), EF-KP, and positive control (PC) groups. After 21 day treatment, the blood leukocyte was checked. Then, the spleen was prepared for the flow cytometry method to measure CD4⁺CD62L⁺ and CD8⁺CD62L⁺ T cells. The relative percentage of CD4⁺CD62L⁺ T cells in EF-KP groups increased insignificantly (p>0.05) (NC=7.91 ± 4.98%, EF-KP = 8.63 ± 3.07%, PC = 12.62 ± 8.09%). The relative percentage of CD8⁺CD62L⁺ T cells increased significantly (p < 0.05) (NC = 6.20 ± 4.91%, EF-KP = 15.94 ± 9.37%, PC = 24.32 ± 17.47%). Additionally, the total leukocytes reduced significantly (p < 0.05) (NC = 6.333 ± 1.078 cells/mm³, EF-KP = 4.967 ± 635 cells/mm³, PC = 4.367 ± 1.102 cells/mm³). The results show that the active compounds in the EF-KP can mainly control the L-selectin regulatory function of CD8⁺ T cells, and then reduce the inflammation process with the point view of reducing its total leukocytes. At last, the main compounds in the EF-KP could control the T-cell activation in lupus.

T cell activation, Ethyl acetate fraction of Kalanchoe pinnata (Lmk) Pers, L-selectin, Leukocyte, Lupus

INTRODUCTION: T cell and B cell activation induces B cells to increase the production of lupus-specific antibodies ^{1, 2}[1,2].

T cell activation is regulated by L-selectin or CD62L as an activation marker. The L-selectin expressed on naïve CD4 T cells is needed for the recirculation between blood and lymph node. The L-selectin is an adhesion molecule that is able to reduce lymphocytes and bind the endothelial venule ligand. This regulation is important to control the traffic of T lymphocyte circulated to peripheral lymph node ^{3 - 8}[3–8]. The naïve T cells in the spleen binds L-selectin, and then the L-selectin will be shedded in the circulation become the memory T cells ^{9, 10}[9]. The low number of L-selectin in the spleen will increase the lupus severity since the high number of T effector, some of them are auto-reactive, cannot be handled results in inflammation.

This severity can be inhibited by using lupus standard treatment. Lupus treatment by rheumatologists is sufficient to increase the quality of life, but it is not in an optimal outcome ^{11, 12}. Most of the drugs used are off-label nonselective immunosuppressive drugs, such as cyclophosphamide, methotrexate, azathioprine, and mycophenolate mofetil that are very toxic in a long term use ^{13 - 16}[12–15]. Many efforts are performed to obtain new lupus drugs which are effective, safe, and acceptable. Many biotechnology manipulations are performed to find the appropriate treatment ^{14 - 17}[16–19], but it needs high cost. The other effort is to find new lupus drugs from potential medicinal plants based on its immunosuppressive effect. However, medicinal plants have multiple effects which can beneficially use to reduce lupus manifestations. In this research, we try to exploit the multiple effects of Kalanchoe pinnata (Lmk) Pers leaves to increase the L-selectin in the spleen of in TMPD-treated lupus mice.[10,11]

According to our previous study, the aqueous extract of Kalanchoe pinnata (Lmk) Pers leaves can reduce the kidney structural damage of TMPD-treated lupus mice. The main flavonoid compounds (mainly found in the ethyl acetate fraction) of this plant are active as immunosuppressant, anti-inflammatory, and antioxidant ^{18 –21}[20–22]. The flavonoids of Kalanchoe pinnata (Lmk) Pers also inhibit T cell mitogenesis ²²[23]. Besides, it inhibits IL-2 and IL-4 production without cell toxicity. Kalanchoe pinnata (Lmk) Pers is not toxic ²³[24] and also safe for maternity since it reduces the uterine contractility ²⁴[25]. This “miracle plant” is widely used as traditional in Africa and Bangladesh for many purposes ^{25, 26}[26,27].

Based on many benefits of Kalanchoe pinnata (Lmk) Pers leaves for lupus, this research provide the data of the effects of flavonoids in the ethyl acetate fraction of Kalanchoe pinnata (Lmk) Pers (EF-KP) leaves in maintaining the L-selectin number in the spleen of lupus mice. The high number of L-selectin in spleen will control the excessive T cell activation in lupus. It could prevent the spread of circulating L-selectin, and then inhibit the systemic inflammation. The high level of leukocyte as an inflammatory outcome found in lupus model would be reduced if the L-selectin regulation was maintained well. Therefore, this research focused on the effect of flavonoids in the ethyl acetate fraction of Kalanchoe pinnata (Lmk) Pers (EF-KP) on L-selectin number in the spleen of TMPD-treated lupus mice.

MATERIALS AND METHODS:

Materials: The Kalanchoe pinnata (Lmk) Pers fresh leaves were obtained from a cultivation farm at Trenggalek, East Java. The botany identity of this plant was performed by the Conservation Unit of Indonesian Institute of Science, Purwodadi with the identification number of 0284/IPH.06/ HM/II/2015.

The female Balb/c mice (80 mice) aged 4 weeks were received from LPPT Gadjah Mada University, Indonesia. These mice were pathogen free species with the certificate number of 352/LP3HP/ 29/VII/2015. They were housed, randomized, and handled using standard maintenance on the Guide of the Care and Use of Laboratory Animal 8^th edition, published by National Research Council 2011 ²⁷[28].

TMPD >98% purity (Pristane) with the code number of Sigma-P2870 was obtained from Sigma-Aldrich supplier in Singapore. Anti-CD4, anti-CD8, and anti-L-selectin antibodies were obtained from Biogenesis, USA. Turk reagent, PBS, and aquadest were obtained from the laboratory of Faculty of Pharmacy, Universitas Airlangga. The chemicals used in the fractionation, i.e. ethyl acetate pro analysis, were obtained from Merck through PT. Dianum as Indonesian supplier. The chemicals used in the profiling were obtained from Biotechnology Laboratory of PUSPIPTEK Serpong, Indonesia.

Methods: The EF-KP was obtained by using a method used on the previous study. The ethyl acetate fraction quality control test was performed by quantitation of one active marker, rutin (a flavonoid compound), by means of LC-MS/MS system. The female Balb/c mice used were 7 weeks old when treated using 0.5mL TMPD. The administration was intraperitoneal injection.

The induction time was lasted for 6 months ^{28 - 31}[29–32]. At the end of the induction time, the proteinuria semi quantitative data showed a severe lupus manifestation. Then, the 30 mice were divided into 3 experimental groups, i.e. the negative control group that consists of lupus mice that received placebo, the EF-KP group that received the EF-KP at a dose which comparable to 400 mg/kgBB of crude extract, and the positive control group that received cyclophosphamide at a dose of 1 mg/kgBW. The compounds tested were orally administered on the daily treatment until 21 days.

Finally, the mice were sacrificed. The blood was prepared as a sample for total leukocyte count (TLC) assay by means of hemocytometer Neubauer-impruved and inverted microscope Olympus CKX41. The fresh spleen of each mouse was prepared to be spleen cells that were ready for flow cytometry assay. The T regulatory cells (T regs) CD4⁺L-selectin⁺ and CD8⁺L-selectin⁺ were measured by using flow cytometer BD FACS Calibur. Then, the data was analyzed by using BD Cell Quest program.

All of the data was analyzed by facilitating of One way ANOVA by means of SPSS Statistics (version 22). The ethical clearance of this research was approved by local ICUC of Veterinary Medicine Faculty of Universitas Airlangga at January 12, 2016, with the certificate number of 526-KE, then the research performed until April, 2017.

RESULTS:

The EF-KP Quality Control: The aqueous extract of Kalanchoe pinnata (Lmk) Pers was made from the fresh leaves. The extract was lyophilized and then fractionated by using liquid-liquid extraction. The EF-KP profile was measured by using LC/MS /MS tandem system. The result is shown in Fig. 1. According to the profile, the rutin is in the retention time of 2.90. The linearity of the rutin standard used for the rutin calculation in the EF-KP sample is shown in Fig. 2. Based on the standard, the rutin concentration in the EF-KP is in Table 1. Then, the EF-KP was prepared as solution form to be administered in each experimental mouse.

A

B

FIG. 1: THE EF-KP CHROMATOGRAM AND THE POSITION OF RUTIN MARKER IN EF-KP (A) AND RUTIN STANDARD (B)

FIG. 2: THE STANDARD CURVE OF RUTIN STANDARD

TABLE 1: CALCULATION OF RUTIN MARKER IN THE EF-KP SAMPLE

The Analysis of L-selectin and the Related Markers: CD62L or L-selectin is a T regulatory marker. The flow cytometry analysis was performed simultaneously on CD4, CD8, and CD62L markers. The relative percentages of CD4⁺L-selectin⁺ and CD8⁺ L-selectin⁺ T cells were calculated. The assessment of CD4:CD8 ratio was done by using the flow cytometry assay. The results are shown in Table 2 and Fig.  3 - 5.

TABLE 2: THE RATIO OF CD4⁺: CD8⁺ T CELLS

(a)                     (b)                   (c)

FIG. 3: THE RELATIVE PERCENTAGE OF CD4⁺CD8⁺ T CELLS AS A RESULT OF FLOW CYTOMETRY ANALYSIS OF THE FRESH SPLEEN OF THE THREE EXPERIMENTAL GROUPS

(a), EF-KP group (b), and positive control group (c) (n = 10 mice per group)

Then, the calculation result of the relative percentage of CD4⁺ L-selectin ⁺ T cells on each group is shown in Fig. 4. This result shows the increase of the percentage of CD4⁺L-selectin⁺ after 21 day administration of the EF-KP. This increase is not significant, but it indicates an inhibition of naïve T-cell differentiation do not become the T cells effector. The flow cytometry analysis was continued to CD8⁺L-selectin ⁺ T-cell parameter. The profile is shown in Fig. 5.

(a)                      (b)                   (c)

FIG. 4: THE RELATIVE PERCENTAGE PROFILE OF CD4⁺ L-SELECTIN ⁺ T CELLS AS A RESULT OF FLOW CYTOMETRY ANALYSIS OF THE FRESH SPLEEN OF NEGATIVE CONTROL GROUP

(a) EF-KP group (b) and positive control group (c) (n = 10 mice per group)

(a)                  (b)                    (c)

FIG. 5: THE RELATIVE PERCENTAGE OF CD8⁺L-SELECTIN⁺ T CELLS AS A RESULT OF FLOW CYTOMETRY ANALYSIS IN THE SPLEEN CELLS IN CONTROL NEGATIVE GROUP

(a) EF-KP group (b), and positive control group (c) (n = 10 mice per group) *significantly different (p < 0.05) to negative and positive control groups

The CD8⁺L-selectin⁺ T cells are activation markers of CD8⁺ T cells (T cytotoxic). The significant elevation of CD8⁺L-selectin⁺ in spleen shows a reduction of circulated T cell cytotoxic. It prevents attachments of auto-antigens and non-self recognition.

The Total Leukocyte Count (TLC) Result: The TLC assay was performed after 2 week treatment. The blood was taken from the tail vein. The mice were kept alive during this procedure for the next treatment. The results are shown in Fig. 6.

FIG. 6: THE TOTAL LEUKOCYTES OF THREE EXPERIMENTAL GROUPS. (n=3 MICE PER GROUP)

* Significantly different to the negative control group

This result shows a significant leukocytes reduction after the treatment. It indicates an anti-inflammatory effect and it is caused by the L-selectin regulation.

DISCUSSION: The EF-KP contains multiple compounds which are active as anti-inflammatory, immunosuppressant, and antioxidant. One of the active compounds quantitated for the quality control in this research was rutin, a flavonoid compound. According to the result on Fig. 1, the rutin compound was found in the retention time of 2.90. Based on a series concentration of rutin standard, the rutin measured in the EF-KP sample is 13.4mg/kg (Table 1). The limitation of the tandem system used was the precision of the molecular weight in the low retention time. It was resulted from the instrument validation which used a standard with the molecular weight of 500 Da. However, the limitation could be handled by comparing the spectrum with other previous publications about the active compounds of Kalanchoe pinnata (Lmk) Pers. Rutin, as one compound of flavonoids in this plant has also identified in other research ^{18, 32, 33}[22,33].

After the treatment finished, the spleen of each mouse was isolated for the L-selectin⁺ T cells assay. CD62L or L-selectin is one of the T regulatory markers. Ermann ³⁴[33] states that T regs CD4⁺CD25⁺ with the co-transfer of L-selectin⁺ is able to prevent tissue damage under the rejection of the immune system. This biomarker could be observed simultaneously by using flow cytometry method on the specific marker of CD4⁺CD8⁺L-selectin⁺. Normally, the mice have the ratio of CD4+ T cells two times higher than CD8 T cells.

In this research, the ratio of CD4:CD8 is shown in Table 2. The results show a mild reduction of the ratio in EF-KP group. Although it did not achieve the normal condition, this reduction was a good sign of the effectiveness of the active compounds in EF-KP as a complementary drug for lupus patients. The effective drugs that could balance the ratio of CD4:CD8 have not been found ^{34 - 36}[34–36]. The balance of the ratio could reduce the auto-reactive cells and then keep the tissue and organ normal. The positive control group showed an opposite effect. The ratio increased higher than the negative control group. It was assumed that the cyclophosphamide used in the positive control group had a toxic effect. It could be a consideration in the cyclophosphamide treatment. For further investigation, we observed the L-selectin⁺ marker.

L-selectin⁺ of T cells, a type 1 membrane protein, regulates lymphocyte homing into peripheral lymph node and accumulates leukocytes in inflammation area ^{3, 8, 37}. This molecule is expressed by naïve T cells CD4⁺ L-selectin⁺ and CD8⁺ L-selectin⁺. The naïve T cells circulate in blood, spleen, and peripheral lymph node and then it is attached by antigen. As a result, the naïve T cells are activated when L-selectin⁺ is shedded. The L-selectin⁺ T cells are expressed more than 80% in the normal condition. On the unhealthy condition, the expression decreases and then the T cells are activated ^{37, 38}.

This research results in the relative percentages of CD4⁺L-selectin⁺ which are shown in Table 2. The EF-KP treatment increases the relative percentage of CD4⁺CD25⁺. The value is about 1% but the statistic calculation results a non-significant value. It suggests that EF-KP is able to inhibit the differentiation of naïve T cells to be the T-cell effector. The role of L-selectin⁺ is as T-cell activation marker. CD4⁺ L-selectin⁺ T cells would be activated as CD4 T cells. The T-cell activation is avoided in autoimmune condition ^{39, 40}[38,39]. The auto-reactive T cells can attach the normal cells and tissues because of a wrong recognition of self-autoantigen. The reduction of the CD4⁺ T-cell activation reduces the immune complex formation. Auto-antigen that could not be eliminated by CD4 T cells will be attached by CD8 T cells.

The flow cytometry analysis results in the increase of the relative percentage of CD8⁺L-selectin⁺ T cells (Table 3). The relative percentage of CD8⁺L-selectin⁺ in the EF-KP group was 15.91%. It was more than two times higher than the negative control group. This increase is statistically significant. The high percentage of the EF-KP group and the positive control group shows the reduction of CD8⁺L-selectin⁺ T-cell differentiation. Thus, the CD8 T cell effector was reduced. This reduction is beneficial since the low number of CD8 has a direct impact on the reduction of CD8 T-cell reactivity ^{37, 41}[40,41].

The low reactivity of CD8 T cell reduces the number of damage normal cells that are recognized as a non-self.

Total Leukocyte Count (TLC): The TLC assay was performed to prove the regulation of L-selectin⁺ that binds the naïve T cells that stayed in the lymphoid organs. After 2 week treatment, we took the mice blood from the tail veins. The results in Table 4 show a significant reduction of leukocytes in the group that received EF-KP and cyclophosphamide. The reduction of the total leukocyte is associated with the reduction of an inflammation. It is consistent with anti-inflammatory effect of EF-KP reported by Ferreira ¹⁹[21]. This effect is benefit for lupus condition. The adhesion molecule L-selectin⁺ might play a role in this effect. However, further research is necessary to prove this suggestion.

CONCLUSION: The active compounds of EF-KP increase the L-selectin⁺ T cells mainly CD8⁺L-selectin⁺ T cells in the spleen cause the inhibition of T-cell activation in the lupus model.

ACKNOWLEDGEMENT: This research was funded by DIPA PNBP 2015 Faculty of Pharmacy Universitas Mulawarman (Grant number 1787/ UN17.12/HK/2015), Hibah Penelitian Disertasi Doktor 2017 (Grant number: 025/E3.4/2017), and BPPDN Scholarship for Indonesian doctorate student (Grant number: 906.8/E4.4/2015).

CONFLICT OF INTEREST: We declare no conflict of interest.

REFERENCES:

1. Moulton VR, Suarez-fueyo A, Meidan E, Li H, Mizui M and Tsokos GC: Pathogenesis of Human Systemic Lupus Erythematosus : A Cellular Perspective. Trends in Molecular Medicine 2017; 23(7): 615–35.
2. Singh N, Kumar B, Aluri V and Lenert P: Interfering with baffled B cells at the lupus tollway: Promises, successes, and failed expectations. Journal of Allergy and Clinical Immunology 2015; 137(5): 1325–33.
3. Mohammed RN, Watson HA, Vigar M, Ohme J, Thomson A, Humphreys IR et al.: L-selectin is Essential for Delivery of Activated CD8+T Cells to Virus-Infected Organs for Protective Immunity. Cell Reports 2016; 14(4): 760–71.
4. Manrique SZ, Correa MAD, Hoelzinger DB, Dominguez AL, Mirza N, Lin H-H et al.: Foxp3-positive macrophages display immunosuppressive properties and promote tumor growth. Journal of Experimental Medicine 2011; 208(7): 1485–99.
5. Domínguez-Luis M, Herrera-García A, Arce-Franco M, Armas-González E, Rodríguez-Pardo M, Lorenzo-Díaz F, et al.: Superoxide anion mediates the L-selectin down-regulation induced by non-steroidal anti-inflammatory drugs in human neutrophils. Biochemistry and Pharmacology 2013; 85(2): 245–56.
6. Bhavsar SK, Merches K, Bobbala D and Lang F: Biochemical and Biophysical Research Communications AKT / SGK-sensitive phosphorylation of GSK3 in the regulation of L-selectin and perforin expression as well as activation induced cell death of T-lymphocytes. Biochemical and Biophysical Research Communication 2012; 425(1): 6–12.
7. El-Samahy MH, Adly AAM, Ismail EA and Salah NY: Regulatory T cells with CD62L or TNFR2 expression in young type 1 diabetic patients: Relation to inflammation, glycemic control and micro-vascular complications. Journal of Diabetes and its Complications 2015; 29(1): 120–6.
8. Marilyn JT: Cellular adhesion and the Endothelium, E-Selectin, L-Selectin, and Pan-Selectin Inhibitors. Hematology and Oncology Clinics of North America 2014; 28: 341-354.
9. Rottman JB and Willis CR: Mouse models of systemic lupus erythematosus reveal a complex pathogenesis. Veterinary Pathology 2010; 47(4): 664–76.
10. Sang A, Zheng YY and Morel L: Contributions of B cells to lupus pathogenesis. Molecular Immunology 2014; 62(2): 329–38.
11. Marceau K, Ruttle PL, Shirtcliff EA, Essex MJ, Susman EJ, Studies A et al.: T cells as a therapeutic target in SLE. Lupus 2015; 24(0): 351–63.
12. Kasjmir Y, Handono K, Wijaya LK, Hamijoyo L, Albar Z, Kalim H et al.: Rekomendasi Perhimpunan Reumatologi Indonesia untuk Diagnosis dan Pengelolaan Lupus Eritematosus Sistemik 2011; 1-54.
13. Ziemer M and Milkova L: Lupus erythematosus Part II : Clinical picture, diagnosis and treatment. Deutsche Dermatologische Gesellschaft 2014; 1204: 285–302.
14. Fiechtner JJ and Montroy T: Treatment of moderately to severely active systemic lupus erythematosus with adrenocorticotropic hormone: a single-site, open-label trial. Lupus 2014; 23(9): 905–12.
15. Thanou A and Merril JT: Treatment of systemic lupus erythematosus: new therapeutics avenues and blind alleys. Nature Reviews Rheumatology 2014; 10: 23-34.
16. Zampeli E, Klinman DM, Gershwin ME and Moutsopoulos HM: A comprehensive evaluation for the treatment of lupus nephritis. Journal of Autoimmunity 2017; http://dx.doi.org/10.1016/j.jaut.2016.12.011.
17. Hahn BH, McMahon MA, Wilkinson A, Wallace WD, Daikh DI, Fitzgerald JD et al.: American College of Rheumatology guidelines for screening, treatment, and management of lupus nephritis. Arthritis Care Research 2012; 64(6): 797–808.
18. Bogucka-kocka A and Zidorn C: Phenolic acid content, antioxidant and cytotoxic activities of four Kalanchoe species. Saudi Journal of Biological Sciences 2016; http://dx.doi.org/10.1016/j.sjbs.2016.01.037.
19. Ferreira RT, Coutinho MAS, Malvar DDC, Costa EA, Florentino IF, Costa SS et al.: Mechanisms underlying the antinociceptive, antiedematogenic, and anti-inflammatory activity of the main flavonoid from Kalanchoe pinnata. Evidence-based Complement of Alternative Medicine 2014; dx.doi.org/10.1155/2014/429256.
20. Julius AI, Charles A, Samuel NE, Ansong AD, Mahmood S, Samuel A et al.: Nutraceutical with Anti-Inflammatory Activity for the Management of Airway Remodeling in Bronchial Asthma: Kalanchoe integra Crenata (Andr.) Cuf Leaf Extract. Pharmacology and Pharmacy 2014; 5: 250–61.
21. Sari Y, Wahyuni R, Maya A, Syukur NA and Dewi ES: Immunomodulation Effects of Bryophyllum pinnatum on Pregnant Pristane-Induced Lupus Mice Model. Research Journal of Life Science 2016; 3(3): 190–200.
22. Coutinho MA, Muzitano MF, Cruz EA, Bergonzi MC, Kaiser CR, Tinoco LW, Bilia AR, Vincieris FF, Rossi-Bergmann B and Costa SS: Flowers from Kalanchoe pinnata are a rich source of T cell-suppressive flavonoids. Natural Product Communications 7(2): 175-8.
23. Ozolua, RI, Idogun SE and Tamafel GE: Acute and Sub-Acute Toxicological Assessment of Aqueous Leaf Extract of Bryophyllum pinnatum (Lam.) in Sprague-Dawley Rats. American Journal of Pharmacology and Toxicology 2010; 5(3): 145-151.
24. Gwehenberger B, Rist L, Huch R and Von Mandach U: Effect of Bryophyllum pinnatum versus fenoterol on uterine contractility. European Journal Obstetry and Gynecology and Reproduction Biology 2004; 113(2): 164–71.
25. Nascimento LBDS, Moreira NDS, Leal-Costa MV, Costa SS and Tavares ES: Induction of wound-periderm-like tissue in Kalanchoe pinnata (Lam.) Pers. (Crassulaceae) leaves as a defence response to high UV-B radiation levels. Annals of Botany 2015; 116(5): 763–9.
26. Furer K, Simoes-Wust AP, Von Mandach U, Hamburger M and Potterat O: Bryophyllum pinnatum and related species used in anthroposophic medicine: Constituents, Pharmacological Activities, and Clinical Efficacy. Planta Medica 2016; 82(11–12): 930–41.
27. NRC: Guide of the Care and Use of Laboratory Animal 2011; 46: 267-268.
28. Indriyanti N, Soeroso J and Khotib J: The immunosuppressant effect comparation between ethyl acetate and n-butanol fractions of Kalanchoe pinnata (Lmk) Pers in 2, 6, 10, 14-tetramethylpentadecane. Journal of Tropical Pharmacy and Chemistry 2017; 4(1): 7-13.
29. Leiss H, Niederreiter B, Bandur T, Schwarzecker B, Blüml S, Steiner G et al.: Pristane-induced lupus as a model of human lupus arthritis: evolvement of autoantibodies, internal organ and joint inflammation. Lupus 2013; 22(8): 778–92
30. Lin Y, Yan Y, Zhang H, Chen Y, He Y, Wang S et al.: Salvianolic acid A alleviates renal injury in systemic lupus erythematosus induced by pristane in BALB/c mice. Acta Pharmaceutica Sinica B 2016; 7(2): 159–66.
31. Zhu W, Xu J, Jiang C, Wang B, Geng M, Wu X et al.: Pristane induces autophagy in macrophages, promoting a STAT1-IRF1-TLR3 pathway and arthritis. Clinical Immunology 2017; 175: 56–68.
32. Nascimento LBDS, Leal-Costa MV, Menezes EA, Lopes VR, Muzitano MF, Costa SS et al.: Ultraviolet-B radiation effects on phenolic profile and flavonoid content of Kalanchoe pinnata. Journal of Photochemistry and Photobiology 2015; 148: 73–81.
33. Milad R, El-ahmady S and Singab AN: Genus Kalanchoe (Crassulaceae): A Review of Its Ethnomedicinal, Botanical, Chemical and Pharmacological Properties. European Journal of Medicinal Plants 2014; 4(1): 86–104.
34. Ermann J, Hoffmann P, Edinger M, Dutt S, Blankenberg FG, Higgins JP et al.: Only the CD62L⁺ subpopulation of CD4 ⁺CD25 + regulatory T cells protects from lethal acute GVHD. Blood 2005; 105(5): 2220–6.
35. Matta MC, Soares DC, Kerstenetzky MS, Freitas ACP, Kim CA and Torres LC: CD4⁺CD25highFoxp3⁺ Treg deficiency in a Brazilian patient with Gaucher disease and lupus nephritis. Human Immunology 2016; 77(2): 196–200.
36. Maldonado A, Mueller YM, Thomas P, Bojczuk P, O’Connors C and Katsikis PD: Decreased effector memory CD45RA⁺ CD62L^- CD8⁺ T cells and increased central memory CD45RA^- CD62L⁺ CD8⁺ T cells in peripheral blood of rheumatoid arthritis patients. Arthritis Research and Therapy 2003; 5(2): 91–6.
37. Hirata T, Usui T, Kobayashi S and Mimori T: A novel splice variant of human L-selectin encodes a soluble molecule that is elevated in serum of patients with rheumatic diseases. Biochemical Biophysical Research Communication 2015; 462(4): 371–7.
38. Bengtsson A, Pettersson Å, Wichert S, Gullstrand B, Hansson M, Hellmark T et al.: Low production of reactive oxygen species in granulocytes is associated with organ damage in systemic lupus erythematosus. Arthritis Research and Therapy 2014; 16(3): R120.
39. Lu MC, Lai NS, Chen HC, Yu HC, Huang KY, Tung CH et al.: Decreased microRNA(miR)-145 and increased miR-224 expression in T cells from patients with systemic lupus erythematosus involved in lupus immune pathogenesis. Clinical and Experimental Immunology 2013; 171(1): 91–9.
40. Zhang Y, Zhao M, Sawalha AH, Richardson B and Lu Q: Impaired DNA methylation and its mechanisms in CD4+T cells of systemic lupus erythematosus. Journal of Autoimmunity 2013; 41: 92–9.
41. Shah D, Sah S and Nath SK: Interaction between glutathione and apoptosis in systemic lupus erythematosus. Autoimmunity Reviews 2013; 12(7): 741–51.
    

    ---

    How to cite this article:

    Indriyanti N, Soeroso J and Khotib J: T-cell activation controlling effects of ethyl acetate fraction of Kalanchoe pinnata (lmk) pers on tmpd-treated lupus mice. Int J Pharm Sci Res 2018; 9(2): 475-82.doi: 10.13040/IJPSR.0975-8232.9(2).475-82.

    ---

    All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

    ---