In global summits, World Health Organization (WHO) encourages evidence-based use of traditional medicine such as Birch bark, with long history of safe human use especially to treat pain and wounds by Native Indians as well as by other cultures. In that context, a double-blind, randomized, vehicle-controlled, phase III study found that topical application of Oleogel S10 containing Birch bark (Betula alba) triterpenes accelerated the chronic skin wound healing of human subjects significantly more than vehicle treated group. Last year, European regulatory agencies granted the approval to Oleogel S10 for clinical use (ref.1). The active ingredients in Birch bark are triterpenes such as Betulinic acid (Fig.1), which has been well characterized and selected as a topical ointment against melanoma (ALS 357) in a Phase I clinical study (NCT00701987). These clinical results on Betulinic acid as a topical drug for wound healing provide valuable insight for developing drugs to heal lesions on bladder lining of Interstitial Cystitis/ Bladder Pain Syndrome (IC/BPS) patients. Prior basic research has shown that Betulinic acid induces apoptosis of immune cells by altering mitochondrial membrane potential. Since Betulinic acid is structurally similar to cholesterol- metabolized in mitochondria- we hypothesized that Betulinic acid exerts its anti-inflammatory effect by targeting the mitochondrial metabolism and metabolic reprogramming of immune cells. To test our hypothesis, we covalently bonded Betulinic acid with a fluorescent probe to study the differences in mitochondrial uptake between fast-replicating immune cells and slow replicating urothelium cells by confocal microscopy.

Cell culture:  Jurkat T immune cells  and non-cancerous,  human urothelial cells (TRT-HU1) were procured from ATCC.  Jurkat T cells were cultured in RPMI-1640 media (Sigma Aldrich) supplemented with 10% Fetal Bovine Serum (FBS) (Life Technologies) with 1% antibiotic solution of Penicillin, Streptomycin and Amphotericin B, followed by maintenance at 37ºC in a humidified atmosphere of 5% CO2 . TRT-HU1 cells were maintained in Keratinocyte Serum Free Medium (Gibco). Cells were grown and passaged on T25 flasks until used for experiments.  Cells were used within 5 passages after initial thawing. 
The covalent bonding of Betulinic acid with fluorescein was confirmed by mass spectrometry. 
Confocal Studies: Synthesized Betulinic acid conjugated to Fluorescein [15micromolar] was exposed to TRT-HU1 and Jurkat T cells for 4 hrs. Exposed cells were washed once with phosphate buffered saline (PBS) before brief 15 minutes treatment with 25nM Mito tracker Red (Invitrogen). The cells were again washed with PBS and examined under Confocal Microscope (Olympus Fluoview FV1000) for acquiring Z-stack images at 60x magnification with Laser HV 454v, 2x Gain and 13% Offset both for green and red fluorescence. Pearson correlation coefficient was determined for the colocalization of green and red fluorescence and signficant differences between immune and urothelium cells were determined by unpaired Student' t test. The mitochondrial action of Betulinic acid was probed further by studying the impact on gene expression of p53, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and phosphatidylinositol 3-kinase (PI3K) relative to beta actin by real-time PCR.

Mass spectra of synthesized compounds confirmed that successful conjugation of Betulinic acid with Fluorescein doubles the molecular weight of free Betulinic acid from 456.7 Daltons to 903 Daltons (Fig.1).  Confocal microscopy not only confirmed that fast-replicating  immune cells  are of smaller size of relative to slow replicating urothelium cells but also that the emission of green fluorescence signifying the internalization of conjugated Betulinic acid was more pronounced in immune cells than in urothelium cells. Since red fluorescence emitted by Mitotracker red displays sub-cellular structure of mitochondria, a significantly higher  Pearson correlation coefficient of 0.86 for the colocalization of green and red fluorescence in immune cells indexes higher co-localization of green Betulinic acid in mitochondria immune cells than in urothelium cell with coefficient of 0.65,  p<0.01(Fig.2). A significantly higher co-localization of green and red fluorescence supports higher accumulation of Betulinic acid in immune cells than in urothelium which is associated with altered gene expression (Fig.3) hallmarked by  signficant upregulation of p53 gene relative to beta-actin in immune cels occuring in conjunction with a 10 to 20 fold downregulation of PI3K and GAPDH genes.

Confocal microscopy supports our hypothesis on significant differences in the mitochondrial entry of Betulinic acid between fast-replicating, small immune cells and slow replicating and larger urothelium cells. The selective accumulation of Betulinic acid into mitochondria of immune cells supports the role of metabolic reprogramming in anti-inflammatory effect of Betulinic acid which is hallmarked by the downregulation of GAPDH gene and PI3K in conjunction with the promotion of apoptosis with p53 gene upregulation. Betulinic acid mediated upregulation of p53 is consistent with the reports of mitochondrial membrane potential changes, production of reactive oxygen species, and the opening of mitochondrial permeability transition pores, that results in the release of mitochondrial apogenic factors, activation of caspases, and DNA fragmentation (ref-2). The mitochondrial accumulation of fluorescent Betulinic acid in immune cells relative to urothelium implicates the preferential uptake of Betulinic acid/cholesterol to generate metabolic energy for cell proliferation in nutrient-poor environments at inflammatory foci (ref.1). Nutrient scarcity is inevitable with the nutrient consumption neccessary to fuel a faster cell division, and nutrient scarcity triggers metabolic signals of pseudo-starvation which in turn induces epigenetic changes to re-route other metabolic pathways or metabolic reprogramming to fuel growth with alternative nutrients such as Betulinic acid. The nutrient scarcity experienced by immune cells infiltrating into inflammatory foci on bladder mucosa can be inferred from venous congestion noted on bladder biopsy specimens of IC/BPS patients, a hallmark of constrained supply of nutrients and oxygen in tissue.

These findings fulfill the WHO mandate for research into traditional medicine of Birch bark with scientific rigor.  The skin wound healing associated with recurrent topical administration of Birch Bark derivatives in recent Phase III study(ref.1) could be partly contributed by the anti-inflammatory effect of Betulinic acid on mitochondria of immune cells. Significantly higher 
co-localization of green fluorescence emitted by fluorescein conjugated to Betulinic acid with the red fluorescence of Mitotracker red in fast replicating immune cells than the slow-replicating urothelium cells substantiate the role of altered gene expression of p53, GAPDH and PI3K in anti-inflammatory effect of Betulinic acid. Findings imply that self-assembling attribute of Betulinic acid into nanoparticles can be capitalized to deliver anti-inflammatory steroids directly to immune cells of urothelium for healing lesions in bladder lining via intravesical administration to IC/BPS instead of cystoscopic guided injection of steroids into lesion (ref.3).

Kern, J. S., Sprecher, E., Fernandez, M. F., Schauer, F., Bodemer, C., Cunningham, T., Lowe, S., Davis, C., Sumeray, M., Bruckner, A. L., Murrell, D. F., investigators, E.: Efficacy and safety of Oleogel-S10 (birch triterpenes) for epidermolysis bullosa: results from the phase III randomized double-blind phase of the EASE study. Br J Dermatol, 188: 12, 2023, 36689495
Ganguly, A., Daugherty, S., Healy, K., Beckel, J., Kanai, A., Tyagi, P.: Anti-inflammatory effect of Betulinic Acid Is Dependent on Metabolic Reprogramming of Immune Cells. J. Urol, 211, 2024
Jiang T, Zhou X, Chen Z, Xiong T, Fu J, Liu Z, Yan D, Zhou Z, Shen W. Clinical efficacy of submucosal injection of triamcinolone acetonide in the treatment of type II/III interstitial cystitis/bladder pain syndrome.  BMC Urol. 2020 Mar 30;20(1):36. doi: 10.1186/s12894-020-00597-3.