Investigation on the action mechanisms of · Bone morphogenetic protein 1 BMP2 NM_001200 Y Y Colony stimulating factor 1 CSF1 NM_000757 XX X Chemokine (C-C motif) receptor 5 CCR5

Cartagena - 2 sept 2017

Investigation on the action mechanisms of

Arnica montana at Cell Level

Paolo Bellavite

Former Professor of General Pathology, School of Medicine,

University of Verona, Italy, Professor of General Pathology at

University of Ngozi, Burundi

© P. Bellavite, Università di Verona

Cartagena, 31 Oct-2 Sept 2017


The research ways: Evidence and Plausibility

Homeopathy

acceptation

and

progress

CLINICAL

RESEARCH

«EVIDENCE»

BASIC

RESEARCH

«PLAUSIBILITY»


Does

it work?

HOW

does

it work?




INTRODUCTION




Caption: Dipartimento di Scienze della Vita, Università di Trieste -

Arnica montana L. (Asteraceae)



Arnica montana L. (Asteraceae)


Arnica montana (Arnica m.), native to the temperate region of Europe, is

an herbaceous perennial plant that belongs to the Asteraceae family

and is widely distributed in mountainous area.

This is one of the most popular remedies traditionally used in

complementary medicine to treat several pathological conditions like

contusion, swelling associated with trauma, pain, inflammation, wounds

and post-operative clinical conditions, but the evidence of clinical

efficacy is debated.

There is some experimental evidence in laboratory animals of anti-

inflammatory action of Arnica m. administered as crude ethanolic extract

or at the homeopathic dilutions.


Composition

The chemical composition of

Arnica m. depends on the

part of the plant that is used,

principally the flowers and

roots, and in general the most

pharmacologically active

compounds are sesquiterpene

lactones, thymol derivatives,

flavonoids, acid

polysaccharides and their

glycoconjugates



Ankle sprains treated with Arnica gel

https://commons.wikimedia.org/wiki/ ( F. Ceragioli)


Widely used in complementary

medicine to treat various

pathological conditions such as

contusion,

swelling associated with trauma,

pain,

inflammation,

postoperative clinical conditions

http://www.google.it/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0CAQQjRw&url=http://ladanzadeisensi.blogspot.com/2013/02/arnica-e-calendula.html&ei=PoZPVKbgOsiBPafEgZgN&bvm=bv.77880786,d.ZWU&psig=AFQjCNGNIevKZ-DGtb_6DIe8p4rvc8LXJQ&ust=1414584255001598

Cartagena - 2 sept 2017 2

Perioperative Arnica montana for reduction of ecchymosis in rhinoplasty surgery

Arnica m. 1M is given preoperatively on the morning of surgery and 2 more later that day

after surgery, Arnica m. 12C is given orally 3 times daily for the next 3 days

Scott and Benjamin, Ann. Plast. Surg. 76(5):477-482, May 2016.




Recent Italian study (2017) - total mastectomy




Aim: This study aimed to evaluate the benefits of Arnica montana on

post-operative blood loss and seroma production in women

undergoing unilateral total mastectomy.

Materials and Methods: 53 women were randomly assigned to

Arnica 1000 Korsakovian dilution (1000 K)or placebo and were

followed up for 5 days. The main end point was the reduction in blood

and serum volumes collected in drainages.

Results: The per-protocol analysis revealed a lower mean volume of

blood and serum collected in drainages with Arnica (−94.40 ml; 95%

confidence interval [CI]: 22.48-211.28; P = 0.11).

Volumes collected on the day of surgery and the following days were

significantly lower with Arnica at days 2 (P = 0.033) and 3 (P =

0.0223).

Conclusions: Arnica 1000 K could reduce post-operative blood and

seroma collection in women undergoing unilateral total mastectomy.

Larger studies are needed with different dilutions to further validate

these data.




-


Cartagena - 2 sept 2017 © P. Bellavite, Università di Verona

A RECENT REVIEW ON CLINICAL EFFECTIVENESS

with my collaboration



Some clinical studies on Arnica m. Endpoints: Pain and/or swelling and/or bleeding

Effective

Osteoarthritis (topical

application, = to ibuprofen)

Ankle sprain (topical)

Cruciate ligand surgery (30x)

Hallus valgus surgery (4x,

swelling, = to diclofenac)

Rhinoplasty, rhytidectomy (low

dose complex)

Rhynoplasty (1M, 12c)

Tonsillectomy (30c)

Mastectomy (1000K) (Per

protocol)

Not effective

Carpal tunnel syndrome (30c)

Wisdom teeth removal (200c)

Arthroscopy (30x)

Hallus valgus surgery (4x,

pain)

Venous surgery (5c)

Aortic valve surgery (5c)

Mastectomy (1000K) (ITT)



Composition

One of the major

components of Arnica m.

with acknowledged biological

activity is the sesquiterpene

lactone helenalin, known for

its antiinflammatory

properties.

In a lymphoid cellular model,

helenalin was found to inhibit

the transcription factor NF-

kappaB e a central mediator

of human immune response




Despite the action of Arnica m. on the wound healing process is acknowledged as a promising

therapeutic property of this plant, the current knowledge of the effects of Arnica m. in laboratory models

of this process is poor.

Wound healing involves different cell types, such as fibroblasts, leukocytes, monocytes/macrophages as

well as endothelial and epidermal cells which cooperate to restore the damaged tissue through

hemostasis, inflammation, angiogenesis and remodelling of the new tissue.

We formulated the hypothesis that one of cellular targets of Arnica m. action is the macrophage, a key

cell in all phases of inflammation and wound healing and we decided to evaluate through an in vitro

model the action of several dilutions of this plant extract on gene expression of key regulatory factors.

We used THP-1 human cell line, that is a widely used cell line to explore the function and regulation of

monocytes and macrophages. These cells resemble primary monocytes but when treated with low doses

of phorbol esters (PMA) differentiate to cells with morphologic and functional features of tissue

macrophages, including adhesion, phagocytosis, free radical production and release of a number of

inflammatory cytokines.

Arnica m. dilutions were tested both on normal PMA-differentiated THP-1 cells (here defined as “Normal

Mac”) and on PMA-differentiated THP-1 cells that were further differentiated by IL4 treatment for 24

hours (here defined as “IL4 Mac”). This T(H)2-type cytokine stimulates an alternative pathway of

macrophage activation with a distinctive phenotype that is consistent with a role in humoral immunity and

wound healing.

As suitable molecular markers of drug activity at cellular level, we have analysed through RT-array PCR

the gene expression of a series of cytokines, receptors, and transcription factors that are involved in

wound healing cellular process. We then evaluated the whole transcriptoe of macrophages exposed to

Arnica m.



Phases of wound healing

Hemostasis

Inflammation

Regeneration

Stop bleeding

Inflammatory

mediators

Chemotaxis

Bacterial killing,

debris removal

Endothelial/epithelial

proliferation

Collagen production,

scarring, maturation

Coagulation, Platelets.

Fibrinopeptides

Cytokines, Neutrophils,

Macrophages, Fibronectin

Microvessels, Fibroblasts,

Collagen, Keratinocytes



Central role of the Macrophage in wound healing

Hemostasis

Inflammation

Regeneration

Stop bleeding

Inflammatory

mediators

Chemotaxis

Bacterial killing,

debris removal

Endothelial/epithelial

proliferation

Collagen production,

scarring, maturation

Coagulation, Platelets.

Fibrinopeptides

Cytokines, Neutrophils,

Macrophages, Fibronectin

Microvessels, Fibroblasts,

Collagen, Keratinocytes





METHODS (Essentials)




THP-1 human macrophages cell line

Macrophages Normal THP-1

(monocyte)


Phorbol

myristate

acetate


Key properties and functions of polarized macrophages.

July 15, 2006; Blood: 108 (2) IMMUNOBIOLOGY. A. Mantovani

M1 Macrophage M2 Macrophage


Bacteria, LPS

Interferon-

Interleukin-4

Interleukin-10


Differential gene expression analysis

with high throughput technologies


Phenotype

Metabolic activity

Environmental

inputs

and

Drugs

• Small changes in the gene

regulation networks have a

relevant impact in the cell

Differential gene expression

Genes

Conditions

Expr. level

RNAs

Quantification of the RNA

expression levels (RT-array,

microarray, RNA-seq)

Compare to control

High sensitivity and many

replicates


Macrophages treatment with Arnica m. and gene expression

(RT-PCR test)


Seeding of

THP-1

cells

PMA

addition

IL-4 addition

RNA

extraction

(24h)

Day2 Day3 Day1

c-DNA

synthesis

Real-time PCR

RT-array

Day4

Arnica 2c, 3c,

5c, 9c, 15c or

placebo

2h or

24h

Normal Mac

IL-4 Mac

Whole transcriptome

sequencing


Special features of RT-Array real-time PCR (custom)

CCL1 CCL2 CCL3 CCL4 CCL5

CCL7 CCL17 CCL22 CCL23 CXCL1

CXCL2 CXCL13

IL1B IL1RN IL8 IL10 TNF

VEGFA

BMP2 CSF1 CCR5 F13A1

MRC1 MMP1

RELA NFKB1 NFKBIA IKBKB

RPLPO (reference gene)






RESULTS: • Real-Time PCR (RT-array)

• RNA-Sequencing of whole transcriptome

• Fibronectin assay and functions






RESULTS:

• Real-Time PCR (RT-array) • RNA-Sequencing of whole transcriptome





Gene NAMES Gene SYMBOL

Gene CODE Change after IL4 (2h)*

Change after IL4 (24h)*

Chemokine (C-C motif) ligand 1 CCL1 NM_002981 ↑ ↑↑ Chemokine (C-C motif) ligand 2; MCP-1 CCL2 NM_002982 ↑↑ ↑↑↑ Chemokine (C-C motif) ligand 3; MIP-1a CCL3 NM_002983 ↑↑ ↑ Chemokine (C-C motif) ligand 4; MIP-1B CCL4 NM_002984 ↑↑ ↑ Chemokine (C-C motif) ligand 5; RANTES CCL5 NM_002985 ↑↑ ↑↑↑ Chemokine (C-C motif) ligand 7; MCP-3 CCL7 NM_006273 ↑ ↑↑↑ Chemokine (C-C motif) ligand 17; TARC CCL17 NM_002987 ↑↑↑ ↑↑↑ Chemokine (C-C motif) ligand 22 CCL22 NM_002990 ↑↑↑ ↑↑↑ Chemokine (C-C motif) ligand 23 CCL23 NM_005064 ↓↓ ↓↓↓ Chemokine (C-X-C motif) ligand 1; GRO-a CXCL1 NM_001511 ↓↓ ↓↓↓ Chemokine (C-X-C motif) ligand 2; GRO-b CXCL2 NM_002089 ↓↓ ↓↓↓ Chemokine (C-X-C motif) ligand 13; BCA-1 CXCL13 NM_006419 ↓↓ ↓↓↓ Interleukin 1, beta IL1B NM_000576 ↑ ↑ Interleukin 1, receptor antagonost IL1RN NM_000577 ↑↑↑ ↑↑↑ Interleukin 8 IL8 NM_000584 ↑ ↓↓ Interleukin 10 IL10 NM_000572 ↓↓ ↓ Tumor necrosis factor TNFA NM_000594 ↓ ↓ Vascular endothelial growth factor A VEGFA NM_003376 ↑↑ ↑↑ Bone morphogenetic protein 1 BMP2 NM_001200 ↓ ↓ Colony stimulating factor 1 CSF1 NM_000757 ↑↑ ↑ Chemokine (C-C motif) receptor 5 CCR5 NM_000579 ↑ ↓↓ Coagulation factor XIII, A1 polypeptide F13A1 NM_000129 ↑↑ ↑↑↑ Mannose receptor, C type 1 MRC1 NM_002438 ↑ ↓↓ Matrix metallopeptidase 1 (collagenase) MMP1 NM_002421 ↑↑ ↑↑↑ V-rel reticuloendotheliosis viral oncogene homolog A (p65)

RELA NM_021975 ↓ ↓

Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (p50)

NFKB1 NM_003998 ↓ ↓

Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha

NFKBIA NM_020529 ↑ ↑

Inhibitor of kappa light polypeptide gene, kinase beta

IKBKB NM_001556 ↓ ↑

Tested genes and changes induced by IL-4



No effects of Arnica m. on cell viability of

human macrophages (no toxicity at any dilution)



Arnica m. effects on Normal Macrophages



Arnica m. effects on Normal Macrophages



Arnica m. effects on IL-4 Macrophages



Arnica m. effects on IL-4 Macrophages



SYNTHESIS OF Arnica montana actions

IL-4 Macrophages (Active in wound healing)

CXCL1

CXCL2

CXCL8 (IL8)

Neutrophil

recruitment

Angiogenic

activity

Bone

morphognetic

protein

Metallo-

proteinases

Regulation of

inflammation

Normal Macrophages

(Active in inflammation phase)

TNFA

IL1B

Connective

tissue protection

and repair







• RNA-Sequencing of transcriptome • Fibronectin assay and functions




Effect of Arnica m. on gene expression by

human macrophages (RNA-seq study)



Effect of Arnica m. on gene expression by

human macrophages (RNA-seq study) - ABSTRACT


Total RNA was isolated and cDNA libraries were sequenced with a NextSeq500

sequencer. Genes with significantly positive (up-regulated) or negative (downregulated)

fold changes were defined as differentially expressed genes (DEGs).

A total of 20 DEGs were identified in Arnica m. 2c treated cells. Of these, 7 genes were

up-regulated and 13 were down-regulated. The most significantly up-regulated function

concerned 4 genes with a conserved site of epidermal growth factor-like region and

three genes of proteinaceous extracellular matrix, including heparin sulphate

proteoglycan 2 (HSPG2), fibrillin 2 (FBN2), and fibronectin (FN1). Protein assay

confirmed a statistically significant increase of fibronectin production.

The down-regulated transcripts derived from mitochondrial genes coding for some

components of electron transport chain. The same groups of genes were also

regulated by increasing dilutions of Arnica m. (3c, 5c, 9c, 15c), although with a lower

effect size.

We further tested the healing potential of Arnica m. 2c in a scratch model of wound

closure based on the motility of bone marrow derived macrophages and found

evidence of an accelerating effect on cell migration in this system.

The results of this work, taken together, provide new insights into the action of Arnica

m. in tissue healing and repair, and identify extracellular matrix regulation by

macrophages as a therapeutic target.


RNAseq

mRNA fragments

cDNA+adapters

libraries

Sequencing and quantification

of all fragments

Bioinformatic

mapping of

expressed genes

Total transcriptome

RNAs


Differential expression (DEGs)

(plus/minus Arnica m.)


Effect

HGC

Nomenclature Mean RPKM

Log2 Fold Change

Description

Mean SE P

value Symbol ID

Up-

reg

CR1 2334 1.8 0.3 0.07 0.0225 complement component

(3b/4b) receptor 1

LRP1 6692 21.5 0.29 0.05 < 0.0001 low density lipoprotein

receptor-related protein 1

FN1 3778 467.9 0.27 0.05 0.0007 fibronectin 1

FBN2 3604 3.3 0.27 0.09 0.0418 fibrillin 2

HSPG2 5273 10.4 0.25 0.09 0.0332 heparan sulfate proteoglycan

2

KMT2D 7133 6.9 0.24 0.06 0.0037 lysine (K)-specific

methyltransferase 2D

MACF1 13664 2.7 0.24 0.08 0.0379 microtubule-actin crosslinking

factor 1

Up-regulated genes of THP-1 macrophages treated

for 24h with Arnica m. 2c (n=5).



Down-regulated genes of THP-1 macrophages treated for

24h with Arnica m. 2c (n=5).

Effect

HGC

Nomenclatu

re

Mean

RPKM

Log2 Fold Change

Description Mean SE P value

Symbol

Down-

reg

COX3 489.2 -0.24 0.052 0.0418 mitochondrially encoded cytochrome c

oxidase III

ND2 1652.3 -0.25 0.041 0.0038 mitochondrially encoded NADH

dehydrogenase 2

COX1 4374.7 -0.25 0.07 0.0418 mitochondrially encoded cytochrome c

oxidase I


dehydrogenase 6

ATP6 369.5 -0.28 0.038 0.0008 mitochondrially encoded ATP synthase 6

ND5 357 -0.28 0.061 0.002 mitochondrially encoded NADH

dehydrogenase 5


dehydrogenase 1

ND4 739 -0.31 0.06 0.0006 mitochondrially encoded NADH

dehydrogenase 4

MTRNR2L8 41 -0.31 0.088 0.0176 MT-RNR2-like 8

CYTB 1374.1 -0.32 0.057 0.0001 mitochondrially encoded cytochrome b

CCDC88B 4.9 -0.32 0.068 0.0078 coiled-coil domain containing 88B

ATP8 31.6 -0.32 0.094 0.0135 mitochondrially encoded ATP synthase 8

ND4L 91 -0.36 0.088 0.0006 mitochondrially encoded NADH

dehydrogenase 4L



Functional classification and gene enrichment

analysis of genes significantly affected by Arnica m.

2c

Database Description Fold

Enrichment

P

value Genes

up-

reg

INTERPRO EGF-like region,

conserved site 32.49 <0.001 LRP1, HSPG2, FBN2, FN1

GOTERM_

CC

Proteinaceous

extracellular matrix 17.12 <0.01 HSPG2, FBN2, FN1

GOTERM_

MF Calcium ion binding 7.06 <0.05 LRP1, MACF1, FBN2

down-

reg

GOTERM_

CC_FAT

Mitochondrial

respiratory chain

complex I

101.44 <0.001 ND1, ND4L, ND4, ND5,

ND2, ND6

KEGG_PAT

HWAY

Oxidative

phosphorylation 26.08 <0.001

ND1, ND4L, ND4, COX3,

ND5, COX1, ND2, CYTB,

ATP8, ND6, ATP6



IL-4 Macrophages (Active in tissue healing)

CXCL1

CXCL2

CXCL8 (IL8)

BMP2

MMP1

Regulation

of inflammation

phase

Normal Macrophages

(Active in inflammation

phase)

TNFA

IL1B

Connective

tissue protection

and repair

Neutrophil

recruitment

Angiogenic

activity

FN1

FBN2

HSPG2

Extracellular

matrix

synthesis


UPDATED SYNTHESIS OF Arnica montana actions



“How small, in other words, must be the dose of each

individual medicine, to effect the best cure?”

“Is, as may easily be conceived, not the work of

theoretical speculation. (...)

Pure experiment, careful observation of the

sensitiveness of each patient, and accurate experience

can alone determine this in each individual case”

Organon, par. 278

HOW SMALL SHOULD BE THE DOSE?

C.F.S. Hahnemann

(1755-1843)


Homeopathy is not always high dilutions

Example of Arnica m. 6X pills

Arnica extract (MT) contains 0.36 grams/L of sesquiterpene active

principles

6x potency = 3.6 x 10-7 grams/L

= 3.6 x 10-10 grams/ml

Since sesquiterpenes MW= 340 Da,

according to Avogadro number

= 1012 molecules/ml

Assuming that 1 ml 6X is used in 500 tablets,

we have about 2x109 (2 BILLIONS) molecules in a single tablet



EXAMPLES OF HIGH DILUTION EFFECTS “IN VITRO”

System Agent Dilution Effect Ref.

Human

basophils

Apis, Histamine 12C-16C

10-24 10-32

Inhibition of activation

markers

Poitevin 1988, Belon

1999-2009 (and Verona

Group)

Human

basophils

Adrenaline 12C-16C

10-24 10-32

Inhibition of activation

markers

Mannaioni et al.

2010

Cicken embrio Bursin 15 C (10-27g) Immunomodulatory and

endocrine activity

Bastide, Youbicier-

Simo 1993-97

Human

neutrophils

Phosphorus 12 D to 30 D Inhibition of superoxide

production

Chirumbolo and

Bellavite 1993

Wheat

germination

Arsenic

Silver nitrate 26 D (10-45 ) Protect from toxicity

Enhances growth

Betti 1997/2015

Pongratz 1998

Rat neurons Glutamate 10-18 10-30 Protection from glutamate

toxicity

Jonas et al., 2001

Neurocytes Cycloheximide 10-27 Increases viability Marotta 2002

Bacteria Arsenicum 30C Protects from toxicity Das et al 2011, De et al

2012

Neurocytes Gelsemium s. 2-30 C Prevalent gene down-

regulation

Marzotto 2014, Olioso

2014

Colon cancer

cells

Ruta grav. MT-30C Decrease viability, apo-

ptotic gene expression

Arora and Tandon 2015

Macrophages Arnica montana 2-15C Modulates inflammatory

genes

Olioso 2016, Marzotto

2016

Epitelial cells Apis mellifera 3-30C Modulates hundreds of

genes

Bigagli 2016



Arnica m. 2c = 10-8 Mol/L of sesquiterpenes


Effect of Arnica m. 2c on gene expression by

IL-4-polarized human macrophages (RNA-seq study)

http://journals.plos.org/plosone/article

?id=10.1371/journal.pone.0166340

fibronectin (p < 0.001)

fibrillin (p < 0.05)

heparan sulfate proteoglycan (p < 0.05)

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0166340

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0166340


3c

10-10 Mol/L sesquiterpene

5c


9c


15c


Effect of Arnica m. increasing dilutions on IL-4-polarized

human macrophages (pool of 5 experiments)



Additional studies on resting and LPS-treated macrophages


C

.

-0.40

-0.20

0.00

0.20

0.40

0.60Arnica m. 2c

-0.40

-0.20

0.00

0.20

0.40

0.60Arnica m. 3c

-0.40

-0.20

0.00

0.20

0.40

0.60Arnica m. 5c

-0.40

-0.20

0.00

0.20

0.40

0.60Arnica m. 9c

-0.40

-0.20

0.00

0.20

0.40

0.60

CR1

LRP1

FN1

FBN

2

HSP

G2

KMT2

D

MAC

F1

Aver

Arnica m. 15c

Control medium

(Rest-Mɸ)

Lo

g F

old

ch

an

ge

(A

rnic

a v

s C

trl)

*

Lo

g F

old

ch

an

ge

(A

rnic

a v

s C

trl)

-0.40

-0.20

0.00

0.20

0.40

0.60Arnica m. 2c

-0.40

-0.20

0.00

0.20

0.40

0.60Arnica m. 3c

-0.40

-0.20

0.00

0.20

0.40

0.60Arnica m. 5c

-0.40

-0.20

0.00

0.20

0.40

0.60Arnica m. 9c

-0.40

-0.20

0.00

0.20

0.40

0.60

CR1

LRP1

FN1

FBN

2

HSP

G2

KMT2

D

MAC

F1

Aver

Arnica m. 15c

IL-4 A

. *

*

*

*

*

B

.

-0.40

-0.20

0.00

0.20

0.40

0.60Arnica m. 2c

-0.40

-0.20

0.00

0.20

0.40

0.60Arnica m. 3c

-0.40

-0.20

0.00

0.20

0.40

0.60Arnica m. 5c

-0.40

-0.20

0.00

0.20

0.40

0.60Arnica m. 9c

-0.40

-0.20

0.00

0.20

0.40

0.60

CR1

LRP1

FN1

FBN

2

HSP

G2

KMT2

D

MAC

F1

Aver

Arnica m. 15c

Endotoxin

(LPS)

Lo

g F

old

ch

an

ge

(A

rnic

a v

s C

trl)

*

*

Effect of Arnica m. increasing dilutions on human macrophages

pre-treated with different agents (pool of 5 experiments)

Arnica 2c

Arnica 3c

Arnica 5c

Arnica 9c

Arnica 15c

Arnica 2c

Arnica 3c

Arnica 5c

Arnica 9c

Arnica 15c

Arnica 2c

Arnica 3c

Arnica 5c

Arnica 9c

Arnica 15c



C

.

-0.40

-0.20

0.00

0.20

0.40

0.60Arnica m. 2c

-0.40

-0.20

0.00

0.20

0.40

0.60Arnica m. 3c

-0.40

-0.20

0.00

0.20

0.40

0.60Arnica m. 5c

-0.40

-0.20

0.00

0.20

0.40

0.60Arnica m. 9c

-0.40

-0.20

0.00

0.20

0.40

0.60

CR1

LRP1

FN1

FBN

2

HSP

G2

KMT2

D

MAC

F1

Aver

Arnica m. 15c

Control medium

(Rest-Mɸ)

Lo

g F

old

ch

an

ge

(A

rnic

a v

s C

trl)

*

Lo

g F

old

ch

an

ge

(A

rnic

a v

s C

trl)

-0.40

-0.20

0.00

0.20

0.40

0.60Arnica m. 2c

-0.40

-0.20

0.00

0.20

0.40

0.60Arnica m. 3c

-0.40

-0.20

0.00

0.20

0.40

0.60Arnica m. 5c

-0.40

-0.20

0.00

0.20

0.40

0.60Arnica m. 9c

-0.40

-0.20

0.00

0.20

0.40

0.60

CR1

LRP1

FN1

FBN

2

HSP

G2

KMT2

D

MAC

F1

Aver

Arnica m. 15c

IL-4 A

. *

*

*

*

*

B

.

-0.40

-0.20

0.00

0.20

0.40

0.60Arnica m. 2c

-0.40

-0.20

0.00

0.20

0.40

0.60Arnica m. 3c

-0.40

-0.20

0.00

0.20

0.40

0.60Arnica m. 5c

-0.40

-0.20

0.00

0.20

0.40

0.60Arnica m. 9c

-0.40

-0.20

0.00

0.20

0.40

0.60

CR1

LRP1

FN1

FBN

2

HSP

G2

KMT2

D

MAC

F1

Aver

Arnica m. 15c

Endotoxin

(LPS)

Lo

g F

old

ch

an

ge

(A

rnic

a v

s C

trl)

*

*

Effect of Arnica m. increasing dilutions on human macrophages

pre-treated with different agents (pool of 5 experiments)

Arnica 2c

Arnica 3c

Arnica 5c

Arnica 9c

Arnica 15c

Arnica 2c

Arnica 3c

Arnica 5c

Arnica 9c

Arnica 15c

Arnica 2c

Arnica 3c

Arnica 5c

Arnica 9c

Arnica 15c


• Since these are three separate series of different cell

treatments and incubations with Arnica compared with

placebo, the fact that the effect of Arnica 2c is

reproduced in all conditions suggests that these are the

«true» genes activated by this remedy in human

macrophages

• IL-4 treatment appears to «sensitize» macrophages to

high dilutions of Arnica, confirming that high dilutions

homeopathic effects are specific for specific target

systems.

• Endotoxin induces non-linear changes of cell sensitivity

to Arnica (responses only to 2c and 5c among tested)





• RNA-Sequencing of transcriptome





Fibronectin in supernatants of cell cultures in the

absence (Control) and in the presence of Arnica m. 2c.



Matrix Biol. Aug. 2016. doi: 10.1016/j.matbio.2016.07.011.

Fibronectin is an

extracellular matrix

protein that is present

during periods of change

within tissues.

It is upregulated and

necessary in a number of

developmental contexts,

and it is also present

during wound healing.

Fibronectin


Fibronectin is the major protein that organizes the connective

tissue during the first phase of wound healing and inflammation

Fibro

nectin

•Fibronectin has the

ability to specifically

bind a large number

of molecules

•Cellular interactions

with fibronectin lead to

bidirectional

crosstalk that directs

cell functions such

as:

•Proliferation

•cell migration

•survival and

growth

Immunofluorescence image

of cells in culture



Scratch test of wound healing





The capacity of macrophages to close an artificial “wound” was assessed using a

culture model in which a portion of a confluent macrophages monolayer was

denuded of cells, after which the number of cells migrating into the open space was

assessed microscopically.

The monolayers were “wounded” by transversely scratching the well with the tip of

a pipette, tracing a 1 mm wide gap.

The medium during the migration test was DMEM (Glutamax, pen-strep) with 2%

FBS, either with or without 20 ng/ml IL-4. Arnica m. 2c or Control solutions were

added 24 h before wounding and maintained, during the migration time, at the

concentration equal to 10% of the culture volume.

The experiments were evaluated by examining microscopically the cell migration

front/border and using a grid composed of 500 small frames to calculate the % of

wounded area occupied by macrophages.





Arnica m. increases migration of macrophages on in

vitro scratch test





GRAPHICAL SYNTHESIS



Cartagena - 2 sept 2017 © Paolo Bellavite

Working hypothesis of the action

of Arnica m. on connective tissue


Evidence and Plausibility: the Arnica contribution!

Homeopathy

acceptation

and

progress

CLINICAL

RESEARCH

«EVIDENCE»

BASIC

RESEARCH

«PLAUSIBILITY»


Does

it work?

HOW

does

it work?


Verona Research Group working on Arnica m. (2013-2017)


We thank

Italian Research Ministry

Boiron Laboratoires-Lyon

Paolo Bellavite

Clara Bonafini

Marta Marzotto

Debora Olioso

Others: Elisabetta Moratti

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