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Effects of Highly Diluted Acetylcholine on Nonneuronal Cholinergic System in Keratinocytes Uberti F, Physiology Lab. & noiVita Spin Off, Dept of Translational Medicine, UPO Novara, Italy Problem of wound Slow healing wounds featuring classic pressure injuries are a major burden to patient suffering but also cost to the healthcare system. Various treatments have been proposed, but full and rapid wound repair remains an open challenge. The main problem of skin re-epithelialization or mucosal wounds is the lateral migration of epidermal keratinocytes (KC). Page 2 Acetylcholine and non neuronal cholinergic system KC functions (viability, proliferation, and migration) depend on a fine balance in control pathways in which the nonneuronal cholinergic system plays a major role This system involves acetylcholine (Ach) and its nicotinic and muscarinic receptors via autocrine and paracrine mechanisms. Ach is not only a neurotransmitter but is an ancient molecule that can be released by and act on non-neuronal cells present in a wide range of living organisms To date, studies on the biological role of Ach have mainly focused nonneuronal cell functions Page 3 Non colinergic system and skin KC are able to synthesize ACh in large amounts: a single KC synthesizes and releases a mean of 2 10-17 mol ACh and 7 10-19 mol ACh per minute, respectively. Skin exhibits molecular components of the NNCS and it is assumed to regulate proliferation, differentiation, apoptosis, adhesion and migration of KC Page 4 Acetylcholine and Keratinocytes Ach or a cholinergic drug exert different effects on KC during their maturation [Grando et al., 2006]. Several important functions, including control of cell viability, proliferation, adhesion, migration, and differentiation, have been also attributed to muscarinic receptors in KC [Beck et al., 2006]. Simultaneous stimulation of nicotinic and muscarinic receptors by Ach may be necessary to synchronize the metabolic and ionic events within cells. Page 5 Acetylcholine a new strategy In recent years, the study of skin trophism, lead to identifying new therapeutic strategies for skin diseases: some evidence is available about the therapeutic capability of cholinomimetics or blockers in two important skin diseases, i.e., pemphigus or psoriatic lesions [Grando et al., 1993, 2006]. The role of nonnervous Ach in KC could have important clinical implications for patients with various skin disorders such as nonhealing wounds Page 6 Therapeutic Application i) Main problem: the therapeutic use. Ach is a highly reactive molecule and consequently, its use can lead to systemic side effects [Beck et al., 2006] ii) Main problem: ACh has a very short half-life being metabolized by acetylcholinesterase Main solution: cholinergic information transmitted by ultra-diluted and activated acetylcholine can provide natural tonic stimulation that can promote the proliferation of keratinocytes in the wound repair process. Page 7 Low Dose Application A promising way to obtain a fine regulation of physiological mechanisms could be the use of highly diluted substances (Low Dose) IL-12 in modulation of Th1 versus Th2 Low dose SKA cytokines was demonstrated in an asthma have been demonstrated to preclinical model suggesting a novel have a significant beneficial therapeutic approach to diseases effect in skin diseases both in which involve a Th1/Th2 imbalance patients and in cultured cells

[Gariboldi et al., 2009] [Barygina et al., 2015; Radice et al., 2015] Page 8 Low Dose SKA Highly diluted forms have been obtained via a sequential kinetic activation (SKA) technique The activated solutions, were kinetically energized by a mechanically applied force via a standardized shaking process called SKA; the applied shaking procedure is characterized by vertical shaking, a 10-cm motion range, and a shaking speed corresponding to 100 oscillations in 10 s. Page 9 Aim of the project Thus, the aim of this study is to evaluate the effects of highly diluted Ach solutions obtained by SKA on cultured normal human KC on an in vitro incisional wound model in order to confirm the hypothesis that a highly diluted form (Low Dose) of Ach could be a greater physiological stimulus for KC viability and proliferation. Page 10 Methods NHEKs cell line: a primary normal human keratinocyte used in scientific research, utilized for their high capacity to proliferate in vitro. They were purchased from Lonza (Basel, Switzerland) and cultured in KBM medium (Lonza, Basel, Switzerland) containing KGM 2 growth supplements (Lonza,Basel, Switzerland) in a incubator at 37C, 5% CO2 and 95% humidity. Experiments were conducted at passage 3-6. Page 11 Methods Cell viability Cell proliferation MTT test Crystal violet (cell counting) Cell migration Woundhealing Intracellular mechanism Western blot Analysis of mitocondria oxygen consumption ROS, potential membrane, Page 12 Methods Ach 1 g/mlg/ml in physiological solution to prepare 10 fg/ml (Ach 5.26x10-14moli/l corrispondenti a 10fg/ml) 1 pg/ml (Ach 5.26x10-12 moli/l corrispondenti a 1pg/ml). NO SKA Page 13 SKA Methods Control In presence or absence of: Ach 1 g/mlg/ml Ach SKA 1pg/ml Atropine (muscarinic antagonist) - Ach SKA 10fg/ml Ach NO SKA 1pg/ml Ach NO SKA 10fg/ml Page 14 - - H89 (inhibitor of PKA) Cheleritrine (inhibitor of PKC) Methods Protocol A Control Ach 1 M Ach SKA 1pg/ml Ach SKA 10fg/ml

Treatment only at the beginnig of experiment and stopped at 6 days (144h). In this condition was also tested the mechanism using the specific inhibitor (30min before the stimulations) Ach NO SKA 1pg/ml Ach NO SKA 10fg/ml Page 15 Protocol B Treatment each 24h till 6 days and stopped at 6 days (144h). h Ac 7 14 ng A SK 10 fg A SK 10 fg A SK 1 pg A SK 1 pg Results: cell viability and proliferation a * * * * * * 20 a a L /m fg L /m fg L /m g p L /m g p 7 1 1 10 10 0 14 A A A A K K h L S L S L SK L SK L c m O m A /m ch /m O /m ch g/ N g/

fg N fg A ng A p p h h 7 1 Ac 1 10 Ac 10 A A 14 KA KA h SK SK c S S h A O h O N Ac N Ac h h c c A A Cell viability at 144 h Increase in viability cell number at 144 at h h (protocol BCell vs. control), %144 vs.(protocol control inBprotocol A, % vs. control), % 30 150 * 30 20 100 20 ** * * * * ** ** b bc c * ** * * * h Ac b ** 50 ** ** * ** * * ** h Ac N O A SK

10 fg h Ac A SK 1 pg h Ac N O SK * 200 * * * 10 50 0 L /m g n L /m fg L /m fg L /m pg L /m pg 7 1 1 10 10 0 14 A A A KA K K L L L h L S L S LL SK LL S c m h m O m ch m O A /m /m //m //m //m c g g g g g g N g g A f f N f f A p nn pp 1 7 11 Ach 1100 Ach 1100 A

AA 1144 AA AA K K K K K K K h S SS c SS SS O AAc chh OO chh N AAc N AAc h h h Ac AAc ** 10 50 * 144 ** h of stimulation with Fig. 1. Cell viability and cell counts after 200 150 different formulations of Ach. a , b Results obtained by MTT and * ** crystal violet staining only at T0 with different * ** * in NHEK treated formulations of highly diluted Ach and maintaine d for 144 h. c, * ** 150 100 * ** * ** d The same agents were adde d every 24 h for 144 h, and then NHEK cell viability*and proliferation were measured. The results ** of the MTT test are expressed as means SD*(% ) of 6 biological 10050 0 0 L L L L L mL mL /m L / mL /m L g//m g// m fg /m fg / m ngg/m p p 7n 1 g 1 g 10 fg 10 fg A 1p A 1p 0 1447 A 10 A 10 h1 L SK A L SK A L SKKA L SKKA L

c K /m h S A ch /m h K /m /m O S /m A ng Acch S fg NOO S fg Acch pg h N O pg A 7 1 Ac h N 1 A 10 Achh N 10 A 14 Ac SKA KA Ac SKA h SK c S h A O h O c c N A N A h h c A Ac * ** 150 100 50 0 h Ac d 1pg/ml * **SKA * induced greater cell viability than others formulations 7 14 L /m ng h Ac A SK 10 L /m fg h Ac N O A SK 10 L /m fg h Ac A SK * ** 1 L /m pg h Ac

N O A SK crystal violet staining in NHEK treated only at T0 with different Fig. 1. Cell viability and cell counts after 144 h of stimulation with * ** 50 0 1pg/ml SKA induced greater proliferation a p p r o p r i a t e , u sthan i n g G r a p h Pothers ad P r ism 5 (G r ap h P ad J o l l a , C A , U S A ) . p < 0 .0 5 w a s c o n s i d e r e d s t a t i s t i c a A l l d a t a f r o m t hformulations e d en sito m etr ic a n a ly sis w er e n o r m replicates normalized to the control. In the crystal tion, cell counts are expressed as means SD (%) replicates. * p <0.05 vs. control. ** p <0.05 vs. 14 Bars indicate significance between the activated and forms and between the2 activated forms. Ach, acety sequential kinetic activation; NO SKA, nonsequenti vation. L L L L L Statistical Analysis /m /m /m /m /m R e s u l t s a r e e x npgr e s s e d a0sfgm e a n s 0 fgS D o f a tpgl e a s t 3 b pi go l o g i c a l 7 r e p l i c a t e s f o 0r e a14c h e x p e r iAm1 e n t a l p rAo1t o c o l , aKnAd1 e a c h r Ke Ap 1l i c a t e w a s K K h r e p r o d u c e d A3c t i m /m e sL cf ho Sr e a c/ mh L eOxSp e r i m/ mLe ncht aS l p r/mo Lt o cOoSl . S /tma Lt i s t i c a l t r o l v a l u e s ( d e fin e d a s 1 ) . A l l o t h e r d a t a f r o m e a c h g N g g g g N f A c o m p a r i s o n s b e7tnw e e n g r o10u p cshw e r e10mf a d eA u s i 1n pg o cnh e - w a1yp A N O V A p r o to c o l w er e n o r m aliz ed to c o n tr o l v a lu es in p er d 4 A ** p<0.05 vs Ach 147ng/ml w i t h B o n f e r crho1n i s p o sStKAh o c At e s tSKoAr t h e M SaKnA n - W h i tSnKAe y U t e*s tp<0.05 , as avs s 0 control; % ). Fig.Page 1. Cellviability and cell counts after 144 h of stimulation with4 16 200 different formulations of Ach. a, b Results obtained by MTT and 4h N h Ac fg 200 10 bc * * * 100 20 100 * L /m ng h

Ac O A SK 10 * ** 150 30 * 40 20 0 N h Ac Increase in cell number at 144 h vs. control in protocol B, % * h Ac O ng 150 * Increase in cell number at 144 h viability 144number h % at 144 h Increase inatcell vs. Cell control in protocol A, (protocol vs. control), % vs.Bcontrol in protocol A, % 60 40 Protocol A Increase in cell number at 144 h Increase in cell number vs. control in protocol B, % at 144 h vs. control in protocol B, % Cell viability at 144 h viability at (protocol ACell vs. control), %144 h (protocol A vs. control), % 60 h Ac 7 14 A d h Ac h Ac N O h Ac h Ac N O Cells Organs In the crystal violet evaluareplicates normalized toTissues

the control. 10.1159/000451023 tion, cell counts areDOI: expressed as means SD (%) of 6 biological replicates. * p <0.05 vs. control. ** p <0.05 vs. 147 ng/mL Ach. replicates normalized to the control. In the crystal violet evalua- Uberti/Bardelli/Morsanuto/G Cochis/Molinari ocol B Wound closure Wound closure Ac (protocol (protocol A vs. control), %A vs. control), % h 14 14 7 Ac 7 Ac ng h ng h /m SK /m A SK L A Ac c L A h 10 h 10 N N f O g/ fg O SK m /m SK L A L A 1 0 10 A fg Ac ch fg /m h /m SK SK L Ac A L Ac A h 1 h 1 N pg N pg O O /m S / m KA SK L L A 1 1 pg pg /m /m L L tocol B Results: wound healing * ** 40 T0

* ** * ** 20 ** 0 * ** 40 20 * 20 * ** * ** ** 0 20 Ac h * Wound closure Wound closure (protocol (protocol B vs. control), % B vs. control), % A Ac ch h 14 14 7 Ac 7 ng h ng ch /m SK /m A SK L A L ch A 1 10 0 N N fg O fg O /m SK /m SK L A L A 10 10 A fg Ac ch fg /m h /m SK SK L A L Ach A 1 1 N p N pg O g/ O m SK /m SK L A L A 1 1 pg pg /m /m L

L 30 20 * ** 10 * ** * ** 30 0 20 10 10 * ** Only one stimulation with 1pg/ml SKA induced a major closure of wound after 6 days * ** * ** Ach 147ng/ m confirmed its negative effects 144 h protocol A 144 h protocol B Control 50m Ach 147 ng/mL Ach SKA 10 fg/mL Ach NO SKA 10 fg/mL Ach SKA 1 pg/mL 0 10 Ach NO SKA 1 pg/mL * p<0.05 vs control; ** p<0.05 vs Ach 147ng/ml Page 17 Fig. 2. Involvement of different formulations of Ach in NHEK wound closure. On the left are representative pictures of wound tocol A and B is sh (%) of wound closu Results: ROS production Ach 147ng/ml induced ROS production on both protocols supporting it negative effects on wound closure in a dose dependent manner Ach 1pg/ml SKA showed a physiological effect on both protocol * p<0.05 vs control; ** p<0.05 vs Ach 147ng/ml Page 18 30 e 40 * 20 Highly Diluted Ach in KC 10 * ** * ** 0 10 20 f Cells Tissues Organs DOI: 10.1159/000451023

* ** O O SK A 1 pg /m L pg /m L fg /m L Ac h N O SK A 1 1 fg /m L pg /m L pg /m L 10 SK A SK A Ac h O * N 1 10 fg /m L SK A SK A N 10 N fg /m L 60 Ac h Ac h 10 10 ng /m L JC-1 ratio (595 nm/535 nm) vs. control (protocol B), % 40 SK A d Ac h SK

A 14 7 pg /m L 0 Ac h Ac h 1 * ** ng /m L pg /m L SK A * 14 7 1 O pg /m L fg /m L fg /m L N O N O SK A 1 pg /m L pg /m L fg /m L fg /m L ng /m L pg /m L 10 1 fg /m L fg /m L pg /m L 10 SK A SK A

Ac h N 1 1 10 14 7 SK A Ac h SK A SK A SK A O Ac h Ac h Ac h O Ac h N ng /m L 10 14 7 SK A Ac h Ac h Ac h Ac h b Ac h SK A N 1 10 SK A SK A 10 * ** Ac h O pg /m L fg /m L O Ac h N Ac

h Ac h SK A ng /m L 60 Ac h N 1 10 fg /m L SK A 10 ng /m L 14 7 JC-1 ratio (595 nm/535 nm) vs. control (protocol A), % * Intensity vs. control (protocol B), % Ac h O SK A N SK A 14 7 Ac h Ac h 20 Ac h Ac h Ac h Ac h c * 40 * 0 * ** * 80 * a 80 * ** * 20 * 0

* ** * ** 30 Page 19 7 1/25/2016 8:45:19 AM Intensity vs. control (protocol A), % Results: Mitochondrial potential and Oxygen Consumption Ach 1pg/ml SKA showed high mitochondrial potential with major evidence with protocol A in a physiological manner explained by the reduction of oxygen consumption 40 * p<0.05 vs control; ** p<0.05 vs Ach 147ng/ml 3 1.0 L L * O N O SK A 1 1 fg /m L fg /m L pg /m L pg /m L 10 SK A SK A Ac h N 10 -act Ac h Ac h -act m L ERK1/2 ng / ERK1/2 SK A pg /m L Protocol A L

6 Co nt ro l 1 pg /m L p-ERK 14 7 SK A Ac h O 1 fg /m L p-ERK Ac h N 10 SK A SK A fg /m L m L KI67 L * ** 9 Ki67 and ERK/MAPK in combination to exclude an uncontrolled 1.5 * ** 8 * ** *proliferation ** of KC 7 Ratio of KI67/ -act vs. control (protocol B) O ng / 10 14 7 SK A Ac h N Ac h Ac h Ac h Ac h Co nt ro l KI67 L

* L 10 L 5 L 3 L 4 L Page 20 2 1 Ratio of KI67/ -act vs. control (protocol A) Results: Western blot analysis Protocol B * ** -act -act Ratio of KI67/ -act vs. control (protocol A) Ratio of ERK1/2/ -act vs. control (protocol A) 9 8 * ** 7 * ** 6 5 4 3 * * 2 1 6 * ** 5 4 3 * * ** * ** * 2 1 g. 4. Western blot and densitometric analysis of ERK/MAPK and 67 in NHEK cells. Protein extracts were analyzed by immunoPagespecifi 21c antibodies against the indicated proteins. otting with he pictures represent 5 biological replicates for each experimenprotocol (A and B). Data areexpressed as means SD of 5 bio- Ratio of ERK1/2/ -act vs. control (protocol B) * ** 10 Ratio of KI67/ -act vs. control (protocol B) Results: densitometric analysis * ** 1.5 * ** 1.0 2.5 * ** 2.0 * *

1.5 1.0 ** ** Ach 1pg/ml SKA induced Ki67 confirming proliferation Ach 1pg/ml SKA induced ERK expression indicating cellular health * p<0.05 vs control; ** p<0.05 vs Ach 147ng/ml logical replicates for each experimental protocol (A and B). * p < 0.05 vs. control. ** p <0.05 vs. 147 ng/mL Ach. Bars indicate significance between the activated and nonactivated form and between the 2 activated forms. Ach, acetylcholine; SKA, sequential kinetic activation; NO SKA, nonsequential kinetic activation. Results: wound with antagonist T0 Control Since the better effects were observed with protocol A than B, the specific mechanisms were analyzed only in Protocol A a Atropine 7.25 g/mL Ach 147 ng/mL Ach 10 fg/mL SKA Ach 147 ng/mL Ach 10 fg/mL SKA Ach 147 ng/mL Ach 10 fg/mL SKA Ach 10 fg/mL NO SKA Ach 1 pg/mL SKA Ach 1 pg/mL NO SKA Ach 1 pg/mL SKA Ach 1 pg/mL NO SKA Ach 1 pg/mL SKA Ach 1 pg/mL NO SKA b Chel 384 ng/mL Ach 10 fg/mL NO SKA c H89 519 ng/mL Ach 10 fg/mL NO SKA d 0 * ** Wound closure vs. control, % Wound closure vs. control, % Wound closure vs. control, % 20 * * ** * ** 15 * 10 * ** 10 *

* ** 5 10 * ** 15 * Page 22 * 5 0 5 * 0 Results: wound with antagonist d 0 * * f Wound closure vs. control, % Wound closure vs. control, % Wound closure vs. control, % * ** 15 20 * 25 * ** * ** 10 * 20 5 * ** g The inhibition of M1, PKA and PKC prevented all wound closure, indicating the importance of M1 receptor, PKA/cAMP and PKC on wound closure Cells Tissues Organs Highly Diluted Ach in KC DOI: 10.1159/000451023 Page 23 * 15 * * ** 10 * * ** * ** 5 * * ** 10 15 e * 5 0 * 0 * p<0.05 vs control; ** p<0.05 vs Ach 147ng/ml

6 11 h Ac h Ac A SK N O A SK h Ac A A 7 e SK SK ch SK SK in A O h O ch p + c N N L A o N A r h h L+ ch /m L+ At Ac Ac m /m +A g / + L g L g 25 /m /m 7. 25 25 g g 7. 7. ne 5 i e e 2 p 25 in 7. in 7. ro e e op op At n r n i r t p A pi At ro ro At At SK

O SK Results: Western blot and densitometric analysis of M1 h Ac 3 -act Fig. 7. Western blot and densitometric L L L L L L L of Lthe M1 L L L ol m m m m m m analysis m m rece mptor in m NHEK cells. tr /m n g/ g/ g/ g/ g/ g/ Protein g/ gextracts g/ were g/ analyze g/ d by immuf f f f o n n p p p p C 7 7 1 1 1 1 10 10 10 10 25 A A 14 14 7. c KA KAantibodies against KA KA KA KAwithSspecifi h h noblotting e SK SK S c c S S S S in h h A A the O O h h indicated proteins. O O N The pictures repreN L+ Ac Ac op N

N Ac Ac h h 3 biological replicates tr m +se h h example L+ c c nt an of / L c A c A A A A /m g /m L+ L+protocol A. g m Ach, acetylcholine; SKA, g for 25 m / 5 / 7. 2 25 kinetic activation; NO SKA, g gsequential 7. 7. ne 5 e e pi 25 nonsequential .2 n . kinetic activation. Data are n i o 7 i 7 r p e op At ne in as means SD of 3 biological ro tr expre ssed i t p A p A ro ro replicates. * p <0.05 vs. control. ** p <0.05 At At 3 * * ** vs. 147 ng/mL Ach. p <0.05 vs. 7.25 g/ mL atropine (specific M1 inhibitor). Bars indicate significance among the different forms of Ach, between activated forms alone and in the presence of pretreatment with atropine. Ratio of M1/ -act vs. control M1 2 1 * * ** * ** * **

* ** 0 1 * * * ** * * * L L L L L L L L L L L /m / m /m /m /m / m g/m g/m g/m g/m g/ m f f ng 0 fg 0 fg pg pg n g p p 0 0 7 1 1 A1 A1 25 147 A 1 A 1 A 1 A 1 14 A A 7. K K K K K K h h e S S SK SK in Ac ch S O S ch Ac h S O S ch O O N L+ Ac A hN A op N N A r + h ch At g/ m L+ Ach mL Ac Ac / +A /m L+ L g g 5 2 m 5 /m 7. 25 g/ .2 g 7. 5 e 7 ne 5 i e 2 p 2 n 7. in 7. ro pi At trop ine tro ne i p A p

A ro ro At At Ratio of M1/ -act vs. control * ** The pre-treatment prevented * ** * p<0.05 vs control; ** p<0.05 vs Ach 147ng/ml * ** the activation of M1, and successive stimulation P K C a with n d P K A k i *n a s e s w e r e a l s o a n a l y z e d i n N H E K c e l l s g r a t i o n o f c e l l s a n d c o n s e q u e n t l y c l o s u r e o f t h e l e s i o n *d u*n d e*r t h e s a m e c o n d i t i o n s a s r e p o r t e d b e f o r e . These * ** t r e a t e a r e a . F i n a l l ydata , a s s h o w confirm n i n F i g u r e 8 , a lthe l o f th e fo r m s ( se* different formulationsA s sof Ach h o w n i n F i g u r e 5 , t h e s p e c i fic b l o c k e r s o f P K C ( c h e - q u e n t i a l l y k i n e t i c a l l y a c t i v a t e d a n d n o n s e q u e n t i a l l y k i involvement of muscarinic ler y th r in e a t 3 8 4 n g / m L ) a n d P K A ( H 8 9 a t 5 1 9 n g / m L ) n e tic a lly a c tiv a ted ) a n d 1 4 7 n g / m L A c h w e r e a b le to in was not able to restore w e r e a b l e this t o p r e v e n t a n y i n c r e a s e i n c e l l v i a b i l i t y o r c e l l receptor d u c e t h e a c t i v aon t i o n oAch f P K C aeffects n d P K A , in d ic atin g th at p r o lifer a tio n , m a in ta in i n g th e c ell a t a b a sa l- lik e lev e l. A c h w a s a b le to in v o lv e th e c a lc iu m in tr a c e llu la r p a th condition. I n th e p r e sen c e o f a ll fo r m s o f seq u en tia lly k in e tic a lly w a y to ex e r t its effec ts. I n p a r tic u la r , 1 4 7 n g / m L A c h 2 1 0 1 L L L L L L L L L L L / m g/m g/ m g/m g/m /m g/m g/m g/m g/m g/m g g f f f f n n p p p p 0 0 7 10 10 A 1 A 1 25 147 A 1 A 1 A 1 A 1 14 A A 7. K K K K K K K K h h e S S S S c S S n Ac ch S O S ch O O ch NO pi +A ch A hN N A

ro mL +A h N L+A t h c ch / L c A c A A g /m L+A g/m L+A g 5 m 2 m 5 7. 25 g/ g/ .2 7. 5 e 7 a c5ti v a t e d a n d n o n s e q u e n t i a l l y k i n e t i c a l l y a c t i v a t e d A c h , ne i e p 2 .2 n in 7. ro pi t7h e s e e f f e c t s w e r e n o t r e v e r t e d . T h e s e d a t a c o n f i r m t h e At trop ine tro ne i p A p A a b i l i t y o f t h e s e b l o c k e r s t o m o d u l a t e s p e c i fic i n t r a c e l ro ro At At Page 24 lu la r m ec h a n ism s a s r ep o r ted in th e liter a tu r e . I n a d d itio n , sim ilar d ata w er e also o b ser v ed in w o u n d h ea lin g ex p er im en ts in w h ic h th e b lo c k er s p r ev en ted th e m i- a n d seq u e n tia lly k in etic a lly a c tiv a te d A c h 1 0 fg / m L sh o w ed sim ilar effec ts. H o w ev er , all o th er fo r m u latio n s w er e ab le to in d u c e th e ac tiv atio n o f th ese k in ases (p < 0 .0 5 ) c o m p a r e d t o t h e c o n t r o l . T h e s e f i n d i n g s w e r e c o n fir m ed b y th e p r etr eatm en ts w ith c h eler y th r in e (3 8 4 n g /m L ) an d H 8 9 (5 1 9 n g /m L ) fo r P K C an d P K A , Ratio of PKC/ -act control Ratio ofvs. PKC/ -act vs. control 2 * 2 1 1 * ** * * ** * ** * ** * ** * ** * ** * ** * * ** * ** * * * * ** * * * * * * * * * ** * * * * 2 1

* * * Ratio of PKC/ -act control Ratio ofvs. PKC/ -act vs. control * Ac Ac Ach h 1 h S 47 N KA ng O S 10 /m Ch Ac Ach KA fg L h S 10 / m Ch Che el 3 N KA f L el l 3 84 g O 38 84 n S 1 /m 4 n g/ Ch KA pg L m / n Ch Ch g/ g/m L+ el 1 p mL el el 3 mL L+ Ac 384 g/ 38 8 +A Ac h n mL 4 4 n ch h 14 g/ ng g S 7 m / m / m NO KA ng L L L+ +A SK 10 /m Ac c A fg L H 8 h h S 10 /m H H8 9 5 N K fg L 89 9 1 O A 5 9 S 1 /m 51 19 n g 9 n / H KA pg L n g m 8 1 / H H89 g/m /m L+ 9 5 pg mL 89 5 L L+ A 1 / 51 19 +A Ac ch 1 9 n mL 9 n ch h S 47 g/ ng g/ N K n m / m mL O A g/ L L+ +A SK 10 mL Ac ch A fg h S 10 /m N KA fg L O / SK 1 p mL A g/ 1 m pg L /m L Ac Ac Ach h 1 h S 47 N KA ng O S 10 / m Ch Ac Ach KA fg L C h S 10 / m Ch he el 3 N KA f L el l 3 84 g O 38 84 n S 1 /m g 4 n / Ch KA pg L m / Ch Ch ng/ g/m L+ el 1 p mL el el 3 mL L+ Ac 384 g/ 38 8 +A Ac h n mL 4 4 n ch h 14 g/ ng g S 7 m / m / m NO KA ng L L L+ +A SK 10 /m Ac c A fg L H 89 H h h S 10 /m H 8 5

N K fg L 89 9 1 O A 5 9 S 1 /m 51 19 n 9 n g/ H KA pg L m n g 8 1 / H H89 g/m /m L+ 9 5 pg mL 89 5 L L+ A 1 / + A 51 19 A c ch 1 9 n mL 9 n ch h S 47 g/ ng g/ N K n m / m mL O A g/ L L+ +A SK 10 mL Ac ch A fg h SK 10 / m N L O A 1 fg/ SK p mL g A 1 /m pg L /m L Co nt ro 14 l 7 Ac ng h /m SK Ac L A h 1 N 0 O fg /m SK A L 1 0 Ac f g h /m SK L Ac A 1 h pg N O /m SK L A 1 Ch pg el /m 38 Ch L 4 Ch e l ng 38 el /m 4 Ch 38 ng L+ 4 el Ac n /m 38 g/ h L 4 m 1 ng L 47 / m +Ac ng

h L+ Ch /m SK Ac el L A h 38 Ch 1 N 0 4 O el f n g/ g/ SK 38 m m A 4 L L+ ng 10 Ac /m fg h /m L+ SK Ac L A h 1 N pg O /m SK H L A 89 1 pg 51 9 /m H H ng 89 L /m 89 5 51 H 19 L+ 9 89 ng Ac ng 51 h /m 14 /m 9 L+ L ng 7 Ac ng /m h / H L+ m SK 89 L Ac A 51 h 1 H 0 9 N 89 fg ng O /m SK 51 /m L 9 A L

+A ng 10 ch /m fg L+ /m SK Ac A L 1 h pg N O /m SK L A 1 pg /m L Ac h Results: Western blot and densitometric analysis PKC PKC p-PKA p-PKA -act -act * 2 0 0 1 1 * ** * * ** * ** * ** * ** ** **** * ** 1 * * ** * * * * ** * * * * * * * * ** * * ** * * * * * * * * g. 8. Western blot and densitometric analysis of PKC and PKA activation; NO SKA, nonsequential kinetic activation; Chel, cheln NHEK cells. Protein were analyzed by immunoblotting * p NO <0.05 vs.nonseque control. *ntial * p< 0.05 vs. 147 ng/mL Ach. Fig. 8.Page Western blotextracts and densitometric

analysis of PKC and PKA erythrine. activation; SKA, kinetic activation; Che l, chel 25 ith c cells. antibodie s against thewere indicated proteins using also a pe< 0.05 vs. *384 ng/mL chelerythrine. <0.05 519 ng/mL inspecifi NHEK Protein extracts analyzed by immunoblotting rythrine. p< 0.05 vs. control. ** p

Page 27 Discussion SKA forms showed a statistically significant decrease in ROS production accompanied by an increase in mitochondrial membrane potential and decreased oxygen consumption indicating the better biochemical energy of the cells The involvement of ERK/MAPK (marker of cell migration) and KI67 (proliferating cells) in the mechanism activated by Ach confirmed the better influence of the single treatment Page 28 Discussion Ach SKA 1 pg/mL confirmed its greatest effectiveness in M1 receptor, PKC, and PKA activations. These findings confirm that low dose Ach acts through the same mechanisms as concentrated Ach and support the hypothesis of beneficial effects exerted by low doses without any negative effect (e.g., oxidative injury). Page 29 Conclusion Low doses of Ach SKA triggers a cascade of dynamic events that results in better regulation of KC functions, cell viability, proliferation, and migration (important for hypothetical application in the clinical). Data suggest that administration of Ach at doses in a physiological range may not only be effective but is also likely to be safe. Low-dose Ach could be important for the development of new therapies for nonhealing wounds. Uberti F., et al., Stimulation of the Nonneuronal Cholinergic System by Highly Diluted Acetylcholine in Keratinocytes. Cells Tissues Organs. 2017;203(4):215-230. doi: 10.1159/000451023. Page 30 Ongoing work Animal wound model to verify the effectiveness on protocol A of 1pg/ml SKA Ach Wound analysis (planimetric area) Hystochemistry (ematoxin/Eosin staining) Muscarinic/nicotinic receptor analysis Matrix and microvascular formation Dunn et al. 2013 Page 31 Plasmatic ROS and Choline quantification Ongoing work: preliminary data Ach SKA 1pg/ml exert a better effect on closure compared to other formulation Page 32 Ach SKA 1pg/ml in a single administration exert an active role during closure THANK YOU FOR YOUR ATTENTION Physiology Lab Prof C. Molinari & GUNA Spa, Milan Italy Dr V. Miranda Page 33

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